Validation of independent component analysis for rapid spike sorting of optical recording data

Abstract

Independent component analysis (ICA) is a technique that can be used to extract the source signals from sets of signal mixtures where the sources themselves are unknown. The analysis of optical recordings of invertebrate neuronal networks with fast voltage-sensitive dyes could benefit greatly from ICA. These experiments can generate hundreds of voltage traces containing both redundant and mixed recordings of action potentials originating from unknown numbers of neurons. ICA can be used as a method for converting such complex data sets into single-neuron traces, but its accuracy for doing so has never been empirically evaluated. Here, we tested the accuracy of ICA for such blind source separation by simultaneously performing sharp electrode intracellular recording and fast voltage-sensitive dye imaging of neurons located in the central ganglia of Tritonia diomedea and Aplysia californica, using a 464-element photodiode array. After running ICA on the optical data sets, we found that in 34 of 34 cases the intracellularly recorded action potentials corresponded 100% to the spiking activity of one of the independent components returned by ICA. We also show that ICA can accurately sort action potentials into single neuron traces from a series of optical data files obtained at different times from the same preparation, allowing one to monitor the network participation of large numbers of individually identifiable neurons over several recording episodes. Our validation of the accuracy of ICA for extracting the neural activity of many individual neurons from noisy, mixed, and redundant optical recording data sets should enable the use of this powerful large-scale imaging approach for studies of invertebrate and suitable vertebrate neuronal networks.

Fig. 1.

Deliberate mixing of signals illustrates the ability of independent component analysis (ICA) to blindly find source signals in mixed and redundant data. Three original signals—2 intracellular recording traces from Tritonia pedal ganglion neurons (A and B) and one white noise trace (C)—were linearly mixed. The mixing coefficient applied to each of the original signals is shown above each of the mixed traces. ICA performed on this 3 detector data set returned 3 independent components that matched the original signals.

Fig. 2.

ICA extracts the spiking activity of individual neurons from mixed and redundant photodiode array optical recording data. A: pre-ICA; 23 traces of a 464 trace photodiode array recording of a 4 cycle swim motor program in the dorsal pedal ganglion of Tritonia, elicited by a stimulus to pedal nerve 3 (10 V, 5 ms pulses, 10 Hz, 1 s; arrow). Typical of such recordings, neurons large enough to be detected by many diodes are recorded redundantly (e.g., red and green traces), whereas many diodes detect signals from more than one neuron, resulting in signal mixtures. Traces were filtered in Neuroplex with a Butterworth band-pass filter (between 5 and 100 Hz). Diode numbers are shown to the left of the diode traces here and in subsequent figures. B: post-ICA; 23 of the 88 neuronal independent components returned by ICA containing recognizable neural activity are shown. The independent components shown in red and green illustrate how redundancy in the optical recordings is eliminated by ICA (see arrows). C: displaying the contribution of each diode to any given component (obtained from the inverse of the ICA weight matrix) indicates the location of the component, and therefore the corresponding neuron, on the photodiode array. The diode maps for the red and green components shown in B are displayed. D: the array locations of all 23 traces shown in A are superimposed at their recording site on the pedal ganglion.

Fig. 3.

Accuracy of ICA spike sorting in a quiescent Tritonia preparation. A: illustration of the central ganglia of Tritonia (Ce, cerebral; Pl, pleural; Pd, pedal) and the optical recording setup. The hexagon over the left dorsal pedal ganglion (bottom) shows the imaged region. A larger view of the imaging area (arrow) with the image of the pedal ganglion superimposed shows the location of 2 neurons that were impaled with electrodes. B: accuracy of ICA spike sorting. Bi: pre-ICA. Current was injected into the 2 pedal ganglion neurons, making them fire trains of action potentials, which were detected optically by several diodes. Diodes 355 and 367 (adjacent diodes) each detected action potentials from both of the impaled neurons. Bii: intracellular recording traces from the 2 impaled neurons. Biii: ICA returned neuronal independent components that represented the spiking activity of individual neurons. Among them were 2 that corresponded exactly to the intracellularly recorded action potentials in the 2 impaled neurons (top 2 traces). In addition, 2 other neuronal components are shown that corresponded to the spiking activity of other neurons observed in the optical data shown in Bi. The third component corresponds to the action potentials of one of the neurons recorded by diode 380 and the fourth component corresponds to action potentials recorded by diodes 367 and 135 (adjacent diodes).

