While the interaction of the cardinal rhythms of non-rapid-eye-movement (NREM) sleep—the thalamo-cortical spindles, hippocampal ripples, and the cortical slow oscillations—is thought to be critical for memory consolidation during sleep, the role spindles play in this interaction is elusive. Combining optogenetics with a closed-loop stimulation approach in mice, we show here that only thalamic spindles induced in-phase with cortical slow oscillation up-states, but not out-of-phase-induced spindles, improve consolidation of hippocampus-dependent memory during sleep. Whereas optogenetically stimulated spindles were as efficient as spontaneous spindles in nesting hippocampal ripples within their excitable troughs, stimulation in-phase with the slow oscillation up-state increased spindle co-occurrence and frontal spindle-ripple co-occurrence, eventually resulting in increased triple coupling of slow oscillation-spindle-ripple events. In-phase optogenetic suppression of thalamic spindles impaired hippocampus-dependent memory. Our results suggest a causal role for thalamic sleep spindles in hippocampus-dependent memory consolidation, conveyed through triple coupling of slow oscillations, spindles, and ripples.

Using closed-loop, optogenetic stimulation of the thalamic reticular nucleus (TRN) to induce spindle activity in mice ( Figure 1 A), we show that only spindles induced during the slow oscillation up-state—that is in-phase coupling—enhance memory for previously learned hippocampus-dependent tasks, whereas spindles occurring out-of-phase with the slow oscillation remain ineffective. Furthermore, in-phase spindle-like stimulation enhanced the co-occurrence of frontal cortical spindles and ripples and, consequently, triplets of slow oscillation-spindle-ripple events.

(E) Freezing level in the last minute of conditioning (left), recall of fear context (middle; average of the first 4 min), and fear cue (right; freezing at the delivery of the tone expressed as percentage of time freezing) were measured during a recall test 24 hr after learning for the in-phase (red bars), out-of-phase (green bars), and no-stimulation condition (black bars). ∗∗∗ p < 0.001, ∗∗ p < 0.01 for post hoc pairwise comparisons. Data are shown as box-whisker plots with box limits representing the first and third quartile and whiskers indicating the data range.

(D) Schematic of the stimulation protocols: mice were optogenetically stimulated with four light pulses (8 Hz, 62.5 ms on/off) as soon as they entered NREM sleep, inducing a spindle-like signal (). Stimulation was applied such that it occurred during a slow oscillation up-state (IN, in-phase, n = 8, red trace) or with a random delay between 0.5 and 1.0 s after a slow oscillation up-state (OUT, out-of-phase, n = 8, green trace) and was then followed by a 1.5 s pause. In the no-stimulation condition, no light pulses were presented, but the time points corresponding to the in-phase (NoSTIM-IN, n = 10, upper black trace) and out-of-phase (NoSTIM-OUT, n = 10, lower black trace) stimulations were marked like for the stimulation conditions.

(C) Mice were tested on a combined cued/contextual fear-conditioning task. Following a learning phase, the animals were allowed to sleep and were subjected to one of three stimulation protocols for 6 hr (red hatched area). Recall of memory started 24 hr after the learning phase.

(B) Schematic depiction of coronal and sagittal (top) positioning of optic fiber cannulas in the anterior ventral region of the TRN (bottom, left; scale bar, 1 mm) and the specifically high expression of mhChR2-EYFP in TRN (bottom, right; scale bar, 100 μm) parvalbumin neurons.

Several studies have demonstrated a causal relationship between increased slow oscillation and memory consolidation in humans (). Using timed electrical stimulation in rats,provided evidence that reinforcing the endogenous temporal coordination between hippocampal sharp wave-ripples, cortical slow waves, and spindles can enhance the consolidation of hippocampus-dependent spatial memory. However, so far, there is no experimental evidence demonstrating a causal influence specifically of thalamic activity on the phase-locking between cortical slow oscillations and the subordinate rhythms—thalamo-cortical spindles and hippocampal ripples—in the process of memory formation. This is even more surprising as thalamic spindles emerging first during ontogeny (), as well as in the course of human nocturnal sleep (), are suspected to play a central role in forming memory during sleep.

