Computational processing of optical measurements of neuronal and synaptic activity in networks

Abstract

Imaging of optical reporters of neural activity across large populations of neurones is a widely used approach for investigating the function of neural circuits in slices and in vivo. Major challenges in analysing such experiments include the automatic identification of neurones and synapses, extraction of dynamic signals, and assessing the temporal and spatial relationships between active units in relation to the gross structure of the circuit. We have developed an integrated set of software tools, named SARFIA, by which these aspects of dynamic imaging experiments can be analysed semi-automatically. Key features are image-based detection of structures of interest using the Laplace operator, determining the positions of units in a layered network, clustering algorithms to classify units with similar functional responses, and a database to store, exchange and analyse results across experiments. We demonstrate the use of these tools to analyse synaptic activity in the retina of live zebrafish by multi-photon imaging of SyGCaMP2, a genetically encoded synaptically localised calcium reporter. By simultaneously recording activity across tens of bipolar cell terminals distributed throughout the IPL we made a functional map of the ON and OFF signalling channels and found that these were only partially separated. The automated detection of signals across many neurones in the retina allowed the reliable detection of small populations of neurones generating "ectopic" signals in the "ON" and "OFF" sublaminae. This software should be generally applicable for the analysis of dynamic imaging experiments across hundreds of responding units.

Fig. 1

Outline of the analysis procedures. (a) The eye of a zebrafish larva 8 dpf expressing SyGCaMP2 in photoreceptor and bipolar cells was imaged on a multi-photon microscope at a resolution of $2.6\times 2.6\times 2\mu$m/pixel. Data shown in Figs. 2, 3, 5 and 6 was recorded from the area delimited by the red box. INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; scale bar $=100$ $\mu$m. (b) Outline of the stages of analysis of optical recordings.

Fig. 2

Synaptic terminals responding to a full field light stimulus. Selected frames of a recording from retinal bipolar cell terminals in the inner plexiform layer of the retina of a 8 dpf zebrafish expressing SyGCaMP2. (a) High resolution ($512\times 512$ pixels) overview of the area imaged. INL, inner nuclear layer; a, sublamina a of the inner plexiform layer; b, sublamina b of the inner plexiform layer. (b) A single frame from the original movie, recorded at $128\times 100$ pixels at 5 Hz in the dark. (c) A single frame recorded while a full field amber light stimulus (13 mW/m²) was presented, indicated by an amber dot. Two terminals responding with an increase in fluorescence are marked by down-facing arrows, and two terminals in close proximity to each other responding with a decrease in fluorescence are marked with an up-facing arrow. The numbers next to the arrows correspond to the numbers of the respective ROIs in Figs. 4 and 6. (d) A single frame recorded 30 s after the light stimulus. Scale bars $=25\mu$m.

Fig. 3

Thresholding using the Laplace operator. (a) Temporal average of the movie shown in Fig. 2. (b) Laplace operator of the image shown in (a). The image has been inverted (i.e. negative values are bright) and values $\ge$ 0 are black. (c and d) Line profiles measured from (a) and (b), respectively, as indicated by the red lines. The dashed lines represent thresholds required to detect the peaks between 40 and 55 $\mu$m. (e) ROI mask generated from the temporal average (a) using the threshold shown in (c). (f) ROI mask generated from the Laplace operator (c) using the threshold shown in (d). Scale bars $=25\mu$m.

Fig. 4

Spontaneous and light-evoked activity in the IPL. (a) Fluorescence data from 59 terminals, identified from the same set of data shown in Figs. 2 and 3. The lower graph shows the duration and intensity of the full field amber light stimulus. The numbers of the ROIs correspond to those shown in Fig. 6b. (b) Selected terminals responding to the light stimulus. ON responses (ROIs #12 and #28) show an increase in Ca²⁺ concentration during the light stimulus, OFF responses (ROI #2) a decrease. (c) Selected terminals showing spontaneous regenerative Ca²⁺ transients.

Fig. 5

Hierarchical clustering to sort traces. (a) Distance matrix showing correlation between the traces shown in Fig. 4. The distance was calculated as $1-$ Pearson’s r. (b) The positions of terminals in six distinctive clusters are overlaid on an overview of the recorded region. Scale bar $=25\mu$m. (c) Traces (grey) and averages (black) of six distinctive clusters. Amber bars indicated when a full-field light stimulus was presented. The symbols in the upper right corner correspond to the symbols in (b).

Fig. 6

Determinig positions within the IPL. (a) Centres of mass of the ROIs determined as shown in Fig. 3 (red circles) and manually outlined borders (white dots). (b) Index numbers of the ROIs, corresponding to the traces shown in Fig. 4a. (c) Interpolated and smoothed borders (bold white lines) and four representative isocontours (thin white lines). (d) Table of relative positions. Scale bars $=25\mu$m.

Fig. 7

Distribution of synaptic terminals in the IPL. (a) Distribution of more than 400 bipolar cell terminals in the IPL, recorded in vivo from zebrafish larvae (8 dpf) expressing SyGCaMP2. (b) Subset of terminals that responded with a depolarising (ON) response to a light stimulus. (c) Subset of terminals that responded with a hyperpolarising (OFF) response to a light stimulus.