Fig. 4.

Accuracy of ICA spike sorting of a Tritonia swim motor program. Ai: pre-ICA. The spiking activity of 12 photodiodes during a swim motor program imaged in the dorsal pedal ganglion elicited by a stimulus to pedal nerve 3 (10 V, 5 ms pulses, 10 Hz, 1 s; arrow). Aii: intracellular recordings from 2 adjacent neurons during the swim motor program. Aiii: 2 of the neuronal independent components returned by ICA corresponded exactly to the 2 intracellular traces. Bi: expanded view of the top intracellular recording trace and its corresponding independent component (see boxes in Aii and Aiii). Bii: expanded view of the bottom intracellular recording trace and its corresponding independent component (see boxes in Aii and Aiii).

Fig. 5.

Accuracy of ICA spike sorting in Aplysia. A: validation of ICA by intracellular current injection and imaging in a quiescent preparation. Ai: pre-ICA. The action potentials from a neuron in the buccal ganglion driven with current injected from an intracellular electrode appear on many diodes, including 2 (369 and 380) that also detected the action potentials of another neuron. Aii: intracellular recording of the impaled neuron, showing 4 firing trains produced by the current injection. Aiii: one of the neuronal independent components (top trace) returned by ICA corresponded exactly to the intracellularly recorded activity of the impaled neuron. Another of the components returned by ICA (bottom trace) represents the activity of the other neuron whose action potentials were also detected by diodes 369 and 380 (shown in Ai). B: validation of ICA by intracellular recording and imaging during an ongoing locomotion motor program in the Aplysia pedal ganglion. Bi: pre-ICA. The action potentials from an impaled pedal ganglion neuron during the motor program are present on many diodes. A large optical artifact is also present in the diode traces. Bii: intracellular recording of the impaled neuron. Biii: one of the neuronal independent components returned by ICA corresponded exactly with the intracellularly recorded activity of the impaled neuron. Note that the optical artifact is not present in the component. Biv: expanded view of the boxes in Bii and Biii showing the correspondence between the intracellularly recorded action potentials and the optical spikes of the matching independent component.

Fig. 6.

By concatenating multiple optical files, it is possible to track the activity of individual neurons over multiple imaging sessions. The top traces show 10 of the 45 neuronal independent components returned by ICA performed on a file concatenated from 5 separate optical files acquired from the dorsal pedal ganglion of Tritonia over 24 min. The different optical recording files are shown separated by spaces. The bottom traces show intracellular recordings from 2 impaled pedal ganglion neurons that were acquired simultaneously with the optical data. The red intracellular recording trace corresponds exactly, spike-for-spike, to the red component and the green intracellular recording trace corresponds exactly, spike-for-spike, to the green component. In optical files 1, 2, and 4, current was injected into the impaled neurons to make them fire action potentials and in optical files 3 and 5, a swim motor program was elicited by pedal nerve 3 stimulation (10 V, 10 Hz, 2 s, 5 ms pulses, stimuli indicated by arrows).

Fig. 7.

ICA removes optical artifacts. Ai: pre-ICA. In an experiment imaging a nerve-evoked rhythm in the pedal ganglion of Aplysia, several diodes contain action potentials as well as large optical artifacts. Diode numbers are shown on the left. Aii: intracellular recording of a pedal ganglion neuron. Aiii: among the neuronal independent components returned by ICA is one containing the activity of a single neuron that generated most of the spikes in the optical data shown in Ai and that corresponds exactly to the intracellular recording shown in Aii. Another component returned by ICA contains the artifacts present in the optical data shown in Ai. The arrow indicates the stimulus given to pedal nerve 9 (10 V, 5 ms pulses, 10 Hz, 2 s).