Simultaneous calcium imaging and extracellular recording from the same neuron

Calcium imaging is a powerful method to record from many neurons simultaneously. But what do the recorded signals really mean?

This question can only be properly addressed by experiments which record both calcium signals and action potentials from the same neuron (ground truth recordings). These recordings are technically quite challenging. So we assembled several existing ground truth datasets, and in addition recorded ground truth datasets ourselves, totaling >200 neuronal recordings.

This blog blog posts contains raw movies together with recorded action potentials (black; also turn on your speakers for the spikes!) and the recorded ΔF/F of the calcium recording (blue). These ground truth data are a very direct way for everybody into calcium imaging to get an intuition about what is really going on. (Scroll down if you want to see recordings in zebrafish!)

Recording from a L2/3 neuron in visual cortex with GCaMP6f, tg(Emx1), from Huang et al., bioRxiv, 2019; a very beautiful recording. Replayed with 2x speed.

Recording from a L2/3 neuron in visual cortex with GCaMP6f, tg(Emx1), from Huang et al., bioRxiv, 2019. Stronger contamination from surrounding neuropil.

Recording from a L2/3 neuron in visual cortex with GCaMP6f, tg(Emx1), from Huang et al., bioRxiv, 2019. Note that single action potentials don’t seem to have any impact at all. – The negative transients in the calcium trace stem from center-surround neuropil decontamination (activity of the surround is subtracted).

Recording from a L2/3 neuron in visual cortex with GCaMP6s, tg(Emx1), from Huang et al., bioRxiv, 2019.

Recording from a L2/3 neuron in visual cortex with GCaMP6s, tg(Emx1), from Huang et al., bioRxiv, 2019.

Recording from a L2/3 neuron in visual cortex with GCaMP6f, virally induced, from Chen et al., Nature, 2013. From the left, you can see the shadow of the patch pipette used for recording of extracellular signals.

Something completely different: recording from a pyramidal neuron in Ca3 with R-CaMP1.07, virally induced, recorded by Stefano Carta, from Rupprecht et al., bioRxiv, 2020. What appears as single events are actually bursts of 5-15 action potentials with inter-spike-intervals of <6 ms.

A recording that I performed myself in adult zebrafish, in a subpart of the homolog of olfactory cortex (aDp) with GCaMP6f, tg(neuroD), in Rupprecht et al., bioRxiv, 2020. Around second 20, it is visible that even a single action potential can be seen in the calcium signal. However,this was not always the case in other neurons that I recorded from the same brain region.

Again a recording that I did in adult zebrafish, in the dorsal part of the dorsal telencephalon with GCaMP6f, tg(neuroD), in Rupprecht et al., bioRxiv, 2020.

What can you do if you want to detect single isolated action potentials with calcium imaging? GCaMP, due to its sigmoid non-linearity, is by often a bad choice and will be strongly biased towards bursts. Synthetic indicators, however, are very linear in the low-calcium regime. – This is a recording that I did myself in adult zebrafish, in a subpart of the homolog of olfactory cortex (pDp) with the injected synthetic indicator OGB-1 in Rupprecht et al., bioRxiv, 2020. Although the temporal resolution of the calcium recording is rather low, the indicator clearly responds to single action potentials. As another asset, the indicator not only fills the cytoplasm of the neuron in a ring-like shape, which makes neuropil-contamination much less of an issue compared to GCaMPs.

Another recording that I performed in adult zebrafish, in a subpart of the homolog of olfactory cortex (pDp) with the injected synthetic indicator Cal-520 in Rupprecht et al., bioRxiv, 2020. This indicator is much more sensitive compared to OGB-1, but also diffuses less well after bolus injection. – These two minutes of recording only contain 4 spikes (this brain region really is into low firing rates in general), but you can clearly see all of them. If this were a GCaMP recording, you would probably see only a flat line throughout the entire recording.

For more information, including all 20 datasets with >200 neurons (rather than these excerpts from 11 neurons), check out the following resources:

This entry was posted in Calcium Imaging, Data analysis, electrophysiology, machine learning, Microscopy, Neuronal activity, zebrafish and tagged , , , , . Bookmark the permalink.

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