In vivo generation of DNA sequence diversity for cellular barcoding

Heterogeneity is a ubiquitous feature of biological systems. A complete understanding of such systems requires a method for uniquely identifying and tracking individual components and their interactions with each other. We have developed a novel method of uniquely tagging individual cells in vivo with a genetic 'barcode' that can be recovered by DNA sequencing. Our method is a two-component system comprised of a genetic barcode cassette whose fragments are shuffled by Rci, a site-specific DNA invertase. The system is highly scalable, with the potential to generate theoretical diversities in the billions. We demonstrate the feasibility of this technique in Escherichia coli. Currently, this method could be employed to track the dynamics of populations of microbes through various bottlenecks. Advances of this method should prove useful in tracking interactions of cells within a network, and/or heterogeneity within complex biological samples

Sequencing the Connectome

Connectivity determines the function of neural circuits. Historically, circuit mapping has usually been viewed as a problem of microscopy, but no current method can achieve high-throughput mapping of entire circuits with single neuron precision. Here we describe a novel approach to determining connectivity. We propose BOINC ("barcoding of individual neuronal connections"), a method for converting the problem of connectivity into a form that can be read out by high-throughput DNA sequencing. The appeal of using sequencing is that its scale--sequencing billions of nucleotides per day is now routine--is a natural match to the complexity of neural circuits. An inexpensive high-throughput technique for establishing circuit connectivity at single neuron resolution could transform neuroscience research

Using high-throughput barcode sequencing to efficiently map connectomes

The function of a neural circuit is determined by the details of its synaptic connections. At present, the only available method for determining a neural wiring diagram with single synapse precision-a 'connectome'-is based on imaging methods that are slow, labor-intensive and expensive. Here, we present SYNseq, a method for converting the connectome into a form that can exploit the speed and low cost of modern high-throughput DNA sequencing. In SYNseq, each neuron is labeled with a unique random nucleotide sequence-an RNA 'barcode'-which is targeted to the synapse using engineered proteins. Barcodes in pre- and postsynaptic neurons are then associated through protein-protein crosslinking across the synapse, extracted from the tissue, and joined into a form suitable for sequencing. Although our failure to develop an efficient barcode joining scheme precludes the widespread application of this approach, we expect that with further development SYNseq will enable tracing of complex circuits at high speed and low cost

High-Side Digitally Current Controlled Biphasic Bipolar Microstimulator

Electrical stimulation of nervous tissue has been extensively used as both a tool in experimental neuroscience research and as a method for restoring of neural functions in patients suffering from sensory and motor disabilities. In the central nervous system, intracortical microstimulation (ICMS) has been shown to be an effective method for inducing or biasing perception, including visual and tactile sensation. ICMS also holds promise for enabling brain-machine-brain interfaces (BMBIs) by directly writing information into the brain. Here we detail the design of a high-side, digitally current-controlled biphasic, bipolar microstimulator, and describe the validation of the device in vivo. As many applications of this technique, including BMBIs, require recording as well as stimulation, we pay careful attention to isolation of the stimulus channels and parasitic current injection. With the realized device and standard recording hardware-without active artifact rejection-we are able to observe stimulus artifacts of less than 2 ms in duration