Inhibitory Parvalbumin Basket Cell Activity is Selectively Reduced during Hippocampal Sharp Wave Ripples in a Mouse Model of Familial Alzheimer's Disease

SWRs are increased in three-month 5xFAD mice. A, SWRs were recorded in acute horizontal slices with the LFP electrode located in CA1. B, Example traces of three-month control (left) and 5xFAD (right) slices. First trace, LFP filtered between 1 and 1000 Hz, raster below indicates detected SWR events as overlap of SW and ripple events. Second trace, SW, filtered between 1 and 30 Hz, raster below indicates detected SW events. Third trace, Low γ, filtered between 20 and 50 Hz. Fourth trace, Ripple, filtered between 120 and 220 Hz, raster below indicates detected ripple events. Bottom, Z-scored time-frequency spectrogram from 1 to 250 Hz. C, Summary plot of SWR event frequency for 5xFAD and littermate controls for both one-month (n_mice = 10 CT, 10 5xFAD; left) and three-month cohorts (n_mice = 27 CT, 29 5xFAD; right). D, SWR event amplitude and duration. E, Peak ripple frequency and the number of ripple cycles within SWR duration. F, Example SWR events for traces displayed in B, with average z-scored time-frequency spectrogram of all events below. G, Average FFT of 200-ms window around all SWR events from characteristic subset of n_slice = 7 CT, 7 5xFAD from n_mice = 4 CT, 5 5xFAD. Shaded region represents SEM. H–J, SWR-locked oscillation power in the low γ (20–50 Hz; H), ripple (120–220 Hz; I), and fast ripple (250–500 Hz; J) ranges. Note that in H, one value was identified as an outlier by the ROUT method but was retained in analysis, as the data point appeared valid, and even with removal did not alter the observed increase (U = 193, p = 0.0015; Mann–Whitney). For all plots, individual data points represent the average of all slices recorded from an animal (n_slice/animal in Table 1). Closed circles represent males, open circles females. Bar plots indicate normal data with mean ± SEM. Box-whisker plots represent non-normal data with Median and IQRs; p values indicated above brackets.

Altered ensembles of PCs are recruited in 5xFAD slices. A, Confocal imaging of PC ensembles were recorded concurrently with SWRs. Scale bars = 50 μm. B, Post hoc IHC for CB of imaged Thy1-GCaMP6f (GC) slices. Dashed gray lines approximately distinguish layers of hippocampus, str. rad. = stratum radiatum, str. pyr. = stratum pyramidale, str. or. = stratum oriens. Scale bar = 50 μm. C, Histogram of the distance from the center of the cell body to the str. pyr./rad. border for 539 GC+ CB–, 292 GC– CB+, and 146 GC+ CB+ cells pooled from n_slice = 4 from n_mice = 2 CT, 2 5xFAD. D, LFP and ΔF/F for identified cells in slices from three-month control (left) and 5xFAD (right) mice. Top trace, LFP trace with identified SWR events in raster below. Middle traces, Individual ΔF/F for each cell, sPCs in magenta, and dPCs in green. Bottom raster, Identified Ca²⁺ events above threshold. Dark colored event indicate concurrence with SWR, light gray indicates spontaneous event. E, The total number of active sPCs and dPCs for CT (n_slice = 25) and 5xFAD (n_slice = 23) slices. F, The number of sPCs/dPCs active during SWRs. Frequency (G), amplitude (H), and duration (I) of Ca²⁺ transient events averaged across sPCs and dPCs for each slice. J, top, Event matrix displaying for one example recording, the SWR events on the x-axis, and cells on the y-axis (magenta = sPC, green = dPC). Bottom left, Pairwise Jaccard similarity between SWR events (columns in event matrix). Similarity matrix is symmetric and the diagonal = 1 by definition - these have not been plotted for clarity. Bottom right, Pairwise Jaccard similarity between cells (rows in event matrix). Dashed lines indicate borders between sPC-sPC, sPC-dPC, and dPC-dPC comparisons. Quantification of cell similarity was performed by grouping all sPC-PC comparisons (sPC-sPC and sPC-dPC), and all dPC-PC comparisons (dPC-dPC and sPC-dPC). Cumulative distribution functions were calculated from all pairwise comparisons for (K) SWR-SWR similarity, (L) sPC-PC similarity, and (M) dPC-PC similar. K–M, All showed significant genotype differences via two-way ANOVAs; asterisks indicate regions surviving multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001. Box-whisker plots represent non-normal data with median and IQRs. Individual data points represent a slice. Closed circles represent slices from males, open circles females; p values of pairwise comparisons indicated above brackets.

