Combined optical tweezers and laser dissector for controlled ablation of functional connections in neural networks

摘要

Neuronal networks in vitro represent an intermediate model situated between single neurons and brain tissue for studying neural development, coding, pharmacology, pathology, and regeneration. Understanding network dynamics requires techniques capable of monitoring the activity of an elevated number of neurons at high temporal resolution. Over the last decade, two core approaches have been favored. One of them is based on optics, as a low invasive tool for imaging the activity of many cells with subcellular resolution over a field of view of a few hundred micrometers.1 The other is the electrical extracellular recording of network activity by means of microelectrode array (MEA) devices,2 which allow the multisite monitoring of several neurons with submillisecond resolution around a limited number of electrodes distributed over a few square millimeters. Several laboratories started to combine these two methods to compensate for their complementary shortcomings in resolving and extracting neural network properties.3nThe properties of the culture substrate represent an important aspect of in vitro systems. The viscoelastic behavior of a neurite is assumed to result from the interaction of cytoskeletal components with the extracellular matrix (ECM).4 Indeed, not only chemical but also mechanical properties of the ECM can regulate cellular properties such as shape, polarity, motility, and morphological phenotypes. Alteration in the interactions between a cell and its surrounding ECM may result in apoptosis, malignant transformation, or loss of tissue architecture.5 The ability of a laser dissection system to selectively ablate subcellular compartments without damaging adjacent structures offers new possibilities for studying cell-ECM interactions6,7 or axonal regeneration.8,9nIn this work, we present the design and evaluate the performance of a system that combines two optical manipulation techniques, holographic optical tweezers (OTs), and a laser micro-dissector (LMD), to directly quantify the mechanical perturbation produced by laser ablation in a neurite, and to study in vitro regeneration of cellular processes after well-calibrated, laser-inflicted damage. Moreover, the system is configured to work in combination with fluorescence imaging and MEA electrophysiology to track and dissect neuronal interconnections for elucidating their local and network-wide role. For this purpose, we used low-density neural cultures to evaluate the architecture of the neural network, and to achieve higher spatial control of the neuronal connections we dissect and the structural changes we impose on the network itself.nThe presented system will be applied to the study of processes involved in the early stages of neuronal differentiation, with particular focus on neurite regeneration and neural network dynamics.