Robust information routing by dorsal subiculum neurons

Abstract

The dorsal hippocampus conveys various information associated with spatial navigation; however, how the information is distributed to multiple downstream areas remains unknown. We investigated this by identifying axonal projections using optogenetics during large-scale recordings from the rat subiculum, the major hippocampal output structure. Subicular neurons demonstrated a noise-resistant representation of place, speed, and trajectory, which was as accurate as or even more accurate than that of hippocampal CA1 neurons. Speed- and trajectory-dependent firings were most prominent in neurons projecting to the retrosplenial cortex and nucleus accumbens, respectively. Place-related firing was uniformly observed in neurons targeting the retrosplenial cortex, nucleus accumbens, anteroventral thalamus, and medial mammillary body. Theta oscillations and sharp-wave/ripples tightly controlled the firing of projection neurons in a target region-specific manner. In conclusion, the dorsal subiculum robustly routes diverse navigation-associated information to downstream areas.

Fig. 1. The subiculum comprises projection neurons targeting distinct downstream areas.

(A to E) After the injection of AAV1/2-EF1α-EGFP into the left dorsal SUB (A), EGFP-labeled axons were observed in the ipsilateral NAC, ipsilateral dorsal tenia tecta (DTT), ipsilateral dorsal peduncular cortex (DP) (B), bilateral AV, anterodorsal thalamic nucleus (AD), ITN, nucleus reuniens (Re) (C), the superficial layer of the ipsilateral granular RSC (D), and MMB (E). All images show coronal sections. Numbers, approximate distances of the sections from the bregma. (F) SUB coronal sections containing cholera toxin B subunit conjugated with Alexa Fluor 488 (CTB488)–labeled projection neurons (left) and the corresponding cell counting (right). Left: Dotted curves, borders of the SUB cell layer. Right: Segmented SUB cell layer (mesh) and detected CTB488-positive neurons (dots). (G to M) Dendritic morphology of SUB projection neurons. (G) Schematic of visualization of projection-specific neuronal morphology. (H to L) SUB coronal sections containing EYFP-labeled projection neurons (top) and apical dendrites (bottom). The AAV6-pgk-Cre was injected in one of the following areas: NAC (H), AV (I), ITN (J), RSC (K), and MMB (L). (M) Dendritic spine density per unit length of the apical dendrite. n = 17, 17, 25, 21, and 20 cells for NAC-p, AV-p, ITN-p, RSC-p, and MMB-p SUB neurons, respectively. *P < 0.05, Tukey test. Means ± SD.

Fig. 2. Optogenetic identification of SUB projection neurons.

(A) Schematic of optogenetic identification. (B) Coronal section indicating ChR2 expression (green) and the estimated recording sites (white dots). Gray curves, borders of the CA1, SUB, and their cell layers. Text, animal ID and silicon probe name. (C to F) NAC (C), AV (D), RSC (E), and MMB (F) coronal sections containing ChR2-expressing SUB axons (green) and the reconstructed optical fiber locations (dotted lines). (G) Left: Spike responses of a SUB neuron upon light irradiation to the NAC. Evoked spikes (top) decreased when spontaneous spikes occurred shortly before the expected latency of evoked spikes (middle and bottom). Shaded blue areas, light stimulation periods. Gray lines, bin ranges of evoked spikes. Right: Mean waveforms of spontaneous (black) and evoked (blue) spikes. (H to J) Latency (H), jitter (I), and fidelity (J) of evoked spikes. (K) Waveform correlation between the evoked and spontaneous spikes. (H to K) Means ± SD. *P < 0.05 and **P < 0.01, Tukey test.

Fig. 3. Place representation on a linear track.

