Inhibition allocates spikes during hippocampal ripples

Sets of spikes emitted sequentially across neurons constitute fundamental pulse packets in neural information processing, including offline memory replay during hippocampal sharp-wave ripples (SWRs). The relative timing of neuronal spikes is fine-tuned in each spike sequence but can vary between different sequences. However, the microcircuitry mechanism that enables such flexible spike sequencing remains unexplored. We recorded the membrane potentials of multiple hippocampal CA1 pyramidal cells in mice and found that the neurons were transiently hyperpolarized prior to SWRs. The pre-SWR hyperpolarizations were spatiotemporally heterogeneous, and larger hyperpolarizations were associated with later spikes during SWRs. Intracellular blockade of Cl−-mediated inhibition reduced pre-SWR hyperpolarizations and advanced spike times. Single-unit recordings also revealed that the pre-SWR firing rates of inhibitory interneurons predicted the SWR-relevant spike times of pyramidal cells. Thus, pre-SWR inhibitory activity determines the sequential spike times of pyramidal cells and diversifies the repertoire of sequence patterns.


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Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy 1 Automated spike sorting was performed using Kilosort1, followed by manual curation in Phy2. All offline computational analyses were performed using MATLABR2017b and Python3. Most custom code for analysis of extracellular unit data is available at https://github.com/buzsakilab/buzcode. Each analysis procedure is described in necessary detail in the Method section for others to execute. Any analysis codes are available by sending a request to the corresponding author.

March
For patch-clamp recordings, sample size (the number of triple patching datasets and single whole-cell recordings in awake condition and from PV-Cre mice) was determined by previous publications that performed in vivo whole-cell recordings from the hippocampus (Gan et al., Neuron, 2017). Single and dual whole-cell recordings in anesthetized condition were obtained more frequently in this order in the process of achieving triple whole-cell recordings, which resulted in larger number of data than triple patching datasets (Jouhanneau et al., 2018(Jouhanneau et al., , 2019. For unit recordings, n=6 animals were used, exceeding the typical cohort sizes (3-4 animals) of studies examining SWRs (Foster and Wilson 2006; Grosmark and Buzsaki 2016). Our larger cohort allows for an examination of individual variability and accurate assessment of population averages.
No further statistical methods were used to predetermine the sample size.
For patch-clamp recordings except for Figure S6, we used male ICR mice (28 to

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Blinding No blinding was done in this study because knowledge of experimental conditions was required during data collection.

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We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. For patch-clamp recordings, data without enough quality (recordings with mean Vm less than -50 mV and action potentials below -20 mV) and enough number of SWRs (recordings with less than 30 SWR events) were excluded for reliable statistical analyses. The criteria is described in the Method section of the manuscript. For extracellular unit recordings, one animal was excluded from further analysis for a lack of preSWR firing interneurons (Figure 7).

Antibodies
All conclusions of this study are based on recordings of populations of cells, which were consistent in all animals we used.
We used a within subject shuffle to generate a null hypothesis (Figure 5b, 7g, 8g). Therefore, the same variability and autocorrelation structure present in each signal is preserved in our null distributions. For Figure 2b inset, which is the only figure with comparison between experimental groups, we randomly allocated mice into control and CsF-DIDS groups.
Chicken anti-GFP was shown to react with YFP by the manufacturer. Guinea pig anti-PV was shown to stain parvalbumin in mice by the manufacturer. Alexa 488-labeled goat anti-chicken IgG and Alexa 594-labeled anti-guinea pig IgG were shown to immunohistochemically stain the corresponding primary antibodies by the manufacture. 3 patch-clamp recordings in Figure S6, we used PV-Cre mice (4 male, 1 female; 5 to 8 weeks old). For unit recordings, adult wild-type C57BL/6J mice (5 male, 1 female; 4 to 6 months old) were used. All animals were housed under a 12/12-h light-dark cycle (light from 07:00 to 19:00) at 22 ± 1 °C with food and water provided ad libitum.
The study did not involve wild animals.
The study did not involve samples collected from the field.
For patch-clamp recordings: Animal experiments were performed with the approval of the animal experiment ethics committee at the University of Tokyo (approval numbers: P29-9) and in accordance with the University of Tokyo guidelines for the care and use of laboratory animals. nature portfolio | reporting summary