Increased Prevalence of Calcium Transients across the Dendritic Arbor during Place Field Formation

Abstract

Hippocampal place cell ensembles form a cognitive map of space during exposure to novel environments. However, surprisingly little evidence exists to support the idea that synaptic plasticity in place cells is involved in forming new place fields. Here we used high-resolution functional imaging to determine the signaling patterns in CA1 soma, dendrites, and axons associated with place field formation when mice are exposed to novel virtual environments. We found that putative local dendritic spikes often occur prior to somatic place field firing. Subsequently, the first occurrence of somatic place field firing was associated with widespread regenerative dendritic events, which decreased in prevalence with increased novel environment experience. This transient increase in regenerative events was likely facilitated by a reduction in dendritic inhibition. Since regenerative dendritic events can provide the depolarization necessary for Hebbian potentiation, these results suggest that activity-dependent synaptic plasticity underlies the formation of many CA1 place fields.

Keywords: branch spike; calcium imaging; dendritic spike; hebbian; hippocampus; memory formation; navigation; place cell; two-photon microscopy; virtual reality.

Figure 1. Switching virtual environments causes global remapping

(A) Examples of familiar (F) and novel (N) virtual environments. (B1) Single mouse behavior showing track position vs time over F to N switch. (B2) Summary across all mice of mean lap velocity over F to N switch (n = 16 mice; n = 32 F to N switches). Mean ± SEM; N.S. Paired t-test, p > 0.05. (C) Bottom left: CA1 field of view indicating place cells encoding F (red). Top left: Mean place fields from cells (red) sorted by track position. Bottom middle: Same field of view and cells as in bottom left. Top middle: Mean activity of indicated cells in N sorted in same order as Top left. Bottom right: Same field of view as Bottom left with all place cells encoding N (red). Top right: Mean place fields from cells (red) sorted by track position. Bottom far right: Same field of view as Bottom left with all place cells colored to indicate F, N or both encoding. (D) Place field spatial correlation within F vs. across F-to-N. p < 0.001, unpaired t-test. (E) Somatic place field transients shown lap by lap in N for 6 example cells; first row represents first traversal mouse ever made across N. Red arrows indicate place field onset lap. (F1 and F2) Histograms of place field onset lap number from all mice in N (F1; n = 4 mice; n = 8 sessions in N) and F (F2; n = 3 mice; n = 8 session in F). (F3) Cumulative fraction plots of the data in F1 and F2. Place field onset lap distribution was shifted (Wilcoxon rank sum test, P < 0.001) such that place fields appeared earlier in F vs N.

Figure 2. Dendritic branch spikes are more prevalent during initial exposure to novel environments and predict future place field location

(A) Top, Cartoon depicting 2-photon imaging planes in the soma and basal dendrites of pyramidal cells and co-acquired images of place cell soma and dendrites from the same cell. Bottom, ΔF/F traces from the (co-acquired) soma and numbered dendritic branches during 3 place field traversals (grey columns, mean place field over session in N) in N. Red traces, transients of P < 0.001 from bootstrapping. (B) Colored plots (left; three different place cells from two mice) show occurrence of detectable branch spiking in each imaged branch (rows) in the somatic place field on each lap in N, each column represents a different lap in N; first column represents first traversal mouse ever made across N. Red, significant transient in the imaged branch; blue, no significant transient in the branch during a co-occurring somatic calcium transient; black, no significant transient in the branch or soma. Right, plots of mean BSP (4 laps binned) vs laps in N from example cells on left. (C) Summary across all mice of mean BSP in F (n = 10 place fields, n = 5 mice) and N (4 laps binned; n = 18 place fields, n = 9 mice). (D) Mean BSP from first 8 laps vs all laps following lap 16 in N (n = 13 place fields, n = 7 mice). **, Paired t-test, p < 0.01. (E) Same as D but using first 16 laps. **, Paired t-test, p < 0.01. (F) Same as (C) but aligned to place field onset lap for each cell (depicted as lap 1). (G) Average BSP in place fields versus outside of place fields in first 8 laps of N. ***,Paired t-test, p < 0.001.