Sleep is known to support the consolidation of memory (). The <1 Hz cortical slow oscillation (), thalamo-cortical spindles () (7–15 Hz), and hippocampal sharp wave-ripples () (100–250 Hz) represent the cardinal rhythms of non-rapid-eye-movement (NREM) sleep, and all these rhythms have been implicated in the consolidation of declarative (i.e., hippocampus-dependent) memory during sleep (). Importantly, it has been proposed that consolidation of hippocampus-dependent memory during NREM sleep essentially relies on the hierarchical nesting of these rhythms (). Phase-locking occurs such that ripples accompanying neural memory reactivation in the hippocampus nest into the excitable troughs of the spindle oscillation, which themselves nest into the excitable up-state of the slow oscillation (). This phase-locking presumably favors the redistribution of the representation from predominantly hippocampal toward neocortical networks that serve as long-term storage sites ().

Together, these results confirm that a modulation of the TRN activity coinciding with a slow oscillation up-state and pre-eminently frequency-specific to the spindle range, with a combined impact on spindle and slow oscillation incidence, is important for hippocampal memory consolidation during sleep.

Finally, to ensure that the promoting effect of spindle-like in-phase TRN stimulation on hippocampal-dependent memory is specific to the stimulation frequency, we performed another control experiment utilizing TRN stimulation at 20 Hz (i.e., a frequency unrelated to the spindle band) in mice expressing ChR2 (n = 7) and in control mice expressing YFP (n = 10; please see legend of Figure S7 and STAR Methods for details). Comparison between these two groups showed no difference in contextual fear memory (F= 0.017, p = 0.899; Figure S7 F) or spindle incidence in the 750 ms post-stimulus interval (FRO: F= 1.361, p = 0.262; Figure S7 D).

As expected, the spindle incidence rate was significantly reduced for the mice that received inhibitory TRN stimulation if compared to the respective no-stimulation control conditions ( Figure 6 G; F= 12.042, p = 0.001, one-way ANOVA, IN versus NoSTIM-IN: p = 0.035, and OUT versus NoSTIM-OUT: p = 0.015). In line with our findings from the main experiments demonstrating that a spindle-like stimulation of the TRN induced slow oscillations, the inhibitory manipulation significantly reduced the incidence of slow oscillations during the 750 ms post-stimulus window for the in-phase and out-of-phase stimulation conditions, in comparison with their respective control conditions ( Figure 6 H; F= 23.921, p < 0.001, one-way ANOVA, IN versus NoSTIM-IN: p = 0.002, OUT versus NoSTIM-OUT: p = 0.019). Inhibitory stimulation had no effect on incidence rates of hippocampal ripples (one-way ANOVA, F= 2.151, p = 0.121; data not shown).

We finally tested whether optogenetic inhibition of TRN Prv neurons could affect memory formation. Prv-cre knockin mice were bilaterally injected in the TRN ( Figure 6 A) with pAAV-CBA-Flex-Arch-GFP ( Figures 6 B–6E). Mice were subjected to stimulation protocols corresponding to in-phase optogenetic inhibition (IN-ARCH; 500 ms yellow laser pulse following the detection of a slow oscillation up-state; n = 8), out-of-phase optogenetic inhibition (OUT-ARCH; 500 ms yellow laser pulse randomly given between 0.5 to 1.0 following the detection of a slow oscillation up-state; n = 7), or no-stimulation condition (NoSTIM; no laser stimulation given; n = 9). We found that in-phase optogenetic inhibition of Prv neurons resulted in a significantly lower contextual memory recall ( Figure 6 F; one-way ANOVA, F= 5.665, p = 0.011) compared to the no-stimulation group (IN-ARCH versus NoSTIM: p = 0.043, Fisher LSD) or OUT-ARCH group (IN-ARCH versus OUT-ARCH: p = 0.003, Fisher’s LSD). The out-of-phase TRN inhibition did not significantly affect memory (NoSTIM versus OUT-ARCH: p = 0.199, Fisher’s LSD). The tone recall was not significantly changed (one-way ANOVA, F= 3.304, p = 0.057).

