Neurons communicate information through fluctuations in the electrical potentials across their cellular membranes. Whole-cell patch clamping, the gold standard technique for measuring these fluctuations, is something of an art form, requiring great skill to perform on only a few cells per day. Thus, it has been primarily limited to in vitro experiments, a few in vivo experiments, and very limited applications in the awake brain. Dr. Forest (and collaborator Dr. Boyden at MIT) developed a robot that automatically performs patch clamping in the living brains of mice by algorithmically detecting cells through analysis of a temporal sequence of electrode impedance changes. Using it, they have demonstrated good yield, throughput, and quality of recording in mouse cortex and hippocampus. With this 'autopatching' robot enabling routine access to electrical and molecular properties of neurons, systematic and scalable in vivo experiments as well as fundamentally new kinds of single-cell analyses have become possible. In the past 12 months, the team has installed 15 autopatchers in academic research laboratories, garnered worldwide media coverage, and led to Dr. Forest's and Dr. Boyden's invitations to President Barack Obama's announcement of the BRAIN Initiative. There are currently no published experiments demonstrating in vivo intracellular recordings of two or more neurons that are synaptically connected. We propose to utilize the autopatcher to target anatomically well-studied sub-circuits to significantly increase the odds of identifying synaptically connected pairs. Specifically, we wil utilize the thalamocortical circuit in the mouse vibrissa/whisker pathway as a model experimental system, where there is a substantial convergence of projections from the thalamus to the input layer in the somatosensory (tactile) cortex. The Stanley Laboratory has extensive experience with stimulation and electrophysiological recordings in this circuit, and is one of only a few laboratories that has successfully recorded from synaptically connected pairs of neurons using extracellular techniques. Thus we aim to demonstrate and characterize the first simultaneous intracellular recording of a functional circuit in the anesthetized and awake living mouse brain to reveal its neural network dynamics. In this 36 month program, the labs of Prof. Stanley and Forest, supported by two postdoctoral researchers, two graduate research assistants, a research engineer and five undergraduates, with assistance from ten graduate students working on related projects, will develop single (Aim 1) and dual (Aim 2,3) autopatching robots for the anesthetized and awake brain. Success will allow, for the first time, quantification of synaptic efficacy in the living brain, crucial for understanding normal and pathological function. Just as molecular biology has greatly benefited from the revolution in in vitro automation, we believe that neuroscience will greatly benefit from the revolution in in vivo automation that we have launched, and here propose to extend.
We will develop powerful, easy-to-use tools that enable intracellular recording and stimulation of functional circuits in the living brain. There are curretly no published experiments demonstrating such in vivo intracellular recordings of two or more neurons that are synaptically connected. This application represents a significant step toward developing and validating novel tools to facilitate the detailed analysis of complex circuits (a BRAIN Initiative high priority research area), thus contributing to the progression of neuroscience from observation to causation.
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