Article Figures & Data
Figures
- Figure 1.
In vivo two-photon imaging of CA3 pyramidal neuron populations during wakefulness and anesthesia. A, Schematic illustration of experimental setup showing the chronic window implant above the corpus callosum and area CA1 of the intact hippocampus for R-CaMP1.07 calcium imaging in CA3. B, Histologic coronal cross-section of the fixed brain after in vivo imaging sessions showing R-CaMP1.07-labeled CA3 pyramidal neurons and the hippocampal window implant. C, Experimental time line. After recovery from the surgery, three consecutive days of anesthetized imaging were conducted. This was followed by 6–10 d of awake imaging including at least three consecutive imaging days. D, Example FOV of an anesthetized two-photon imaging session. Example neurons are labeled by red asterisks and their respective ΔF/F traces are displayed on the right. Detected calcium transient peaks are labeled with black asterisks. E, Example FOV and ΔF/F traces of neurons recorded during wakefulness. An example large calcium transient is labeled in blue. Run speed of the animal is shown below.
- Figure 2.
Calibration of in vivo spiking activity of CA3 pyramidal neurons using simultaneous two-photon calcium imaging and juxtacellular voltage recordings in acute experiments. A, Schematic of experimental setup. B, Maximum-intensity projection of two-photon images of R-CaMP1.07-labeled CA3 pyramidal neurons together with the recording pipette in juxtacellular configuration. C, Example traces of simultaneously recorded calcium transients (ΔF/F) and spontaneous APs (Vpip) during isoflurane-anesthesia. The number of APs per burst is indicated below. Insets show two example burst events magnified. D, Average calcium transients caused by 1–10 APs (within 200 ms) aligned to the occurrence of the first AP. E, Relationship between peak ΔF/F changes and number of APs (mean ± SEM). F, Relationship between the calcium transient decay time constant and the number of APs (exponential fits; error bars indicate 90% confidence interval). The heterogeneity of burst events is further analyzed in Extended Data Figure 2-1.
- Figure 3.
Large and prolonged calcium events in CA3 pyramidal neurons during wakefulness. A, Example ΔF/F traces (black) with corresponding estimated spike rates (SR, red). B, Distribution of mean ΔF/F level in a 3-s time window around calcium transient peaks (dashed line in A) under anesthesia (red) and during wakefulness (blue). Wakeful transients that exceeded the 95th percentile of anesthetized mean ΔF/F levels (37% mean ΔF/F) were classified as large events. Arrows indicate the respective mean of each distribution (awake: 23 ± 24%, anesthetized: 11 ± 10%, mean ± SD, p < 0.001, two-sided Wilcoxon rank-sum test). The lower panel, as a calibration look-up table for the upper panel, shows the relation of mean ΔF/F level to the number of APs extracted from the ground-truth dataset. The linear reference line is fitted to data points with mean ΔF/F values <40%. The 95% confidence corridor (gray shading) is based on a model-free fit of all data points. C, Average shape of calcium transients classified as anesthetized events, awake small and awake large events (mean ± SEM). D, left, Mean frequency of small calcium transients per neuron and session under anesthetized, awake resting, and awake running conditions (mean ± SEM; resting vs anesthetized: 0.70 ± 0.04, running vs anesthetized: 1.53 ± 0.08, running vs resting: 2.19 ± 0.12; ratio of means ± error propagation of SEM). Right, Mean frequency of large calcium transients across neurons during awake resting and awake running condition (running vs resting: 3.67 ± 0.83; ratio of means ± error propagation of SEM); ***p <0.001, one-way ANOVA. E, Frequency distribution of 1859 small awake events (blue) and 947 large awake events (black), normalized to the times spent at specific speeds, as a function of running speed.
- Figure 4.