(H) Slow oscillation incidence rate (in percent of the total number of stimulations) in frontal recordings during in-phase (IN, blue), out-of-phase (OUT, light-blue), and no-stimulation conditions (NoSTIM-IN, black; NoSTIM-OUT, gray). Incidence rates were determined by the number of spindles/slow oscillations occurring within a 750 ms interval following the onset of the inhibitory stimulus and normalized by the total number of stimulations. ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05 for post hoc pairwise comparisons; data are shown as box-whisker plots with box limits representing the first and third quartile and whiskers indicating the data range.

(G) Spindle incidence rate (as percentage of total number of stimulations) in frontal (FRO) recordings following TRN inhibition in-phase (blue) and out-of-phase (light-blue) with detected slow oscillation and their corresponding control conditions (NoSTIM-IN, black; NoSTIM-OUT, gray).

(B2–E2) Magnification of the area marked by the white dashed square in (B1) for the corresponding staining/fluorescence as shown in (B1)–(F1). Scale bar represents 100 μm.

(B1–E1) Representative coronal section of the thalamus stained against NeuN antibody (B1), viral expression of ARCH-GFP virus in the TRN region (C1), staining against PV antibody (D1), and merged ARCH-GFP/PV staining (E1). Scale bar represents 100 μm.

(A) Schematic showing a typical coronal section used for bilateral injection of pAAV-CBA-Flex-Arch-GFP.WPRE.SV40 virus and the implantations scheme of optic fiber cannula in the TRN of Prv-cre knockin mice.

Since sleep spindles are known to be spatially distributed oscillations, we further examined the cross regional occurrence of spindles during stimulation. In-phase stimulation was accompanied by a significant increase in the rate of co-occurring (i.e., temporally overlapping) spindle events between FRO and PAR recordings (F= 13.298, p < 0.001, one-way ANOVA), between FRO and CA1 recordings (F= 6.602, p = 0.010, Welch test), and between all three recording sites (FRO-PAR-CA1, U= 7.175, p = 0.028, Kruskal-Wallis one-way ANOVA) compared to the out-of-phase and no-stimulation conditions ( Figure S6 ). We further examined whether these coherent spindle events also included a hippocampal ripple. Consistent with the increase in (detected) slow oscillation, frontal spindle, and hippocampal ripple events during in-phase stimulation (reported above; Figure 5 D), this analysis showed that in-phase stimulation, in comparison with the other two conditions, was specifically associated with an increased rate of coherent frontal-hippocampal spindle events, including a ripple ( Figure 5 E; U= 13.008, p = 0.001, Kruskal-Wallis one-way ANOVA; IN versus NoSTIM: p = 0.004, IN versus OUT: p = 0.001, and OUT versus NoSTIM: p = 0.605; rank-sum test). In sum, these results indicate that in-phase stimulation produces a unique temporal-spatial pattern of the three oscillatory phenomena of interest, characterized not only by an increased triple coupling of slow oscillation, spindle, and ripple events, but also by an increased co-occurrence of spindles in anterior and posterior cortical and hippocampal regions, where, in particular, spindles co-occurring in frontal cortex and hippocampus also entrain ripples.

We found that optogenetic stimulation did not affect the ripple incidence rate under any condition ( Figure 5 B; one-way ANOVA, F= 1.258, p = 0.306). However, an analysis of the temporal overlap of spindle and ripple events revealed that the proportion of hippocampal ripples co-occurring with spindle events during optogenetic stimulation was increased in the in-phase stimulation condition (one-way ANOVA, F= 14.679, p < 0.001) compared with the no-stimulation condition ( Figure 5 C; NoSTIM-IN versus IN: p = 0.001 and NoSTIM-OUT versus OUT: p = 0.162, Fisher’s LSD). Notably, 20% of induced spindles were accompanied by a ripple, with no significant difference among conditions (one-way ANOVA, F= 1.277, p = 0.3). Importantly, the percentage of online-detected slow oscillations co-occurring with a spindle (within 750 ms after detection) that also exhibited at least one ripple co-occurring within a spindle was significantly increased in the in-phase group compared to the no-stimulation and out-of-phase groups ( Figure 5 D; Kruskal-Wallis one-way ANOVA, U= 9.974, p = 0.007; NoSTIM versus IN: p = 0.024, NoSTIM versus OUT: p = 0.372, and IN versus OUT: p = 0.002, Fisher’s LSD).

