The functional activity and dysregulation of neuronal circuits relies critically on the physiology of neuronal synapses, which are challenging to analyze because they appear in great numbers, and they are difficult to record in vivo, especially in relation to the dynamic neural codes generated by specific neurons. To make things even more complex: synapses are incredibly dynamic in fashions that are dependent on recent history, sensory stimuli, disease state, and other behaviorally relevant contexts. Ideally there would be a technology that would allow for individual investigators to rapidly analyze synapses between neurons exhibiting neural codes in a behavioral context, so that it is possible to understand how information is trans formed at synapses. We here propose to develop a simple, easily deployable toolbox for achieving this, building from several recent discoveries. First, we have found (manuscript in preparation) that it is possible to automatically perform whole cell patch clamp neural recording of cells in the living mouse brain that have been identified via two-photon fluorescence microscopy (e.g., cells of a given type that express a genetically encoded fluorophore). We here propose to invent a multiple-neuron patching version of this ?imagepatching? robot, to enable the simultaneous characterization of the neural codes in multiple neurons, as well as the synaptic connections between them (Aim 1). We will also develop miniaturized and optimized hardware capable of performing imagepatching, neurosurgery, and patch clamp electrode reuse for improved yield and throughput of synaptic assessment.
(Aim 2). Also, we have discovered that it is possible to physically expand preserved neural circuits, by embedding them in swellable polymers, and then chemically expanding those polymers, a technology we call expansion microscopy (ExM), which enables nanoscale imaging of 3-D tissues and organisms. We propose to optimize ExM for the analyses of synapses (Aim 3). We here propose a fast-paced, 4-year grant, to create a powerful, easy-to-use toolbox that makes the critical task of in vivo synaptic physiology into a routine, automated procedure. We will distribute all tools and datasets as freely as possible, sharing all algorithms, circuit designs, and assembly instructions, and hosting visitors to learn these technologies ? for which we have an extensive track record.

Public Health Relevance

Synapses are key to neural computation, and compromised in many brain disorders. A technology that would empower scientists to characterize synaptic functions in vivo could greatly accelerate our ability to understand how synapses change in disease, and to pinpoint new clinical targets. By enabling a robotic approach to characterizing synaptic properties, this proposal will empower high-throughput analyses of synapses in vivo.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS102727-02
Application #
9495764
Study Section
Bioengineering of Neuroscience, Vision and Low Vision Technologies Study Section (BNVT)
Program Officer
Langhals, Nick B
Project Start
2017-07-01
Project End
2021-03-31
Budget Start
2018-04-01
Budget End
2019-03-31
Support Year
2
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Biology
Type
University-Wide
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
Lee, John; Kolb, Ilya; Forest, Craig R et al. (2018) Cell Membrane Tracking in Living Brain Tissue Using Differential Interference Contrast Microscopy. IEEE Trans Image Process 27:1847-1861
Asano, Shoh M; Gao, Ruixuan; Wassie, Asmamaw T et al. (2018) Expansion Microscopy: Protocols for Imaging Proteins and RNA in Cells and Tissues. Curr Protoc Cell Biol 80:e56
Kodandaramaiah, Suhasa B; Flores, Francisco J; Holst, Gregory L et al. (2018) Multi-neuron intracellular recording in vivo via interacting autopatching robots. Elife 7:
Karagiannis, Emmanouil D; Boyden, Edward S (2018) Expansion microscopy: development and neuroscience applications. Curr Opin Neurobiol 50:56-63
Stoy, W A; Kolb, I; Holst, G L et al. (2017) Robotic navigation to subcortical neural tissue for intracellular electrophysiology in vivo. J Neurophysiol 118:1141-1150
Suk, Ho-Jun; van Welie, Ingrid; Kodandaramaiah, Suhasa B et al. (2017) Closed-Loop Real-Time Imaging Enables Fully Automated Cell-Targeted Patch-Clamp Neural Recording In Vivo. Neuron 95:1037-1047.e11
Singer, Annabelle C; Talei Franzesi, Giovanni; Kodandaramaiah, Suhasa B et al. (2017) Mesoscale-duration activated states gate spiking in response to fast rises in membrane voltage in the awake brain. J Neurophysiol 118:1270-1291
Suk, Ho-Jun; van Welie, Ingrid; Kodandaramaiah, Suhasa B et al. (2017) Closed-Loop Real-Time Imaging Enables Fully Automated Cell-Targeted Patch-Clamp Neural Recording In Vivo. Neuron 96:244-245
Kolb, I; Stoy, W A; Rousseau, E B et al. (2016) Cleaning patch-clamp pipettes for immediate reuse. Sci Rep 6:35001