Combining voltage-sensitive dye, carbon fiber array, and extracellular nerve electrodes using a 3-D printed recording chamber and manipulators

Background: The analyses of neuronal circuits require high-throughput technologies for stimulating and recording many neurons simultaneously with single-neuron precision. Voltage-sensitive dyes (VSDs) have enabled the monitoring of membrane potentials of many (10-100 s) neurons simultaneously. Carbon fiber electrode (CFE) arrays allow for stimulation and recording of many neurons simultaneously, including intracellularly.New method: Combining CFE with VSD leverages the advantages of both technologies, allowing for stimulation of single neurons while recording the activity of the entire network. 3-D printing technology was used to develop a chamber to simultaneously perform VSD imaging, CFE array recording, and extracellular recording from individual glass electrodes.Results: Aplysia buccal ganglia were stained with VSD and imaged while also recording using a CFE array and extracellular nerve electrodes. Coincident spiking activity was recorded by VSD, CFE, and extracellular nerve electrodes. Current injection with CFE electrodes could activate and inhibit individual neurons as detected by VSD and nerve recordings.Comparison to existing methods: The large size of traditional manipulators limits the number of electrodes used and the number of neurons recorded during an experiment. Here we present a method to build a 3-D printed recording chamber that includes a 3-axis micromanipulator to position a CFE array and eight 2-axis manipulators to position eight extracellular electrodes.Conclusions: 3-D printing technology can be used to build a custom recording chamber and micromanipulators. Combining these technologies allows for the direct modulation of the activity of neurons while recording the activity of 100 s of neurons simultaneously.