We next examined whether spindles induced optogenetically using our protocols are similar to spontaneous spindles in terms of their influence on hippocampal oscillations ( Figures S5 A and S5B). Previous studies have shown a high propensity for sleep spindles to synchronize hippocampal ripples into their troughs () ( Figure 5 A). A nesting of ripple activity into the troughs of the spindle cycle in particular for spindles recorded at PAR and CA1 ( Figures S5 C and S5D) was observed and was closely comparable among all stimulation conditions, as confirmed by more fine-grained time-event correlation histograms of discrete ripple events time-locked to the spindle troughs ( Figure S5 C; p < 0.001). Consistent with findings in humans and rodents (), grouping of ripples to spindle troughs was evident for parietal cortical and hippocampal spindles ( Figure S5 D) but was absent during frontal spindles. This result indicates that the spindle stimulation did not affect the naturally weak spindle-ripple nesting in FRO recordings and mostly preserved known nesting properties of CA1 ripples into PAR and CA1 spindles troughs.

(E) Incidence rate (in percent of total number of stimulations) of offline detected spindle events co-occurring in frontal and parietal (FRO & PAR), frontal and hippocampal (FRO & CA1), parietal and hippocampal (PAR & CA1), or all three recording sites (triple) and with at least one hippocampal ripple event within 750 ms following stimulation onset. ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05 for post hoc pairwise comparisons; data are shown as box-whisker plots with box limits representing the first and third quartile and whiskers indicating the data range.

(D) Proportion (per online detected slow oscillations) of frontal spindles co-occurring with at least one hippocampal ripple within 750 ms following an online detected slow oscillation (which corresponds to the proportion of slow oscillations-spindle-ripples triples) for in-phase (IN, red bar), out-of-phase (OUT, green bar), and no-stimulation control conditions (NoSTIM, black).

(C) Proportion of hippocampal ripples co-occurring with FRO spindles following in-phase (IN, red) and out-of-phase (OUT, green) stimulation and the corresponding periods for no-stimulation controls (NoSTIM-IN, orange; NoSTIM-OUT, light green).

(B) CA1 ripple incidence rate (in percent of stimulations) induced by in-phase (red) and out-of-phase (green) optogenetic stimulation and the corresponding periods for no-stimulation controls (NoSTIM-IN, orange; NoSTIM-OUT, light green). An induced ripple was defined as a ripple event occurring 750 ms after stimulation onset.

(A) Representative 10 s traces of raw frontal (FRO) EEG and hippocampal (CA1) LFP recordings filtered between 100 and 250 Hz. Periods with discrete spindle events in the EEG are framed by dashed lines. Black asterisks indicate CA1 ripple events. Below is a magnification of the second detected spindle, illustrating ripples coinciding with spindles (red asterisks) during light stimulation (blue bars).

We then asked whether thalamic spindle stimulation exerts a bottom-up effect on cortical slow oscillations. Averaging EEG responses filtered in the 0.5–4.0 Hz band time-locked to stimulation onset did not reveal any difference in amplitude of slow oscillations occurring during in-phase stimulation and the corresponding interval of the no-stimulation condition ( Table S3 ), excluding an immediate effect of in-phase stimulation on ongoing slow oscillation activity. However, an analysis of slow oscillation negative half-wave peaks in the FRO within a 500 ms interval following stimulus onset ( Figure 4 A) revealed a distinctly increased probability of the emergence of a slow oscillation during this interval in the out-of-phase condition compared with all other conditions ( Figure 4 B), including a comparison with an interval of the no-stimulation condition that covered the respective out-of-phase stimulation period (one-way ANOVA, F= 76.356, p < 0.001; OUT versus NoSTIM-OUT: p < 0.001, Fisher’s LSD). The stimulation-induced slow oscillations emerged mainly during the first 250 ms of the out-of-phase spindle stimulation ( Figure 4 C, top right), but not in-phase spindle stimulation ( Figure 4 C, top left). The enhancing effect of out-of-phase stimulation on slow oscillation incidence was also found to be significant in the PAR region (one-way ANOVA, F= 15.277, p < 0.001; OUT versus NoSTIM-OUT: p = 0.011, Fisher’s LSD). In control analyses, we confirmed that out-of-phase stimulation was performed without a particular phase preference to continuing or newly appearing slow oscillation ( Figure S4 ), whereas in-phase stimulation was consistently performed during a slow oscillation up-state ( Figure S4 ). Overall, these results indicate that spindle stimulation of the TRN can induce slow oscillations in the out-of-phase condition but did not affect the consolidation of contextual memory in this group compared with the no-stimulation control group.

