| We have perfected a culture system that allows us to grow dissociated neurons in a dish where they can be directly observed. The neurons form connections and self-assemble into functional networks, allowing us access to the same phenomena of plasticity that occur in vivo. The system is also amenable to genetic transfections, and provides a very short incubation period for studying the role of various genes. Best of all, the cultured cells can be viewed, measured and manipulated without dissection, providing what is perhaps the most convenient preparation for plasticity studies. |  |
| The lab incorporates a wide variety of staining and dynamical imaging techniques for locating synapses and characterizing their functional capabilities. Functional dyes such as FM1-43 and FM4-64 are used in nearly all of our experiments to depict which synapses on a given cell or network are functional, as well as providing some index of the synapses’ strengths. We have also been experimenting with methods for using the exchange of these dyes to visualize synaptic activity, and have recently fostered collaborations to develop new staining molecules for better functional assays. These methods can combine with calcium-sensitive fluorescence and immunostaining to provide a visual description of the synapses along a cultured cell or neural network. |  |
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A major challenge in studying the synapse is how to tell what observed effects are due to presynaptic factors and which to postsynaptic ones. A large proportion of the lab’s efforts have gone towards finding a technique to directly control the behavior of one side of the synapse – the presynaptic one – by replacing it with an artificial terminal under our control. By using a carefully optimized form of iontophoresis, we are able to deliver specified profiles of neurotransmitter directly to single synapses. A slight holding potential can retain transmitter inside a quartz micropipette that can then be robotically positioned directly alongside a single synapse (located using dyes such as those described above). A specialized amplifier and software program then delivers an iontophoretic current to eject transmitter from the pipette, generating a time course that exactly mimics that observed during real synaptic transmission. A patch-clamped electrode on the receiving cell can then record any post-synaptic activity that results from this simulated presynaptic release. This method therefore allows the researcher to study the synapse one side at a time, by effectively replacing the presynaptic terminal with an artificial “terminal” capable of delivering arbitrary profiles of neurotransmitter. |
 
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| One of our recent techniques is to provide a complete functional description of a given neuron’s dendrites. After labeling functional synapses with FM1-43, automated computer software drives robotic manipulators to guide iontophoresis pipettes to synaptic sites. Applying iontophoretic pulses while patch clamping the postsynaptic cell then yields a functional description of each synapse’s strength. After approximately five minutes a dendritic tree of many synapses can be precisely mapped, giving information that can be useful for describing poly-synaptic interactions within a given neuron. |  |
| The use of cultured neurons permits direct visualization that we try to realize via the best optical equipment. The lab is equipped with three confocal microscopes, many with muiltiple lasers, that provide high-resolution imaging of cells and fluorscent markers during recording and stimulation. The lab has also recently gained access to a two-photon microscope setup, as well as acquired our own high-resolution digital video microcamera for rapid, real-time visualization of network activity. |  |
| We are also equipped with full facilities for transfecting our cell cultures with inserted genes, with up to 50% of all cells within a culture successfully transfected. Also, thanks to a collaboration with Prof. Susumu Tonegawa, we are able to probe questions of synaptic plasticity in vivo at the physiological and behavioral levels, by creating mice overexpressing or deficient in particular genes. |  |
| The lab is unusual among groups working at a similar level of experimental biology in the number of students with computational backgrounds that we employ. Our group has numerous members with degrees in computer science and engineering, and we hope to continue to attract more. Modeling studies are currently underway to predict the transmitter release and binding kinetics at single synapses, while other models seek to discern equations governing the homeostasis of synaptic input. We especially hope to require computational analysis in the coming years as our cultured neural network technology matures. Towards this end, collaborations are currently underway with students in the Seung Lab for Theoretical Neurobiology. |  |
| One of our recent acquisitions is a 64-channel multi-electrode array, which supports recording and inducing the electrical activity of many cultured neurons. The system can be used in conjunction with patch clamp and iontophoresis, as well as our visualization protocols. Commercial software allows for in-depth analysis of network activity in real time, and stimulation protocols that automatically respond to the system's observations. We have been working on ways to deploy this system in an incubator to extend our interface with the cells from hours to days. |  |
