In vivo two-photon [Ca2+] imaging from head restrained mice during spatial navigation in a virtual corridor

(A) Double transgenic mice (GCaMP6s and Cre-dependent td-Tomato) were injected with a diluted AAV expressing Cre-recombinase for sparse td-Tomato labelling. Following craniotomy and cannula implantation above the left dorsal CA1 area, animals were trained and imaged with a two-photon (2P) microscope during navigation in an ∼8-meter-long virtual corridor. Timeline shows the minimum, (median), and maximum number of days between the procedures.

(B) Mean 2P image of an imaging field of view. Example cells shown in panel (E), (G) and (I) are indicated by colored circles.

(C) Wall pattern of the virtual corridor consisting of low contrast random checkerboard background with 6 high contrast vertical visual landmarks.

(D) Average running speed (orange: mean; ± SD: grey) decreased and lick propensity (blue) increased before the animal reached the reward zone (green area).

(E) ROI masks and [Ca2+] signals during a single session of the cells marked in panel (B). Gray segments correspond to odd laps in the virtual environment.

(F) Position of the animal along the corridor aligned with the fluorescence activity shown in panel (E)

(G, H) Same as panel (E) and (F) on an extended time scale showing a single lap (indicated by * in (E)). Time periods when the animals’ running speed was below 5 cm/s are shown in red and were excluded from the analysis. Black vertical bars are inferred activity.

(I) Raster plots showing the inferred neuronal activity of three cells (color coded cells in panel (B), (E) and (G)) as a function of the lap number and spatial location of the animal.

(J) Coverage of the virtual corridors by place fields (PFs). Tuning curves from cells with at least one significant PF are included. The tuning curves are ordered by the location of their largest peak activity. Left panel: Data from a single session. Right panel: Data from all experiments (22,325 place cells recorded across 163 sessions from 45 mice) was randomly down sampled to match the sample size of the single run.

(K) Distributions of the number of PFs of place cells, PF widths, and PF coverage of the virtual corridor. Data are presented for all sessions and for a single session (insets).

Comparison of [Ca2+] transients underlying different place field formation events

(A) BTSP-like PFFs. Activity raster plots (left) show three example cells with BTSP-like PFFs (red arrows). The middle panels display the isolated activity of newly formed PFs marked by red arrows. Fluorescence traces (right) are shown before, during (red traces), and after PFF (± 5 laps).

(B) Same as (A), but for non-BTSP-like PFFs (blue arrows and traces).

(C) Comparison of [Ca2+] transients before, during, and after PFF. Fluorescence traces for BTSP- (left) and non-BTSP-like (right) PFFs are shown for each lap spanning from the pre-formation lap (FL-1) to the fourth lap after formation (FL+4). Traces were aligned to the midpoint of the steepest rising slope for detectable [Ca2+] transients, or to the timepoint at which the place field center was reached for traces lacking transients. The mean fluorescent transients are shown in red for BTSP and blue for non-BTSP-like events. Insets: Peak-normalized and peak-aligned mean fluorescence traces are superimposed. Bar graphs depict the mean raw fluorescence activity (± SD, BTSP: n = 310 events, non-BTSP: n = 59 events from 30 mice) in the given lap. Statistical analysis revealed that the amplitude of the [Ca2+] transients in the formation lap is significantly higher for the BTSP compared to the non-BTSP-like events and the amplitudes in the other laps are not significantly different (Mixed ANOVA: main effect for group (BTSP/non-BTSP): p = 0.033; lap: p < 0.001, interaction: p < 0.001; Tukey post hoc test: formation lap: p < 0.001, all other laps: p > 0.787).

Newly formed place fields cover the entire virtual corridor and have non-uniform birth rates

(A) The tuning curves of BTSP- (left) and non-BTSP-like (right) PFs were ordered by the center position of their newly born PFs (white space bins).

(B) The width of BTSP-like newly formed PFs is correlated with the animal’s running speed during PFF. Scatter plot shows individual BTSP- (red symbols) and non-BTSP-like (blue symbols) PFF events (Spearman correlations: BTSP: R = 0.40, p = 3.4×10-13; non-BTSP: R = 0.15, p = 0.25).

(C) Histogram of the number of newly formed PFs per single session in the virtual corridor. Note that a few sessions have unusually high (>20) new PFF events.

(D) Number of newly formed PFs by laps. Example sessions showing high number of BTSP- (red) and non-BTSP-like (blue) PFFs (upper panels, two of the largest three sessions of the histogram in panel (C)) and sessions with moderate number of PF formations (lower panels). The histograms are aligned with lap-by-lap population vector correlograms, which reveal sudden change in population activity for the upper panels and lack of such representational switch for the lower panels.