(C) Mean ± SEM event-correlation histogram of slow oscillation events (slow oscillation incidence rate, defined as the number of detected slow oscillation events, characterized by their negative peaks, divided by the total number of stimulations during the 6 hr intervention) in EEG recordings over frontal (FRO, top) and parietal (PAR, bottom) cortical areas, time-locked to stimulation onset for in-phase (left, red) and out-of-phase (right, green) stimulation and their respective no-stimulation control periods (NoSTIM-IN and NoSTIM-OUT, black). Horizontal blue bar indicates stimulation interval. Note that the distinct increase in frontal slow oscillation events during out-of-phase stimulation concentrated at 0.1–0.25 s post-stimulation onset.

(B) Slow oscillation incidence rate in frontal (FRO, left) and parietal (PAR, right) following in-phase (IN, red) and out-of-phase (OUT, green) stimulation and the corresponding no-stimulation control conditions (NoSTIM-IN, orange; NoSTIM-OUT, light green). ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05 for post hoc pairwise comparisons; data are shown as box-whisker plots with box limits representing the first and third quartile and whiskers representing the minimum and maximum range of the data.

(A) Three representative 3.5 s traces of frontal EEG recordings illustrating slow oscillations evoked by out-of-phase stimulation (marked by green arrows). Dashed vertical line indicates the beginning of the stimulation period (horizontal blue bar).

Determining discrete spindle events within a 750 ms time window following the onset of stimulation revealed for FRO recordings a significantly higher spindle incidence rate for in-phase than out-of-phase stimulation conditions ( Figure 3 A, left; one-way ANOVA, F= 60.243, p < 0.001; IN versus OUT: p < 0.001, Fisher’s LSD), as well as a distinct elevation compared with the corresponding no-stimulation conditions, no-stimulation in-phase (IN versus NoSTIM-IN: p < 0.001, Fisher’s LSD), and no-stimulation out-of-phase conditions (OUT versus NoSTIM-OUT: p < 0.001, Fisher’s LSD). Increases in spindle incidence were closely comparable between in-phase and out-of-phase stimulation with reference to the rates during the respective NoSTIM-IN and NoSTIM-OUT control conditions. In the NoSTIM-IN condition, spindle density within 750 ms following detection of a slow oscillation up-state was significantly higher than that for the delayed interval corresponding to the out-of-phase stimulation protocol (NoSTIM-OUT versus NoSTIM-IN: p < 0.001, Fisher’s LSD; Figure 3 A), confirming endogenous phase-locking between slow oscillations and spindles. No difference was found for the incidence of PAR and CA1 spindles following stimulation or slow oscillation detection (one-way ANOVA, PAR: F= 1.275, p = 0.297; CA1: F= 0.615, p = 0.610). Importantly, whereas the ratio of the total spindle count to the total slow oscillation count (for the 6 hr period of sleep monitoring) was comparable in all three conditions ( Figure 3 B; Kruskal-Wallis one-way ANOVA, U= 3.640, p = 0.162), slow oscillation-spindle coupling (i.e., the proportion of detected slow oscillations that are coupled with a spindle within 750 ms) was distinctly increased for the in-phase condition compared with both out-of-phase and no-stimulation conditions ( Figure 3 C; Kruskal-Wallis one-way ANOVA, U= 16.923, p < 0.001). Time-event correlation histograms also confirmed that spindle modulation during optogenetic stimulation, as observed not only in the cortical EEG but also in local field potential (LFP) recordings from CA1, derives from spindle cycles that are highly synchronized to the imposed stimulation rhythm ( Figure 3 D), with this synchrony being absent in the no-stimulation condition. Finally, we examined the influence of the TRN stimulation on inter-event intervals by determining the spindle incidence for a time window of 1.5 to 2 s post-stimulus. This analysis did not reveal any persisting effect on the generation of spindles (F< 1.170, p > 0.170).