(A) Peak-normalized rate maps of CA1 (top) and SUB (bottom) neurons sorted by peak positions. (B to F) Cumulative distribution of I_spike (B), mean firing rate (C), peak firing rate (D), I_sec (E) (gray curve, the distribution of shuffled data; number and dotted line, its 99th percentile), and mutual information (F) of CA1 and SUB neurons. P values: Wilcoxon rank sum test. (G) Example position decoding from simultaneously recorded CA1 (top) or SUB (bottom) neurons in three consecutive trials. Color maps, probabilities of estimated animal positions. Dotted curves, observed animal positions. Numbers, decoding errors of the trials. (H) Error of position decoding averaged across all animals. Decoding errors were calculated either from all time bins irrespective of the number of spikes in the bins (solid lines and shaded areas, means ± SDs) or from time bins containing one or more spikes (dotted lines, mean). Dashed-dotted line, SUB decoding error calculated after randomly removing spikes from SUB neurons to match the CA1 mean firing rate (mean). Black line, decoding error obtained from the shuffling procedure. (I) Difference in decoding errors after randomly removing (negative x values) or adding (positive x values) spikes to each neuron. Group data were averaged across all animals. P < 0.01, Bonferroni test after two-way repeated-measures ANOVA. Means (solid lines) ± SD (shaded areas). (J) Peak-normalized rate maps of SUB projection neurons sorted by peak positions. (K and L) Percentage of place cells in the CA1 and SUB (K) and SUB projection neurons (L). Numbers, number of place cells to the total number of neurons. Dotted line, percentage of place cells in SUB neurons.

Fig. 4. Speed representation in an open field.

(A) Rate maps (left), z-scored running speed (gray) and firing rate (color) (middle), and mean firing rate as a function of running speed (right). Each row shows the firing patterns of a single neuron. Numbers, peak firing rates in the figure (left and middle) and speed scores (right). (B to E) Cumulative distribution of I_spike (B), mean firing rates (C), I_sec (D), and speed scores (E). P values: Wilcoxon rank sum test. (D and E) Gray curves, numbers, and dotted lines, the distribution of shuffled data and its 1st (E) and 99th (D and E) percentiles. *P = 0.019 and ****P < 0.0001, CA1 versus SUB neurons exceeding either the 1st or 99th percentile threshold, Wilcoxon rank sum test. (F) Example decoding of speed from simultaneously recorded CA1 (top) and SUB (bottom) neuron activity. Gray, observed speed. Color, decoded speed. Numbers, decoding accuracy. (G) Decoding accuracy averaged across all animals. Dashed-dotted line, SUB decoding accuracy calculated after randomly removing spikes from SUB neurons to match the CA1 mean firing rate (mean). Black line, decoding error obtained from a shuffling procedure. (G and H) Means (solid lines) ± SD (shaded areas). (H) Normalized decoding accuracy after randomly removing (negative x values) or adding (positive x values) spikes to each neuron. Group data were averaged across all animals. P < 0.05, Bonferroni test after two-way repeated-measures ANOVA. (I and J) Percentage of n-speed (left) and p-speed (right) cells in the CA1 and SUB (I) and among SUB projection neurons (J). Numbers, number of speed cells to the total number of neurons. (I) ***P = 0.0002, χ² test. (J) Dotted lines, percentage of n-speed (left) and p-speed (right) cells in SUB neurons. *P < 0.05, bootstrap analysis.

Fig. 5. Trajectory-dependent firing during an alternating T-maze task.

(A) Schematic of the behavioral task. (B) Rate maps of a CA1 and two NAC-p SUB neurons while rats were in the start box (normalized position, 0.02 to 0.07) and stem (normalized position, 0.07 to 0.38) of the T-maze. Numbers, auROC of the neuron. Red, left-arm trials. Green, right-arm trials. Means (solid lines) ± SD (shaded areas). (C to G) Cumulative distribution of I_spike (CA1, 0.034 ± 0.064 bits/spike; SUB, 0.011 ± 0.023 bits/spike) (C), rate change ratio (CA1, 0.24 ± 0.19; SUB, 0.15 ± 0.13) (D), mean firing rate (CA1, 2.3 ± 3.2 Hz; SUB, 6.8 ± 6.8 Hz) (E), I_sec (CA1, 0.034 ± 0.063 bits/s; SUB, 0.040 ± 0.089 bits/s) (F), and auROC (CA1, 0.601 ± 0.084; SUB, 0.609 ± 0.091) (G) in the start box. P values: Wilcoxon rank sum test. (H) Accuracy of trajectory decoding averaged across all animals. Dashed-dotted line, SUB decoding accuracy calculated after randomly removing spikes from SUB neurons to match the CA1 mean firing rate (mean). Black line, chance level of decoding error estimated by a shuffling procedure. *P < 0.05, CA1 versus SUB solid lines, Bonferroni test after two-way ANOVA. (H and I) Means (solid lines) ± SD (shaded areas). (I) Normalized decoding accuracy after randomly removing (negative x values) or adding (positive x values) spikes to each neuron. Group data were averaged across all animals. *P < 0.05, Bonferroni test after two-way repeated-measures ANOVA. (J and K) Percentage of trajectory-dependent cells in the CA1 and SUB (J) and SUB projection neurons (K). Numbers, number of trajectory-dependent cells to the total number of neurons. Dotted line, percentage of trajectory-dependent cells in SUB neurons. *P < 0.05, bootstrap analysis.

