A hippocampal circuit mechanism to balance memory reactivation during sleep

Abstract

Memory consolidation involves the synchronous reactivation of hippocampal cells active during recent experience in sleep sharp-wave ripples (SWRs). How this increase in firing rates and synchrony after learning is counterbalanced to preserve network stability is not understood. We discovered a network event generated by an intrahippocampal circuit formed by a subset of CA2 pyramidal cells to cholecystokinin-expressing (CCK+) basket cells, which fire a barrage of action potentials ("BARR") during non-rapid eye movement sleep. CA1 neurons and assemblies that increased their activity during learning were reactivated during SWRs but inhibited during BARRs. The initial increase in reactivation during SWRs returned to baseline through sleep. This trend was abolished by silencing CCK+ basket cells during BARRs, resulting in higher synchrony of CA1 assemblies and impaired memory consolidation.

Fig. 1.. A hippocampal network pattern with distinctive cellular contribution.

(A) Histology of bilateral electrophysiological recordings of all hippocampal subregions. DiD, 1,1′-dioctadecyl-3,3,3′3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt; Dil, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; DiO, 3,3-′dioctadecyloxacarbocyanine perchlorate; PCP4, Purkinje cell protein 4. DiD, Dil, and DiO were used for electrode localization; PCP4 was used as a CA2 marker. (B) Recording of a BARR and a SWR (delimited by dashed lines). (Top) CA1 and CA2 pyramidal layer wide-band LFP traces. (Bottom) Raster plot of firing from different neuronal subtypes (each row shows a different neuron, and each tick mark is one spike). Int, interneuron; Pyr, pyramidal cell. (C) Averaged cross-correlogram between BARRs and SWRs (n = 52 sessions from n = 10 mice). Dashed lines indicate 95% confidence interval (CI) from shuffled data (P < 0.001, Wilcoxon rank sum test). (D) Averaged peri-event firing rate curves for CA1 (n = 998), CA2phas (n = 1650), CA2ramp (n = 339), and CA3 (n = 105) pyramidal cells during SWRs (left) and BARRs (right), from n = 10 mice. (E) Firing rate ratio [(BARR FR − SWR FR)/(BARR FR + SWR FR)] between BARRs and SWRs for cells from one session (red indicates higher firing during BARRs; blue indicates higher firing during SWRs) noted at their soma location. FR, firing rate. (F) Same as in (D) for putative PV+ (n = 157) and BARR active (n = 72) interneurons. (G) Wavelet spectrogram of SWRs (left) and BARRs (right) for CA1 (left two panels) and CA2 (right two panels) for one session. (H) Increase in the rate of SWRs (left) and BARRs (right) in postlearning sleep compared with prelearning sleep for the three different memory tasks (n = 23 sessions from n = 8 mice for object displacement, n = 16 sessions from n = 7 mice for social memory, n = 14 sessions from n = 4 mice for T-Maze. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant; signed-rank test). Data are shown as mean ± SEM.

Fig. 2.. BARRs inhibit task-active cells and assemblies.

(A) Firing rate gain of cells most active during learning (“task active”) versus the rest of pyramidal cells (“others”), during SWRs and BARRs (n = 124/241, n = 102/130, and n = 140/237 task-active/other cells for Objects, Social, and T-Maze task, respectively). (B) Firing rate gain during SWRs and BARRs for CA1 PCs (n = 34/141, n = 36/128, and n = 313/447 PCs/non-PCs for Objects, Social, and T-Maze task, respectively). PC, place cell. (C) CA1 pyramidal cell firing rate gain during SWRs sorted by their firing rate gain in BARRs (quartiles) for each task (n = 185, 164, and 327 cells for Objects, Social, and T-Maze task, respectively; *P < 0.05; **P < 0.01; ***P < 0.001; linear regression). (D) Firing rate evolution over time in SWRs (gray) and BARRs (blue) for cells that showed a higher increase in SWRs after the task [P < 0.001, analysis of variance (ANOVA) of type of event, SWRs and BARRs, versus time interaction]. For (A) to (D), n = 22 sessions, n = 8 mice for Objects task; n = 15 sessions, n = 7 mice for Social task; n = 16 sessions, n = 4 mice for T-Maze task. (E) Averaged CA1 assembly reactivation during postlearning sleep SWRs (left) and BARRs (right) compared with shuffled periods (light gray) (n = 72, 41, and 42 assemblies for Objects, Social, and T-Maze task, respectively). (F) CA1 explained variance ratio [(EV−REV)/(EV+REV)] in postlearning sleep SWRs (n = 59 sessions) and BARRs (n = 14 sessions). EV, explained variance. REV, reversed explained variance. (G) Schematic of optogenetic SWR generation. (H) Example of artificially generated CA1 SWR. (I) SWR and BARR rate during the poststimulation period compared with the preceding baseline period (P > 0.05 and P < 0.01 for SWR and BARR rate increase, respectively; n = 4 mice, n = 9 sessions). (J) CA1 pyramidal cells firing rate gain during BARRs versus SWRs from pre– to postartificial SWRs (quartiles) (n = 148 cells, P < 0.05, linear regression). *P < 0.05; **P < 0.01; ***P < 0.001; Wilcoxon rank sum test unless otherwise noted.

Fig. 3.. Sncg+ cells are entrained by BARRs.