(D) Means ± SEMs for event-correlation histogram of identified spindle troughs in frontal EEG (FRO, top) and hippocampal LFP recordings (CA1, bottom), time-locked to stimulation onset (t = 0) for in-phase (IN, red histogram), out-of-phase (OUT, green), and corresponding no-stimulation control conditions (NoSTIM, black). The spindle trough density percentage was estimated as the number of detected spindle troughs per 10 ms bin, divided by the total number of stimulations during the 6 hr intervention (set to 100%). Left column: comparison of in-phase and out-of-phase stimulation. Middle and right columns: comparison of in-phase (middle) and out-of-phase (right) stimulation with corresponding periods of the no-stimulation control. Significant differences (p values) are indicated at the bottoms of panels.

(C) Proportion of online-detected slow oscillations which coincided with a spindle within 750 ms after slow oscillation detection for in-phase (red bar), out-of-phase (green bar), and no-stimulation conditions (black bar). ∗∗∗ p < 0.001, ∗∗ p < 0.01 for post hoc pairwise comparisons; data in (A)–(C) are shown as box-whisker plots with box limits representing the first and third quartile and whiskers indicating the data range.

(B) The ratio (set to 100%) of the total number of spindles to that of slow oscillations identified offline during the entire 6 hr sleep period for in-phase (red bar), out-of-phase (green bar), and no-stimulation (black bar) groups.

(A) Spindle incidence rate (in percent of total stimulations) in frontal (FRO, left) and hippocampal (CA1, right) recordings following in-phase (IN, red) and out-of-phase (OUT, green) stimulation and for the corresponding periods in the respective no-stimulation control conditions (NoSTIM-IN, orange; NoSTIM-OUT, light green). An induced spindle was defined as a spindle event occurring within a 750 ms interval following the start of stimulation.

The effects of in-phase stimulation on memory formation were not conveyed by gross changes in sleep architecture. Sleep onset and time spent in different sleep stages during the 6 hr interval were closely comparable among the three stimulation conditions ( Table S1 ). Also, the overall density of slow oscillation, spindle, and ripple events did not differ among conditions ( Table S2 ). However, optogenetic spindle stimulation altered the fine-tuned interplay between the three rhythms ( Figure 2 A). Averaging electroencephalograms (EEGs), time-locked to stimulation onsets, confirmed the emergence of spindle-like activity coalescing with a large negative slow oscillation half-wave during in-phase stimulation; notably, this negative half-wave was absent in the out-of-phase condition ( Figure 2 B). Moreover, this analysis revealed that the optogenetic spindle-like generation of the anterior TRN induced predominantly frontal cortex (FRO) spindles, which, on average, synchronized to the stimulation. An entrainment by the optogenetic stimulation was also observed for spindles in the hippocampal CA1 region, which is consistent with observations that spindles invade hippocampal structures (). However, surprisingly, spindle-like stimulation did not entrain spindles recorded in parietal cortex (PAR). Overall, this distribution indicates that the stimulated TRN regions mediate a specific topographical pattern of spindle activity and influence the hippocampal local field potential within short delays.

(B) Mean ± SEM. EEG signals (filtered at 0.3–30 Hz) over frontal (FRO, top) and parietal (PAR, middle) cortical areas and averaged LFP signals from the hippocampal CA1 (bottom), time-locked to stimulation onset, across all mice under in-phase (red lines), out-of-phase (green), and no-stimulation (black) conditions. Vertical dashed black lines indicate stimulation onset (t = 0). Left column: comparison of in-phase and out-of-phase stimulation. Middle and right columns: comparison of in-phase (middle) and out-of-phase (right) stimulation with corresponding periods in the no-stimulation control. Significant differences (p values) are indicated at the bottoms of panels. Please note that the SEM was generally smaller than 0.2 mV and is thus not discernable from the trace of the mean in some cases.

