1 INTRODUCTION

The hippocampus has long been known to play an important part in a variety of cognitive functions. Over recent years many research groups have advanced in identifying the neural activity patterns that are instrumental for maintaining these functions. The hippocampal sharp-wave ripple (SWR; 100−200 Hz), an oscillatory event connected to both highly synchronous neural firing within the hippocampus and modulation of neural activity in various brain regions, is one of those patterns (Buzsáki, 2015).

Of note, the sharp-wave and the ripple are two separate components of the SWR, that occur in different layers of the hippocampus (Figure 1). Most studies discussed in this review identify and manipulate the ripple component of the SWR (for overview of studies see Tables 1 and 2). For more details on online and offline detection of ripples see Box .

Ripples and sharp waves: (a) shown are local field potential recordings from different layers in the hippocampus. The stratum oriens shows an upward deflection during a ripple, the stratum pyramidale the actual ripple oscillations (100–200 Hz) and below the stratum radiatum shows the simultaneously occurring sharp wave. The sharp wave is due to the input coming from CA3 exciting the dendrites of the CA1 pyramidal cells. On the right a cartoon pyramidal cell with the cell body at stratum pyramidale, dendrites both up and down in stratum oriens and radiatum, and the axon leaving upwards. (b/c) Shown are examples for electrical ripple disruption (left) and control disruption with the electrical pulse send with a delay after the ripple (right; from (Aleman-Zapata et al., 2020)) TABLE 1. Overview of experimental details across different ripple disruption and extension studies Authors Model N Behaviour Ripple-d timepoint (sleep/wake) All ripples or selection Stim. type Girardeau et al. (2009) Rat (male)

17 (test method)

26 (task)

Spatial memory task (8-armed radial maze) Post training; rest/sleep (1 hr) All ripples Single pulse (0.5 ms; 5~30 V) stim. through bipolar electrodes in vHC. Ego-Stengel and Wilson (2010) Rat (male) 6 (only 5 in results; 1 of those is stopped after 3 days) Two identical spatial navigation tasks (4-arm radial maze in one single, large wagon-wheel structure) Post training after one of test maze tasks; rest/sleep (1 hr) All ripples during NREM and wake (though ripples could occur in the 2-s recovery interval) Electrical stim. (two biphasic pulses (20–60 µA) with 10 ms interval) of vHC via 3–6 tetrodes. Jadhav et al. (2012) Rat (male)

6 ripple-d

6 control stim

4 un-implanted

Spatial alternation task (W-track task) During the task; wake All ripples Single pulse stim. by a bipolar electrode in vHC. Girardeau et al. (2014) Rat (male) 6 (within animal control; though not all rats were tested in each condition) Spatial memory task (8-armed radial maze) and exploratory locomotor task (circular arena) Post training in 8-arm radial maze, locomotion in familiar circular arena or home cage; rest/sleep (<1 hr) All ripples Stim. electrodes in vHC + recording electrodes in CA1 pyramidal layer. Single pulse stim. (0.5 ms). de Lavilleon et al. (2015) Mouse (male) 10 (of 40 implanted animals) Open field During task or post exploration; wake and sleep (1 hr)

