High precision spatial and temporal control of neural circuitry using semi-automated, multi-wavelength nanopatterning system

The ideal way to manipulate neurons would be to reversibly activate and inactivate neuronal firing and thus enable a bi-directional control. Many attempts have been made in the past to have such a tool. The traditional approach for controlling neural circuits is to generate a local electric field which has been shown to be highly effective at triggering action potentials in neurons [53]. Although the timing of the electrode stimulation is very precise, its specificity and spatial control are poor. Also, silencing neuronal activity is as important as stimulating it. Ed Boyden described a protein that switches off nerve firing when activated by light. Karl Deissoroth described the fuller story of the protein called NpHR (Natronomonas pharaonis)[62]. This allows for a spatially and temporally precise neural silencing. Earlier, Boyden worked on a channel for positively charged ions (such as calcium) that is found in green algae and is activated by blue light. This channel, ChR2 was transplanted into mammalian neurons and it was possible for the first time to stimulate a nerve remotely at speeds closer to normal neuronal transmission [52]. The newly discovered NpHR protein is a chloride pump which silences physiological activity, when activated by yellow light. NpHR, in spite of it being a pump rather than a channel, operates at a speed close to that of ChR2 leading to a close to perfect temporal precision. Using these two proteins to alternately switch the neurons on and off, a bi-directional optical control switch could be obtained. In Zhang, et al's work published in April 2007, groups of neurons (cholinergic motor neurons) were targeted to control bi-directional locomotion. This opened a new gateway for a multimode, high-speed, genetically targeted, all-optical interrogation of living neural circuits [50].;However, there is a need for spatial precision along with temporal precision. This need, leading to a method for precise spatial control of neuronal circuitry along with temporal controls will be discussed in this thesis. With spatial resolution, one could go a step further and interrogate inter neuronal connectivity instead of targeting clusters of neurons.;The aim of the work presented here was to target individual neurons and their inter-connectivity. For this purpose, a novel instrument was built which is an inter-disciplinary project that includes cutting edge technologies such as the DLP (Digital Light Processing) technology, genetically engineering the embryonic neurons to express two protein channels (NpHR and ChR2); and isolation and culture of these neurons. This work also used standard laboratory techniques such as the fluorescence microscopy and optics.