(A) Three representative EEG traces from frontal (FRO) recording electrode for each of the three experimental protocols (3 s raw signals and signals filtered in the 7–10 Hz spindle band): in-phase, out-of-phase, and no-stimulation. The top two rows show examples with co-occurring spindle, whereas the bottom row shows cases with no identified spindle events. Online-detected slow oscillations that trigger the stimulation are highlighted in green. Intervals corresponding to light stimulation are indicated by blue bars; the starts of intervals for the no-stimulation condition corresponding to in-phase (NoSTIM-IN) and out-of-phase (NoSTIM-OUT) protocols are indicated by green filled and empty triangles, respectively. Offline-identified discrete spindle events are framed by dashed rectangles.

The hippocampal dependency of the effect of in-phase spindle stimulation was assured in 26 additional mice using an object-place recognition task (OPR; Figure S2 ) known to be sensitive to the effects of sleep (). Mice receiving stimulation in-phase with the slow oscillation up-state during slow wave sleep (SWS) after learning showed enhanced place memory (in terms of enhanced exploration time for the displaced object) on retrieval testing 24 hr later (F= 4.338, p = 0.025; Figure S2 C). Place memory after out-of-phase stimulation did not differ from memory in the no-stimulation control group. Together, these results suggest that spindle stimulation phase-locked to the slow oscillation up-state specifically enhances consolidation of hippocampus-dependent memory during sleep.

We tested transgenic mice expressing channelrhodopsin2 (ChR2) in parvalbumin (Prv)-expressing inhibitory neurons (Prv-mhChR2-EYFP; n = 26), a dominant subpopulation of the TRN that is sparse in surrounding thalamic nuclei () ( Figure 1 B; see also Figure S1 ), on a combined cued/contextual fear-conditioning (FC) task in which a 30 s tone followed by a 2 s shock was delivered in context A during conditioning () (see STAR Methods for details; Figure 1 C). Retrieval was assessed 24 hr later by measuring freezing (1) to the same context A, as a readout of hippocampal contextual fear memory, and (2) to a tone delivered in a different context B, as a readout of cued fear memory that is not essentially dependent on the hippocampus. During the first 6 hr following conditioning, the mice were subjected to one of three stimulation protocols ( Figure 1 D; Figure S3 ; see also Method Details section). In-phase mice (IN) received spindle-like optogenetic stimulation (62.5 ms on/off, 4 pulses) to the TRN during NREM sleep, which occurred in synchrony with the up-states of online-detected slow oscillations. Out-of-phase mice (OUT) were likewise stimulated during NREM sleep but with a random delay between 0.5 and 1.0 s following identification of a slow oscillation up-state. Control mice received no stimulation (NoSTIM). At retrieval, contextual fear memory was selectively enhanced in in-phase mice, whereas out-of-phase mice showed no improvement in contextual fear memory compared with no-stimulation controls ( Figure 1 E; F= 8.358, p = 0.002, for one-way analysis of variance [one-way ANOVA]; stimulation condition main effect; NoSTIM versus IN: p = 0.001, NoSTIM versus OUT: p = 0.438, and IN versus OUT: p = 0.007, Fisher’s least significant difference [LSD]). Collectively, these results demonstrate that spindles are not effective per se, but instead enhance hippocampus-dependent memory only if they coincide with the more excitable depolarizing up-state of a slow oscillation (). Neither in-phase nor out-of-phase stimulation changed memory for cued fear compared with that observed in no-stimulation control mice (F= 0.051, p = 0.950), indicating that our stimulation in-phase during NREM sleep preferentially benefits hippocampus-dependent memory ().

Discussion

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Born J. The consolidation and transformation of memory. We found that thalamic-induced spindles nest hippocampal ripples to their troughs both in optogenetically induced and spontaneous conditions. This pattern suggests that spindles, rather than slow oscillations, are the major factor synchronizing the occurrence of hippocampal ripples and possibly memory replay, in the first place, to the excitable phase of the spindle cycle and, in the case of slow oscillation driven spindles, to the excitable slow oscillation up-states (). Whereas the mechanism of spindle oscillation-induced nesting of ripples remains unclear, our results support the view that the increased spindle-ripple co-occurrence in the presence of a slow oscillation up-state represents a condition promoting the systems consolidation of contextual memory. Indeed, given that ripples enwrap reactivated hippocampal memory information, our observations are consistent with the notion of spindle-ripple events facilitating the hippocampal-to-neocortical transmission of memory information for longer-term storage in neocortical networks ().