Figure 3. Putative local dendritic spikes predict the location of delayed onset somatic place fields

(A) Cartoon depicts co-recording from soma and basal dendrites of CA1 pyramidal cells. 3 Example place cells and their dendritic branches are shown (red regions, all branches belonging to the co-imaged soma; green regions, dendrites that displayed a dendrite-localized calcium transient; non-selected dendrites from different cells). Bottom, somatic place field transients lap by lap in N; first row representing first traversal mouse ever made across N. Green stars, track location where dendrite-localized calcium transient detected in indicated branch (at top) in the absence of a detectable somatic calcium transient (time-series ΔF/F traces from the branch and soma shown at right). B) Histogram of the area of significant ΔF/F increase for each dendrite-localized calcium transient detected during the delay period of delayed-onset place fields. The area of significant ΔF/F increase for known single spine-restricted calcium transients (likely single synaptic inputs, not shown) are all less than dotted line. (C) 3 example dendrites showing dendrite-localized calcium transients from different place cells during delay period. Left: Same as (A) top. Middle: magnified view of the indicated dendrites. Right: ΔF/F image of the indicated dendrites during dendrite-localized calcium transients. Area of significant ΔF/F increase (>3 s.d.) at right. (D) Histogram of track locations of all dendrite-localized calcium transients that occurred during the delay period of delayed-onset place fields relative to the location of the later forming somatic place field (somatic field center at 0; running direction from negative to positive; only transients > threshold in (B) included). ***, Chi Squared proportionality test, p < 0.001. (E) Max ΔF/F and transient duration for each dendrite-localized calcium transient (only transients > threshold in (B) included).

Figure 4. Dendritic inhibition is transiently reduced and somatic inhibition is transiently increased following exposure to novel environments

(A) GCaMP6f-expressing SOM+ interneuron cell bodies (left) and axons (right) in Stratum Oriens of CA1 in vivo. (B) Mean SOM+ interneuron axonal ΔF/F (average over all axons in field) during running vs resting in F (Paired t-test). Open circle, means from 6 sessions in F from 3 mice. (C) Mean velocity vs mean axonal ΔF/F on each lap in F (6 sessions). N.S., Linear regression, slope not significantly different from 0. (D) Bottom: Single mouse behavior of track position vs time during F to N switch. Middle: axonal ΔF/F during switch. (E) Mean axonal ΔF/F during running on each lap (normalized to the mean in F in each case) during F to N switch (6 switches; n = 3 mice). Mean from all mice in red. (F) Normalized Mean axonal ΔF/F during running in F, laps 1–8 in N and laps 15–22 in N. Open circles represent each F to N switch. Repeated measures ANOVA with Tukey’s post-test, N.S., p > 0.05. (G) GCaMP6f-expressing PV+ interneuron cell bodies and axons in CA1 Pyramidal cell layer in vivo. (H) Mean PV+ interneuron axonal ΔF/F (average over all axons in field) during running vs resting in F (Paired t-test). Open circle, means from 9 sessions in F from 5 mice (one mouse had only one F to N switch). (I) Mean velocity vs mean axonal ΔF/F on each lap in F (9 sessions). N.S., Linear regression, slope not significantly different from 0. (J) Bottom: Single mouse behavior of track position vs time during F to N switch. Middle: axonal ΔF/F during switch. (K) Mean axonal ΔF/F during running on each lap (normalized to the mean in F in each case) during F to N switch (9 switches; n = 5 mice). Mean from all mice in red. (L) Normalized Mean axonal ΔF/F during running in F, laps 1–8 in N and laps 15–22 in N. Open circles represent each F to N switch. Repeated measures ANOVA with Tukey’s post-test, N.S., p > 0.05.

Figure 5. NMDA receptors in CA1 pyramidal neurons are required for the formation of a subset of place fields

(A, B) GCaMP6f-expressing neurons in CA1 of WT (A) and NR1 (B, NMDA receptors functionally knocked out in GCaMP6f-expressing neurons) mouse. Top, all active neurons in the field of view in N (green). Bottom, All place cells encoding N (red). (C) Fraction of the population of active cells in the field of view with a place field in N for WT vs NR1 mice. Open circles, fraction from individual fields of view. Bars, mean across all fields (n = 8 fields from WT n = 5 from NR1; from n = 4 WT, n = 3 NR1 mice). *, Chi Squared proportionality test, p < 0.05. (D and E) Somatic place field transients shown lap by lap in N in WT mice (4 in D) and NR1 mice with functional NMDA receptors knocked out of GCaMP6f-expressing cells (4 in E); first row represents first traversal mouse ever made across N. Somatic place field calcium transients have higher dF/F amplitudes during initial laps and for cells lacking NMDA receptors, fields tend to disappear towards session end (E). (F) Mean number of significant calcium transients detected from all active cells in WT mice (n = 4 mice, n = 1882 transients; blue) and NR1 mice with functional NMDA receptors knocked out of GCaMP6f-expressing cells (n = 3 mice; n = 835 transients; red). Blue to red, Unpaired t-test, ***, p < 0.001; N.S., p > 0.05; Within color, paired t-test, **, p < 0.01, * p < 0.05; all t-tests with Bonferroni correction. Bars, mean from all transients across all mice. Open circles, means from each field of view.