A micro-optrode for simultaneous extracellular electrical and intracellular optical recording from neurons in an intact oscillatory neuronal network

Abstract

Fluorescent dyes and proteins have become ubiquitous tools for the study of intracellular function. However, standard techniques used to visualize these indicators in the brain usually require cellular dissociation or fine sectioning of the specimen into slices for viewing under a fluorescence microscope. These actions remove cells from their natural physiological environment and in the case of neurons, sever synaptic connections from other regions of the central and peripheral nervous system. Even with the use of multi-photon excitation microscopy, resolution of neurons in the intact brain beyond depths of around 500 μm is technically difficult to achieve. We have developed a relatively inexpensive small fiber optic probe (‘micro-optrode’) to simultaneously record extracellular electrical activity and intracellular fluorescence changes in real-time from structures deep within the intact brain. The micro-optrode was tested during experiments in the in situ working heart–brainstem preparation (WHBP) of rat, which allows the study of the intact ponto-medullary neuronal network that controls breathing. In conjunction with calcium-sensitive indicators, we successfully validated the utility of the micro-optrode by recording, for the first time, intracellular calcium dynamics in respiratory neurons of the intact respiratory network under normal conditions and during physiological and pharmacological manipulations.