Separation of single neurons from optical recordings in Tritonia diomedea using ICA

Abstract

Event Abstract Back to Event Separation of single neurons from optical recordings in Tritonia diomedea using ICA Optical recordings obtained with photodiode arrays and voltage-sensitive dyes allow firing of large numbers of neurons to be recorded during behaviorally-relevant motor programs. Although this technique has tremendous potential for network studies, the resulting datasets can be challenging to interpret; one detector may record multiple neurons and one neuron may appear on many detectors. Earlier studies applied independent component analysis (ICA), a blind source separation technique, to optical traces from Tritonia diomedea's escape swim behavior, to recover components from individual neurons [1] and identify unreported neurons involved in the behavior [2]. Using intracellular and optical methods [3], we evaluate the accuracy of neuronal activity that ICA returns and demonstrate an application of these methods, measuring the change in individual neurons’ activity following stimulus adaptation. To evaluate accuracy, we recorded from neurons in Tritonia with intracellular electrodes while imaging with a 464-element photodiode array using the fast voltage-sensitive absorbance dye RH-155. ICA was run on the optical traces, transforming them into statistically independent components. ICA returned one component for neurons detected by multiple diodes and separated neurons mixed on a single diode. Intracellular recordings confirmed the accuracy of this technique. For each intracellular recording, we found a component that corresponded exactly. Also, the location returned by ICA matched that of the electrode. Additionally, these methods allow us to drive an identified neuron and image its functional connectivity to large numbers of other individual neurons, before and after a treatment of interest. We imaged the responses of several pedal ganglion neurons to a test train of action potentials in CPG neuron C2 driven with an intracellular electrode. Then we applied a sensitizing nerve shock stimulus. Two minutes later we imaged the responses of the same neurons to a second test train. Combining the records and applying ICA, we observed how responses of individual neurons changed after the sensitizing stimulus. Optical recordings in combination with ICA allow quick identification of functional synaptic connections onto previously unknown neurons. Using location maps from ICA, those neurons can be penetrated with intracellular electrodes within the same experiment, enabling new experiments to probe the Tritonia brain over a wide range of conditions and time scales. Given our intracellular validation of ICA's unmixing, these methods promise to be a powerful tool for discovery of unreported neurons and studies of functional connectivity and network plasticity.