Wake: during spikes of place cell

Sleep: reactivation-related spiking activity of the selected place cell

Polytrodes in CA1, stim. electrode in MFB. Train lasting 100 ms and composed of fourteen 1 ms negative square-wave pulses (140 Hz). Kovács et al. (2016) Mouse (female) 3 (within animal control) (initial) Novel environment, passive exploratory behaviour Post training, between 1st and 2nd exposure to same enclosure; rest/sleep (3 hr) All ripples Microdrive with 15 tetrodes and bilateral 2 × conical optic fibre (stable, placed in stratum oriens) covering dorsal CA1. Closed-loop optogenetic disruption (laser light pulses, green light 200 ms) only affecting Arch-ECFP pyramidal cells in CA1. Maingret et al. (2016) Rat (male) 15 out of 23 (12 implanted and 3 un-implanted) Spatial object recognition task + flower pot for sleep Post training; sleep (lasted until 1,000 stims had been delivered [~4,000 s of SWS]) All ripples in HPC followed stimulation of motor cortex Microdrive with six or 16 tetrodes in right mPFC and CA1, incl. bipolar electrode in left motor cortex. Monophasic single-pulse (0.1 ms) of deep layers of motor cortex, delivered by a constant current stimulator. Optimal stim. voltage (17.5–22.5 V) was the minimum necessary to reliably induce propagating delta waves. Novitskaya et al. (2016) Rat (male) 22 implanted (divided over three diff. stim. groups), unknown number un-implanted (total reported: 48) Radial maze Post training; rest/sleep (1 hr) All ripples in HPC followed stim. of LC Monopolar electrical stim. applied unilaterally to LC using trains of bi-phasic square pulses (0.4 ms, 0.05 mA) at 20-100 Hz for 100–200 ms. Low freq. group: five pulses at 20 Hz, 200 ms. High freq. group: 50/100 Hz, 100/200 ms. Papale et al. (2016) Rat (male) Spatial adjusting delay discounting task During the task; wake van de Ven et al. (2016) Mouse (male) 8 (within animal control; only 7 for ripple-d) Novel (irregular shaped) and familiar (round) box Post training after novel or familiar environment; rest/sleep (1 hr) Selected ripples during sleep (1: identification of assembly patterns during first exploration, 2: track expression of these during subsequent sleep and re-exposure, 3: ripple-d) Bilateral: 5 tetrodes, 1 optic fibre in CA1. Closed-loop optogenetic disruption (50 or 80 ms light on). Ensemble recordings of CA1 principal neurons. Roux et al. (2017) Mouse (male) 5 (within animal control) Cheeseboard maze (water reward); new set of 3 goal locations daily. Extra test: cue-guided version of the task. During the task, at goal location; wake All ripples in a subset of pyramidal neurons in CA1 Drive with silicone probe (4 or 8 shanks, 32 or 64 sites); one or more shanks equipped with etched optical fibres coupled to head-mounted laser diodes for focal stim. Implanted unilateral (2 mice), bilateral (3 mice). Closed-loop optogenetic disruption (60 ms square pulses, approx. 200 µW, 1 per detection). Rangel Guerrero et al. (2018) Mouse (female) 3 (pilot study) Cheeseboard maze In-between training sessions; sleep (3 hr) All ripples Microdrive array (15 tetrodes) combined with 4 optic fibres, targeting dorsal CA1. Closed-loop optogenetic disruption via 200 ms long green laser pulses (561 nm). Fernandez-Ruiz et al. (2019) Rat (male) 5 out of 20 (15 control rats) W-maze During the task; wake All ripples Microdrive with silicone probes and optic fibres. Closed-loop optogenetic stim. For prolongation: tapered-onset 100 ms-long light stimulus. For truncation: 10 ms pulses. Michon et al. (2019) Rat (male) 8 out of 23 used for ripple-d Dual-environment reward-place association task In-between training in the two environments; rest/sleep (2 hr) All ripples Microdrive array with 24 tetrodes and 3 stim. electrodes in vHC. Biphasic electrical pulses (0.2 ms) varied from 100–500 mA. Aleman-Zapata et al. (2020) Rat (male) 6 (within animal control) (baseline, track, novelty) plusmaze for ripple-d Post training; sleep (4 hr) All ripples Stimulation electrode in vHC. 1/3 of detected ripples were followed by two stim. pulses at 200 and 400 ms. Gridchyn et al. (2020) Rat (male) 4 (within animal control) (double) cheeseboard maze Post training; rest/sleep (4 hr) HSEs 128-channel, independently movable electrode arrays + 4 optic fibres in CA1. Assembly detection performed during initial phase of HSEs, and a laser pulse triggered disruption of HSE firing pattern if HSE encoded for target maze. Oliva et al. (2020) Mouse (male) 17 (7 ripple-d, 10 control) Social-recognition task Post training, between learning and test; sleep (1 hr) All ripples Microdrive with unilateral silicon probe (4 or 5 shanks, 64 or 60 sites) targeting HPC and bilateral optic fibre in CA2. Closed-loop optogenetic disruption via 10 ms high-intensity (5–10 mW) light pulses. Igata et al. (2021) Rat (male) 17 (5 rats recording electrodes only, 12 recording and stim. electrodes) Open field, spatial learning task (start, checkpoint 1, goal; update checkpoint - orig. goal) within 1 hr During the task, after reward relocation; wake All ripples Drive with 16 tetrodes (incl., in some, bipolar electrodes in vHC); right dorsal CA1. Closed-loop electrical stim. (single pulse of 100 µs, 140 to 180 µA, stim. rate max. 4 Hz) applied to vHC. Abbreviations: freq., frequency; HPC, hippocampus; HSE, high synchrony event; LC, locus coeruleus; max., maximum; MFB, medial forebrain bundle; mPFC, medial prefrontal cortex; NREM, non-rapid eye movement; stim., stimulation; SWS, slow-wave sleep; vHC, ventral hippocampal commissure. TABLE 2. Overview of online and offline detection techniques, detection assessment and controls across studies Authors Online detection Offline detection Detection assessment Controls Girardeau et al. (2009) Filtering in the ripple band and thresholding Band-pass filtering (100–200 Hz), squaring and normalising, then thresholding the field potential recorded in CA1 pyramidal layer. Ripples were defined as events peaking at >5 SD and lasting <100 ms. Average online detection rate was 86.0 ± 1.3% (SEM) of post hoc detected ripple Delayed trigger (random 80–120 ms) Ego-Stengel and Wilson (2010) Ripple events detected through hardware; filter 100–400 Hz Causal online filtering delay was determined and corrected by shifting all signals −7 ms. No use of zero-phase FIR filter to avoid premature stimulation. Double-threshold crossing method on absolute value of LFP (mean ripple power + 3 SD and + 10 SD). Minimum duration of 30 ms. Gaps smaller than 50 ms were discarded. No stimulation Jadhav et al. (2012) Monitored power (100–400 Hz) simultaneously across multiple tetrodes (5–6) in CA1. Threshold had to exceed on at least 2 tetrodes. Speed filter with threshold of 5 cm/s to 10 cm/s to prevent false positives. LFPs filtered between 150–250 Hz. Ripples detected when a smoothed ripple envelope was above 3 SD of the mean for at least 15 ms on at least one tetrode. Delayed trigger (random 150–200 ms) Girardeau et al. (2014) Filtering in the ripple band and thresholding See Girardeau et al., 2009