Fig. 6. Head-direction representation in an open field.

(A) Directional rate maps of CA1 and SUB projection neurons. Each map shows the firing patterns of a single neuron. Numbers, peak firing rates in the figure. (B to D) Cumulative distribution of I_spike (B), mean vector length (C), and I_sec (D). P values: Wilcoxon rank sum test. (E) Example head-direction decoding from simultaneously recorded CA1 (top) or SUB (bottom) neurons. Color maps, probabilities of estimated head directions. Dotted curves, observed head directions. (F) Angular error of head-direction decoding averaged across all animals. Solid lines and shaded areas, means ± SDs. (G and H) Percentage of head-direction cells in the CA1 and SUB (G) and SUB projection neurons (H). Numbers, number of head-direction cells to the total number of neurons. Dotted line, percentage of head-direction cells in SUB neurons. ***P = 0.001, χ² test.

Fig. 7. Projection-specific theta phase locking during RUN periods.

(A and B) Distribution of theta power (A) and theta phase deviations (B) in an open field (left). White lines, borders of the CA1, SUB, and dentate gyrus (DG). Values along the CA1 (black dotted lines) and SUB (solid black lines) are plotted in the right. Red dots, the reference recording site with the maximal theta power in the SUB cell layer. (C) Wide-band local field potentials (LFPs) simultaneously recorded from the CA1 (top) and SUB (bottom). (D) Spike phase distribution along the reference SUB theta oscillations (left) and corresponding PPC (right) in the CA1 (top) and SUB (bottom). P values, one-way ANOVA. Means (solid lines) ± SD (shaded areas). (D and G) Top gray traces, idealized reference theta cycles. (E) Distribution of the preferred theta phases (left y axis, solid lines) and spike theta phases (right y axis, dotted lines). (F) Distribution of preferred theta phases for neurons located at proximal (green), distal-deep (yellow), and distal-superficial (purple) part of the SUB cell layer. (G) Spike phase distribution along the reference SUB theta oscillations in SUB projection neurons. (H and I) Distribution of the spike theta phases (H) and PPC (I) of SUB projection neurons. (J) Preferred theta phase plotted against cell location and projection target. Normalized positions indicate the anatomical cell location within the SUB cell layer.

Fig. 8. Projection-specific firing modulation by SPW-Rs during SWS.

(A) Wide-band LFPs simultaneously recorded in the CA1 (top) and SUB (bottom) showing SPW-Rs. so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum; slm, stratum lacunosum-moleculare. (B) Cross-correlogram (CCG) of ripple events detected in the CA1 and SUB cell layers. Inset: The same CCG at a finer temporal scale. Means (solid line) ± SD (shaded area). (C) Ripple-triggered average of firing rates (z-scored) of CA1 (top) and SUB (middle) neurons and the firing rates averaged over CA1 or SUB neurons (bottom). Ripple events detected in single recording sites at the center of the SUB cell layer were used as the reference in (C) to (J). (C and F) Topmost trace, example SUB LFP showing SPW-Rs. (D and E) Mean z-scored firing rate averaged around the negative ripple peaks (−10 to 10 ms) for CA1 and SUB neurons (D) and for SUB neurons located at the proximal (green), distal-superficial (purple), and distal-deep (yellow) part of the SUB cell layer (E). (F) Ripple-triggered average of z-scored firing rates of individual SUB projection neurons. (G) Ripple-triggered average of z-scored firing rates of SUB projection neurons. **P < 0.01, Tukey test for peak height. Means (solid lines) ± SEM (shaded areas). (H to J) Mean z-scored firing rate averaged around negative ripple peaks (−10 to 10 ms) as a function of ripple power (H), ripple duration (I), and ripple frequency (J). The plot colors are the same as in (G). Means ± SEM.