(A) (Top) Average waveforms of PV+, BARR+ interneurons, and pyramidal cells (for comparison) (n = 157, 72, and 998 for PV+, BARR+, and pyramidal cells, respectively; n = 10 mice; P < 0.001 for PV+ versus BARR+ waveform width comparison). (Bottom) Averaged normalized autocorrelograms of PV+, BARR+ interneurons, and pyramidal cells (P < 0.001 for PV+ versus BARR+ burstiness comparison). (B) Firing rate difference during periods of running and immobility for PV+ and BARR+ interneurons (P < 0.001). (C) Firing modulation of Sncg+ cells and putative PV+ cells (n = 20/33, respectively, from n = 10 sessions in n = 3 mice) during SWRs (left) and BARRs (right). (D) Firing rate difference during periods of running and immobility for PV+ and Sncg+ interneurons. (E) t-SNE of waveform and firing properties of different interneuron subtypes (from n = 13 mice: PV+, n = 172; BARR+, n = 94; Sncg+, n = 20; other interneurons, n = 510). *P < 0.05; **P < 0.01; ***P < 0.001; Wilcoxon rank sum test unless otherwise noted.

Fig. 4.. CA2 to Sncg+ cells form an anatomically defined circuit.

(A) Schematic of viral strategy used to label monosynaptically projecting cells onto Sncg+ cells. First, an AAV-TVA (AAV, adeno-associated virus; TVA, tumor virus A) cocktail targeting specific cell types (Sncg+) was injected. Next, a G protein–deleted rabies-dependent virus was injected to retrogradely label the presynaptic partners of Sncg+ cells. EGFP, enhanced green fluorescent protein; Str. Lac., stratum lacunosum moleculare; Str. Or., stratum oriens; Str. Pyr., stratum pyramidale; Str. Rad., stratum radiatum. (B) Example of cells labeled as starter neurons (yellow cells), with additional labeling of input neurons (green cells) and Sncg+ interneurons (red cells). Blue: 4′,6-diamidino-2-phenylindole staining. (C) Histology showing AAV-infected cells (red), starter cells (yellow), and input cells (green). Dashed lines delimit hippocampal regions. CA2 is labeled in cyan with PCP4. Horizontal bar = 300 μm. (D) Quantification of cells identified as input cells (EGFP+/mCherry−) from different subregions of the hippocampus (*P < 0.05; **P < 0.01; ***P < 0.001; ANOVA with Dunn’s post hoc test, n = 11 slices from n = 4 mice). (E) (Left) Cross-correlograms of putatively monosynaptically connected CA2 pyramidal cell (green) and BARR+ interneuron (peach color), with their respective waveforms. Dashed line indicates CIs (α = 0.001). (Right) Relative proportion of CA2 pyramidal cell functional monosynaptic inputs into CA1 BARR+ and PV+ interneurons (P < 0.001; n = 170 CA2-BARR+ pairs and 70 CA2-PV+ pairs). (F) (Left) CA1 pyramidal cell (purple) and PV+ interneuron (brown) cross-correlogram and waveforms. (Right) Relative proportion of CA1 pyramidal cell inputs into CA1 BARR+ and PV+ interneurons (P < 0.001; n = 276 CA1-BARR+ pairs and 666 CA1-PV+ pairs). *P < 0.05; **P < 0.01; ***P < 0.001; Wilcoxon rank sum test unless otherwise noted.

Fig. 5.. BARRs are necessary for memory consolidation.

(A) Example recording showing ~2-s delay between an SWR and BARR. (Top) CA2 wide-band LFP traces. (Bottom) Raster plot of firing from CA2 pyramidal cells (each row shows a different neuron, and each tick mark is one spike). (B) Quantification of average distance from BARRs to the previous preceding SWR, restricted to a maximum of 10-s lag time (n = 45,301 BARRs and n = 464,828 SWRs from 52 sessions in n = 10 mice). (C) SWR rate increase from pre- to postlearning sleep for control and Sncg+ silencing groups (n = 16 sessions in n = 8 mice; P > 0.05; Wilcoxon rank sum test). (D) Mean CA1 assembly reactivation strength during SWRs for control and Sncg+ silencing groups (n = 50 and 52 assemblies in control and Sncg+ groups, respectively; P > 0.05; Wilcoxon rank sum test). (E) Memory recall performance (discrimination index) for mice in the object displacement task without manipulation (control, light gray), Sncg+ silencing during BARR periods (BARR Sncg+ silencing, blue), and Sncg+ silencing during SWRs (SWR Sncg+ silencing, dark gray) (**P < 0.01; ***P < 0.001; ANOVA with Dunn’s post hoc test for between-group comparison and Wilcoxon signed-rank for within-group comparison; n = 16 sessions per group in n = 8 mice). (F) Firing rate gain of CA1 pyramidal cells during SWRs in postlearning sleep for control and Sncg+ silencing sessions (P < 0.001 for between-group comparison and P < 0.05 for time effect; repeated-measures ANOVA; n = 273 and 210 cells in control and Sncg+ groups, respectively; n = 13 sessions in n = 8 mice for control and n = 11 sessions in n = 8 mice for Sncg+ group). (G) CA1 population synchrony in SWRs (P < 0.01; t test; n = 13 sessions in n = 8 mice for control and n = 11 sessions in n = 8 mice for Sncg+ group). (H) Interspike interval within SWRs for control and Sncg+ silencing groups (P < 0.01; Wilcoxon rank sum test; n = 509 and 426 events in control and Sncg+ groups, respectively; n = 13 sessions in n = 8 mice for control and n = 11 sessions in n = 8 mice for Sncg+ group). *P < 0.05; **P < 0.01; ***P < 0.001; Wilcoxon rank sum test unless otherwise noted.