Detection rate >83 ± 2%.

False detection rate <19 ± 2%

Delayed trigger (random 80–120 ms) de Lavilleon et al. (2015) Spike detection based on voltage threshold on a polytrode channel with Spike2 software. Threshold manually adjusted to detect highest action potentials using hexa/octrodes. Semi-automatic cluster cutting using KlustaKwik and Klusters. Sensitivity of 43.2% (true positive rate). High specificity (false negative rate) Non-rewarded stimulation Control wake-pairing protocol Kovács et al. (2016) Use of an analogue ripple detector circuit as Nokia et al., 2012. Band-passed filtered differential signal (subtracted stratum radiatum channel from stratum pyramidale channel). Delayed trigger (1.32 s) Maingret et al. (2016) Threshold crossing on the ripple band (100–250 Hz) LFP recording in CA1 pyramidal layer was band-pass filtered (150–250 Hz), squared, low-pass filtered and normalised. Ripple detected when signal was above 2 SD for 30 ms to 100 ms and peaked >5 SD. Delayed trigger (random 160–240 ms) Novitskaya et al. (2016) Threshold crossing on band-passed (140–240 Hz) CA1 LFP LFP band-passed (120–240 Hz). Ripple detected when ripple-RMS exceeded 4 SD threshold of the mean. Start and end 2 SD with min. duration of 0.02 s. Random high freq. stim. with 2–11 s interstimulus intervals. Ripple-triggered low freq. stim. No stimulation. Papale et al. (2016) See Jadhav et al., 2012 See Jadhav et al., 2012 See Jadhav et al., 2012 van de Ven et al. (2016) Every 5 ms a copy of the last 20 ms was filtered (125–250 Hz) and another copy was convolved with a Morlet wavelet (160 Hz central freq.). Detection of average power in the last 10 ms was 4 SD above baseline and the maximum of the wavelet-convolved signal was 3 SD above baseline. Same as McNamara et al. 2014. Signals were band-pass filtered (135–250 Hz) and a signal from a reference electrode was subtracted. The power (RMS) was calculated per electrode and summed across all CA1 pyramidal cell layer electrodes. In rest sessions without light delivery 80.1% ± 1.0% of the ripples were detected. Average latency of 7.68 ± 0.30 ms before peak power. Random silencing independent of ripples. Total number of random pulses equal or higher than the average number of pulses delivered in the ripple disruption condition. Roux et al. (2017) The root mean square of a CA1 pyramidal layer signal (80–250 Hz) was computed in two running windows (RMS1 = 2 s and RMS2 = 8 ms). Ripples detected when RMS2 exceeded 3 × RMS1 for at least 8 ms. Filtered signal (80–250 Hz) and instantaneous power was computed. Events exceeding 2.5 SD from the mean were selected. Events shorter than 15 ms were discarded and those closer to 15 ms were merged. 83 ± 4% of visually identified ripples were targeted. 63 ± 4% of all online detected ripples were considered false positives by visual scoring. Ripple-delayed place cells (random 100–300 ms) and non-silenced place cells Rangel Guerrero et al. (2018) See Kovács et al., 2016 Delayed trigger (1.32 s) Fernandez-Ruiz et al. (2019) Detection of sharp wave (8–40 Hz), ripple (80–300 Hz) and neocortex noise (80–300 Hz). Ripple detected when both events co-occurred in the absence of a noise signal in the neocortex. Ripple detection: LFP filtered (80–250 Hz) and instantaneous power (clipped at 4 SD) was rectified, and low pass filtered (55 Hz). Power of non-clipped signal exceeded 4 SD with a min. duration of 15 ms. Sharp waves detection: LFP from str. radiatum filtered (5–40 Hz) with a duration of 20–400 ms. Events exceeded 2.5 SD. SWR: Simultaneous sharp waves and ripples. Delayed trigger (random 400–1,000 ms). No stimulation. Michon et al. (2019) Ripple power was summed across electrodes and ripples were detected when it exceeded a linear combination of mean and mean absolute deviation estimates of the ripple power. Detection of noise in the cortex to discard spurious ripples. The ripple envelope (140–225 Hz) was averaged across recording sites, smoothed and detrended. A ripple was detected when crossing 8 SD above the mean. Detection rates above 1 Hz led to increased false positives. A maximum