(E) [Ca2+] transients of PFF events during representational switches (switch, isolated from sessions with the three highest number of PFF event in the histogram in panel (C)) are compared to PFF events outside representational switches (no switch) for BTSP- (left) and non-BTSP-like (right) events. Fluorescence traces are shown from the lap preceding PFF (FL-1) to the lap following PFF (FL+1). Traces were aligned to the midpoint of the steepest rising slope, or to the timepoint at which the PF center was reached (for traces without detectable transients). The mean traces are shown in orange, red, cyan and blue. Insets: peak-normalized and peak-aligned mean fluorescence traces. Bar graphs depict the peak of the mean fluorescence waveform (mean ± SD; BTSP no switch: n = 270, BTSP switch: n = 40, non-BTSP no switch: n = 36, non-BTSP switch: n = 23 events from 30 mice) in the given lap. The peak amplitudes of [Ca2+] transients are not significantly different for the BTSP-like events (Mixed ANOVA: main effect for group (switch/no switch): p = 0.463; lap: p < 0.001, interaction: p = 0.256) and for the formation lap of the non-BTSP-like events but are significantly higher for non-BTSP-like events during switches than outside switches in the lap after the formation lap (Mixed ANOVA: main effect for group (switch/no switch): p = 0.001; lap: p < 0.001, interaction: p < 0.001; Tukey’s post hoc test: formation lap −1: p = 0.999, formation lap: p = 0.847, formation lap+1: p < 0.001).

Similar [Ca²+] transients underlie place field formation in familiar and novel environments

(A) Place field formation (PFF) in a familiar environment with a novel extension. A familiar corridor was extended with a new 6-m-long segment. New PFs appear already during the first traversal (top) by both BTSP- (red arrow) and non-BTSP-like (blue arrow) dynamics. Bottom, fluorescence [Ca2+] transients during and after the PFFs marked on the raster plot.

(B) Histogram of BTSP- and non-BTSP-like PFFs during the first session in the extended maze region (n = 6 mice).

(C) [Ca2+] transients of the formation lap and the following lap (FL+1) of BTSP- (left panels) and non-BTSP-like (right panels) PFF events in the new extension. The mean fluorescent activities of BTSP- (red) and non-BTSP-like (blue) PFFs are also displayed. Insets show peak-normalized, peak- aligned mean fluorescence traces. Bar graphs show the mean fluorescence activities (peak amplitude ± SD) during initial encounter (BTSPNEW, red, n = 24, non-BTSPNEW: blue, n = 16 events) of the novel environment (n = 6 animals), and familiar environment (BTSP- and non-BTSP: black; data from Figure 2). The amplitudes of [Ca2+] transients in the formation lap and the lap after formation were not significantly different between the new and familiar environments for both BTSP- and non-BTSP-like events (two-way mixed ANOVA: main effect for PFF type (BTSP/non-BTSP): p < 0.001, corridor (familiar/new): p = 0.996, type vs. corridor: p = 0.226, lap: p < 0.026, lap vs type: p < 0.001, lap vs. corridor: P = 0.692, lap vs. type vs. corridor: p = 0.049; Tukey post hoc tests: lap0: BTSP new vs. familiar: p = 0.999, non-BTSP new vs. familiar: p = 1; lap1: BTSP new vs. familiar: p = 0.540, non-BTSP new vs. familiar: p = 0.939).

Large somatic [Ca2+] events are often not sufficient to evoke novel place field

(A) Raster plots of neuronal activity (top) and [Ca2+] transients (bottom) vs. spatial location vs. laps for three example cells reveal BTSP-like new PFFs (red arrows) as well as large amplitude activities (magenta arrows) which did not evoke new PFs (solitary events).

(B) Fluorescence traces around the same BTSP-like PFF events (red) marked in panel (A) and larger amplitude [Ca2+] transients not associated with PFF (magenta) within the same sessions are shown.

(C) Trace pairs from the lap before (EL-1), during (Event Lap) and the lap after (EL+1) for BTSP- like PFF events (left traces) and solitary non-PF yielding events (right traces) identified within a session. In EL-1, traces were aligned to the timepoint of crossing the center of the PF. Pre and post solitary event traces were aligned to the crossing time of the first space bin of the corresponding solitary event. Inset: peak-normalized and peak-aligned mean fluorescence traces. Bar graphs depict the peak amplitudes of [Ca2+] transients (mean ± SD; BTSP: n = 60, solitary: n = 60 events, n = 23 mice) in the given laps. The amplitudes of [Ca2+] transients of the solitary events in the event lap were significantly higher, whereas in the EL+1 they were smaller compared to those of BTSP-like events (Mixed ANOVA: main effect for group (BTSP/solitary): p = 0.681, lap: p < 0.001, interaction: p < 0.001; Tukey post hoc test: lap −1: p = 0.999, event lap: p < 0.001, lap+1: p < 0.001).