Longitudinal functional imaging of CA3 population activity over consecutive recording days. A, Example FOV (left) and calcium transients of selected neurons over three consecutive imaging days during anesthesia. B, Example FOV and across-days calcium transient examples during wakefulness. Example large calcium events are labeled blue. Periods of locomotion are highlighted in gray. C, Comparison of mean ΔF/F transient amplitude, mean FWHM, and mean IEI per neuron over consecutive imaging days (dashed lines represent unity lines; for Pearson correlations see main text). For a plot of these calcium transient properties across all neurons and days, see Extended Data Figure 4-1. D, Percentage of cells firing large events for each day and over consecutive days (number of neurons: nday1 = 21, nday2 = 21, nday3 = 30, nday1+day2 = 9, nday2+day3 = 12, nday1+day3 = 11, nday1+day2+day3 = 7). E, Percentage of neurons in a FOV that show co-activity within a 1-s time window surrounding a detected event, compared with randomly shuffled event times (***p < 0.001; box displays median and 25th and 75th percentiles, whiskers indicate first and 99th percentiles; resting vs anesthetized 2.15 ± 0.05, running vs anesthetized 2.57 ± 0.06, resting vs running 1.20 ± 0.02; ratio of means ± error propagation of SEM). F, Percentage of coincident events between all neuron pairs of one example FOV over 2 d. Hierarchical link clustering was performed on the co-activity matrix of day 1, and the resulting order of neurons was maintained for day 2. G, Comparison of percentage of coincident events per neuron pair over consecutive imaging days. For this plot all data from all FOVs were pooled (experimental data: ρ = 0.34, shuffled data: ρ = 0.06). Dashed line represents unity line. H, Normalized 2D density plot based on G. For temporal aspects of co-activity and its dependence on neuron pair distance see Extended Data Figures 4-2, 4-3, respectively.
Extended Data
Extended Data Figure 2-1
Heterogeneity of burst events in CA3 pyramidal neurons from cell-attached ground-truth recordings. A, Electrophysiological recordings of three example bursts. Example two and three show intermittent bursts. B, Overview of all recorded bursts with up to 17 APs. Amplitudes were normalized to the maximum AP amplitude within the burst. C, Distribution of inter spike interval times pooled from all recordings (four neurons, three mice). The bimodal distribution can be split into two clusters using k-means clustering with one cluster around 5.3 ± 7.3 ms (mean ± SD) and the second cluster at 0.81 ± 1.51 s. D, Mean ΔF/F transient and peri-event histogram of underlying spikes for all ΔF/F transients detected as in Figure 3 (see detection criteria in Materials and Methods). In addition, we show the mean instantaneous SR, estimated from the deconvolved ΔF/F traces. The SRs were temporally smoothed compared to the peri-event histogram since our method to estimate SRs was trained with temporally smoothed ground truth data in order to be more resilient against noise (Materials and Methods). Averages of 173 detected events; shaded corridors show SEM. Download Figure 2-1, EPS file.
Extended Data Figure 4-1
Properties of calcium transients of CA3 neurons over consecutive imaging days. Neuron-wise analysis of (A) mean ΔF/F amplitude, (B) mean IEI, and (C) mean transient FWHM calculated over one session for each neuron on three consecutive days in the anesthetized, awake resting and awake running condition. Neurons that were recorded on all 3 d were sorted according to their properties on the first imaging day (nanesth. = 91 cells, nawake = 181 cells, 6 mice, 6 FOVs imaged under anaesthesia, 10 FOVs imaged during wakefulness). Download Figure 4-1, EPS file.
Extended Data Figure 4-2
Temporal aspects of neuronal co-activity in CA3. A, Effect of window size on the Pearson’s correlation measure of stability of co-activity across days (compare Fig. 4G). The analysis was performed with ΔF/F traces in the awake and anesthetized condition as well as with estimated SRs in the awake condition (SR). For the 0.1-s window, the analysis was performed on the single imaging frame for which the transient peak of the event was detected. Windows up to a duration of 2 s started 0.5 s before the transient peak. From a window size of 3 s onwards, windows started 1 s before the transient peak. Dashed vertical line indicates the1-s window chosen for analysis in the main text. B, Average temporal profile of ΔF/F traces (top) and estimated SR (bottom) in co-active neurons aligned to the peak of detected events in reference neurons. Mean traces (±SEM) for events co-active to large events are shown in red and events co-active to small events in blue. Note that the average traces do not include the original detected events (as in Fig. 3C) but only calcium transients in co-active neurons. C, Peri-event histograms of the calcium transient peaks for small (top) and large (bottom) events in co-active neurons, aligned to the calcium transient peak of the originally detected reference event. Download Figure 4-2, EPS file.
Extended Data Figure 4-3
Influence of physical Euclidean distance between neurons on neuron-pair co-activity. A, left, Correlation between ΔF/F traces of neuron pairs (y-axis) as a function of the Euclidian distance between neuronal ROI centroids (x-axis). Right, The values for neighboring neuron pairs (distances ≤10 μm) show higher mean correlation values than more distant neuron pairs (0.30 ± 0.28 and 0.14 ± 0.30; mean ± SD). B, Pearson correlation values of neuron-pair co-activity across multiple days for variable temporal windows (windows defined as in Extended Data Fig. 4-2A) from all neuron pairs (black) and from neuron pairs separated by more than 10 μm (orange), showing similar results. Dashed vertical line indicates the 1-s window chosen for analysis in the main text.1 Download Figure 4-3, EPS file.
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