detection rate of 1 Hz was set in the online detection. Delayed trigger (random 100–250 ms) Aleman-Zapata et al. (2020) Bandpass-filtered the LFP signal (100–300 Hz). A user-defined threshold was applied to the rectified band-passed signal. LFP band-pass filtered on the ripple spectrum (100–300 Hz). Thresholding of voltage peaks with a minimum duration of 30 ms. Two detected ripple peaks closer than 50 ms were merged. Delayed trigger (200 and 400 ms) No stimulation. Gridchyn et al. (2020) High synchrony events were detected when, in a 20 ms window, the number of detected spikes exceeded a threshold of 3.5-times the mean spike numbers in a 20-ms windows during the pre-rest session. Threshold adjusted to achieve a 1 Hz detection rate. Real-time decoding determined which environment was being encoded. Spike sorting and pyramidal unit discrimination performed similarly to Csicsvari et al. 1999. Environment preference was determined based on unit firing rate increase, coherence, and sparsity of place fields. Most events occurred earlier than ripples and ~50% of events were followed by a ripple within 50 ms. The time interval between the threshold crossing and the deflection in the LFP caused by the light pulse was 1.04 ± 0.09 ms No stimulation if high synchrony event encoded the control maze. Oliva et al. (2020) Bandpass-filtered the LFP signal between 100 and 300 Hz. Detection of noise in the neocortex. LFP filtered (100–300 Hz) and instantaneous power (clipped at 4 SD) was rectified, and low pass filtered (55 Hz). Power of non-clipped signal exceeded 4 SD with a minimum duration of 15 ms. Delayed tigger (random 500–1,000 ms). Cre (negative) animal. Igata et al. (2021) A smoothed envelope of a tetrode band passed signal (100–400 Hz) was computed. Ripples detected when the animal’s running speed was <5 cm/s and the envelope exceeded the detection threshold of 3–4 SD above the mean computed during periods in the rest box. LFP filtered at 150–250 Hz. Smoothed envelope. Ripple power during periods with a running speed of <5 cm/s in the task periods were computed per tetrode. Only considered detections with a duration of 50–500 ms. Delayed trigger (250 ms). No stimulation. Abbreviations: FIR, finite impulse response; LFP, local field potential; min., minimum; RMS, root mean square.

Hippocampal ripples are thought to play a role in both the consolidation and retrieval of memories but have also been proposed to contribute to planning of future behaviour (Buzsáki, 2015). Moreover, hippocampal neuronal firing during ripples has been shown to contain elements from both past, i.e. memory reactivations, and future experiences (Buzsáki, 2015; Genzel et al., 2020; Girardeau & Zugaro, 2011; de la Prida, 2020). While early studies provided correlative links between hippocampal ripples, memory reactivations and memory performance (Dupret et al., 2010; Mölle et al., 2006), it is only in the last decade that a select number of studies started to demonstrate the causal relationship. Among others, the use of closed-loop ripple disruption, by which ripples during either rest/sleep (i.e. targeting memory consolidation) or wakefulness (e.g. targeting memory retrieval, working memory, planning or consolidation) are online detected and subsequently disrupted, can alter subsequent memory performance. Both electrical stimulation to the ventral hippocampal commissure as well as direct optogenetic inactivation of selected cells in the hippocampus have been used as disruption methods.

In the present review, our aim is to provide an overview of the different ripple-related, closed-loop intervention methods used in rodent research. We will summarise findings these studies have produced and how they have contributed to our understanding of the function of this oscillation. We will further discuss closed-loop applications used in rodents targeting either other oscillations or more specific cell activity. Finally, we will highlight how detection methods (Box ), controls (Box ) and other methods differ across studies (Tables 1 and 2), and discuss the implications of these differences.