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Destexhe A. Why do we sleep?. Taking this systems consolidation perspective, our finding is of interest that in-phase stimulation appeared to increase spindles phase-locked to slow oscillations mainly over frontal cortical regions, whereas nesting of ripples within spindle troughs revealed to be stronger for parietal cortical and hippocampal networks, a pattern which has been likewise revealed to occur spontaneously under unstimulated conditions in humans (). Simultaneously, in-phase stimulation increased the co-occurrence of spindles between frontal and parietal cortical regions as well as between frontal as hippocampal sites. Taken together, this topographical pattern suggests that the putative hippocampal-to-neocortical transmission of reactivated memory information during spindle-ripple events involves a fine-tuning between anterior and posterior spindle activities in thalamo-cortical networks, with the former primarily impacting frontal cortical networks and the later primarily serving to synchronize memory reactivations in hippocampal networks. While this fine-tuning of spindle activities and its function remains to be elucidated, we speculate that the frontal spindles are particularly relevant for redistributing the representation toward respective neocortical networks. This view is supported by our finding that the enhancing influence of in-phase stimulation on the incidence of slow oscillation-spindle-ripple triplets was specifically linked to the frontal cortical recordings. Generally, spindles that occur during the excitable up-state of the slow oscillation can be considered to be particularly effective in inducing plastic synaptic changes in cortical networks that mediate the more persistent storage of the memory information in neocortical networks ().

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Morris R.G.M. Schema-dependent gene activation and memory encoding in neocortex. Moreover, the view of slow oscillation-spindle-ripple triplets reflecting systems consolidation of hippocampal memories, in light of our findings, implicates that silencing frontal cortex during sleep within 6 hr after fear conditioning should impair memory consolidation which, at a first glance, contrasts with the rather slow temporal gradient in which memories become independent of the hippocampus (). However, although a direct test of this implication is missing, there is evidence that, depending on pre-existing knowledge, new memories can become rather quickly independent from hippocampus (within 24 hr) and at this stage can be also disrupted by impairing prefrontal cortex function (e.g.,).

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Born J. Driving sleep slow oscillations by auditory closed-loop stimulation-a self-limiting process. In addition, we found that the thalamic-induced spindle-like stimulation can promote the generation of a cortical slow oscillation, whereas an inhibition of the TRN resulted in a reduced incidence of slow oscillations. Here, we confirmed that the effect on slow oscillation incidence alone was not sufficient to alter the consolidation of memory since enhancing versus suppressing slow oscillation events in the respective out-of-phase conditions did not express itself in corresponding changes in memory performance. Although a contribution of TRN activation to the emergence of slow oscillations has been reported by others (), our findings are the first to show that TRN stimulation that mimics natural thalamo-cortical spindle input suffices to generate a cortical slow oscillation. The mechanisms mediating this effect remain obscure. The effect might reflect a phase-resetting of cortical networks, which spindles produce by facilitating the transition of network activity into the slow oscillation down-states. However, a mere resetting would not explain that spindles induced out-of-phase increased the absolute number of slow oscillation events. The state of readiness of TRN neurons, i.e., the number of recruitable neurons for population spiking, and on-going thalamo-cortical oscillations might be another factor determining whether stimulation produces spindles () together with slow oscillations () or no response. In natural conditions, thalamo-cortical spindle activity might contribute to the development of longer trains of slow oscillatory activity ().

Importantly, we found that both in-phase and out-of-phase inhibition of TRN reduced the occurrence of spindles; however, only mice subjected to an in-phase inhibition of the TRN showed an impairment during contextual fear recall, corroborating the view that a reduction in spindles alone is not sufficient to disrupt memory formation. Instead, an effective impairment requires that the inhibition of spindles occurs in the presence of a cortical slow oscillation up-state, obviously representing the time window during which, under normal conditions, the hippocampal-cortical dialog underlying systems consolidation is vulnerable.