Combined Atomic Force Microscopy/Fluorescence Spectroscopy approach for measuring adhesion, connectivity and electrical activity of neurons patterned on 2-dimensional protein subst

Tufts University, Medford MA

Investigators

Abstract

1067093 Staii Intellectual merit: The objective of this proposal is to gain a deeper understanding of the basic rules that neuronal cells use to form functional connections with one another. Understanding the brain is of tremendous fundamental importance, but it is immensely challenging because of the complexity of both its architecture and function. The central nervous system consists of many different spatially localized and yet highly interconnected regions. To date the processes involved in forming functional neuronal connections, the mechanisms of axonal navigation to their target region and their specific interactions with guidance factors such as chemical gradients and mechanical cues are still largely unknown. The scientific goal of the current project is to understand the fundamental processes governing the development of connections and communications between neurons in living systems by studying the growth and interconnectivity of small numbers of neurons patterned in simplified, well-controlled geometries. The central hypothesis is that simplifying the neuronal growth environment by creating highly controlled neuronal circuits in vitro will allow the basic rules that underlie neuronal development and the formation of neural connections to be elucidated. Simple neuronal networks will be created on two dimensional substrates, guiding the formation of synapses and measuring their electrical activity using a) atomic force microscope nanolithography; b) atomic force imaging and atomic force based electrical force microscopy; c) fluorescence spectroscopy. Specifically, one aims to: 1) pattern different types of proteins/growth factors at precise locations on surfaces and use them as growth templates for fluorescently labeled neurons; 2) guide the formation of neuronal synapses by controlling the type and geometry of the underlying protein patterns; 3) systematically investigate the adhesion and growth of neuronal processes using both atomic force and fluorescence spectroscopy measurements; 4) map the electrical activity of the network by combined electrical force microscopy and fluorescence microscopy. The crucial aspect for this last step is the use of a voltage-biased atomic force tip as a movable electrode to both stimulate and record the electrical activity of patterned neurons, both at the synapse level and along the neuronal pathway. Simultaneous fluorescence monitoring will identify the specific signaling molecules released during synapse formation as well as during the propagation of the electrical signal. By performing these experiments one seeks to a) quantify the role that different types of biochemical and geometrical cues play in neuronal growth and development; b) to measure under what conditions synaptic junctions are functional and c) to learn to control the formation of functional synapses in neuronal circuits having well-defined geometries. Broader Impacts: The proposed research may lead to great insights into diseases that result when the growth of neuronal processes fails, including birth defects, mental disorders, and sensory-motor deficits. Further, options to direct nerve-material interfaces have broad applicability for prosthetic devices to better mimic human functions. A specific goal for broader impact will be to use the research in the grant as a focused teaching tool for the undergraduates. Specifically, the investigators will establish a Research Mentorship Team which will provide undergraduate students with: a) research intensive experience b) multidisciplinary teams and projects (integration between physics, biology and engineering) such that the students gain exposure to broader thinking outside of their own discipline; c) mentorship experience at the undergraduate level, as senior students will serve as the upper class mentors to the second and third year undergraduate students helping to prepare them for their senior year. The postdoctoral researcher and graduate student involved in the grant will be part of the mentorship team. As part of this activity the investigators will also work directly with Tufts Center for Engineering Education Outreach to explore how to modularize the tools and teaching for use in the broader outreach activities.