2 RIPPLE DISRUPTION AFTER TASK LEARNING

Ripple events associated with short bouts of neural firing activity in the hippocampus during post-learning rest or sleep have been linked to the reactivation of neural activity of preceding learning experiences and these reactivations have been proposed to be needed for the memory consolidation process (Buzsáki, 2015). While various studies had shown convincing evidence for a link between hippocampal ripples and offline memory consolidation (e.g. Dupret et al., 2010), it was not until the study of Girardeau et al. (2009) that a causal relationship was demonstrated.

Rats were trained in an eight-armed radial maze (Figure 2) daily over 15 days and the authors used closed-loop electrical stimulation upon online detection of hippocampal ripples during the first hour of rest/sleep after learning. This ripple disruption affected performance in the task, while global sleep architecture remained unchanged. In contrast, control rats that either received a delayed stimulation (i.e. stimulation was triggered by the detection of a ripple but delivered with an 80–120 ms delay) or no stimulation, did not show any performance deficits.

Tasks used in ripple disruption experiments targeting rest/sleep (a) and targeting the task period (b). (a.1) In the radial arm maze animals learn which of the eight arms are baited with food (usually three). Animals need to be trained for multiple days before performing above chance. (a.2.) In the wagon wheel maze animals learn to navigate from one fixed start location to one fixed goal location. Animals need to be trained for multiple days to perform well. (a.3) Plusmaze as used by (Aleman-Zapata et al., 2020). Each day (and therefore for each condition) a new arm is baited and each trial in that day has a different start location (different start arm). Each day also will include new external cues for orientation. Animals only need to be trained for 20 min to perform above chance at the 24-hr test. (a.4) For the novel open field environment an animal is placed in a new box, with a new shape and new cues on the wall and is free to explore. (b.1) In the cheese board maze each day (and therefore for each condition) the animal learns three new goal locations (baited ports), which are tested after a short (~1 hr) delay period on the same day. After pre-training the animals perform stable over a long time period; the external cues are stable over time. (b.2) For the w-maze (also known as m-maze) the animal learns the rule to go from outside arm in (1), then from the inside arm to the other outside arm (2) and then back from the outside arm in (3). After pre-training the animals perform stable over a long time period; the external cues are stable over time

Following similar closed-loop electrical stimulation Ego-Stengel and Wilson (2010) disrupted all ripples occurring in the hour after the rats had learned to navigate one of two four-armed radial mazes arranged on a single, large wagon-wheel structure (Figure 2) daily over 8–10 days. Like Girardeau et al. (2009), they found that although stimulation disrupted the ongoing ripples and suppressed further ripples within 1 s of stimulation, no change in sleep/wake structure was observed. Furthermore, rats learned the control maze (not followed by ripple disruption) significantly faster than the test maze (followed by ripple disruption), indicating that ripple disruption during the post-training rest/sleep period impaired memory consolidation and thus slowed down learning the memory task.

Both these studies used a hippocampal-dependent spatial memory task and thus showed that elimination of ripples during the 1-hr post-training consolidation period results in a performance impairment in rats. However, Ego-Stengel and Wilson (2010) also showed that differences in performance by the rats in the test and control maze disappeared little over a week into training. This “delayed” learning could be due to the fact that ripple disruption only took place in the first hour post-training and that for learning to be completely eliminated, longer periods of ripple elimination are needed. Likewise, in the study by Girardeau et al. (2009) rats with disrupted ripples did perform above chance at the end of training. Indeed, Aleman-Zapata et al. (2020) using a one-session learning paradigm in contrast to the previous multi-day learning paradigms showed that a 4-hr post-learning closed-loop electrical stimulation protocol does eliminate learning in the plusmaze (Figure 2) similarly to the performance of rats that received sleep deprivation post-learning. In contrast, the rats performed above chance in the 24-hr test when delayed or no stimulation was applied during this 4-hr period, which is in line with the earlier mentioned studies.

While these studies point towards an important role for hippocampal ripples during the post-learning sleep period, it remained unclear how learning and subsequent consolidation requirements might influence and regulate ripples during sleep. Hence, Girardeau et al. (2014) trained rats on a spatial memory task and showed that when ripples were disrupted by closed-loop electrical stimulation during the subsequent sleep period, ripple occurrence was upregulated compared with control sleep periods where stimulations were delayed in time and thus did not interfere with ripples. Moreover, upregulation of ripples did not occur when ripple disruption was applied during sleep following random foraging in a familiar environment. These results indicate that ripple occurrence during post-training sleep is triggered by lear