PC spiking is relatively unchanged in 5xFAD mice. A, Diagram of a loose cell-attached recording of spikes from a Thy1-GCaMP6f+ PC paired with LFP recording of SWRs. B, Example traces from three-month control (left) and 5xFAD (right) slice. Top trace, LFP with SWR events in raster below. Gray shaded events indicate spontaneous SWR. Green shading indicates SWR coincident with at least one spike. Dark green shading indicates SWR coincident with burst, defined as three or more spikes each within 60 ms. Bottom trace, Cell-attached recording from PC. Detected spikes and bursts indicated in raster below. Dark shading indicates they are coincident with SWR event, light gray shading indicates spontaneous events. Spike rates were calculated separately during spontaneous and SWR periods for (C) sPCs, n_sPC = 13 CT, 9 5xFAD, and (D) dPCs, n_dPC = 26 CT, 35 5xFAD. The peri-SWR spike probability was calculated for (E) sPCs and (F) dPCs, by binning spikes into 100 2-ms bins and normalizing by total number of SWRs. Dark lines indicate average of all cells within a genotype, shaded regions indicate SEM, 0 ms indicates time of SWR peak. G, The rate of bursts for sPCs and dPCs, defined as at least three spikes each within 60 ms. H, Intraburst interval for sPCs and dPCs, defined as the average time between successive spikes in a burst. I, The fraction of spikes that occurred during SWRs. J, The fraction of SWRs that had one or more spikes. K, Example of SWR (top trace), with filtered slow γ (20–50 Hz) and ripple (120–220 Hz) signals (middle traces). The phase of oscillations was set at 0° at peaks and 180° at troughs. Bottom trace, Cell-attached recording, with spike times marked by vertical dashed lines. L–O, Polar phase plots of phase-locking of spikes to SWR-nested slow γ (L–M) and ripple (N–O) for sPCs (L, N) and dPCs (M, O). The angles of individual data points represent the average phase of all spikes for a cell. Length from origin (0–1) indicates the degree of phase-locking. A length of 1 signifies perfect phase-locking (every spike at same phase); a length of 0 indicates random or no phase-locking. Lines with arrowheads represent cell average, solid colored = CT, dashed gray = 5xFAD. Asterisks indicate result of Raleigh's test for non-uniformity, *p < 0.05, **p < 0.01, ***p < 0.001. p Values indicate results of circular mean comparison. For all plots, individual data points represent a cell. Closed circles represent cells from males, open circles females. Box-whisker plots represent non-normal data as median and IQRs; p values of pairwise comparisons indicated above brackets.

PCs receive increased synaptic E/I ratio. A, Example traces of LFP + whole-cell recording voltage-clamped at −70 mV in Thy1-GCaMP6f littermate controls and 5xFAD/+;Thy1-GCaMP6f mice. Excitatory postsynaptic potentials (EPSCs) were sorted as sEPSCs or swrEPSCs. B, Example swrEPSCs. C, Example traces of LFP + whole-cell recording voltage-clamped at 0 mV to detect sIPSCs and swrIPSCs. D, Example swrIPSCs. E–H, Summary plots of sEPSC frequency (E), amplitude (F), rise tau (G), and decay tau (H) for sPCs and dPCs in control and 5xFAD mice (n_sPC = 7 CT, 5 5xFAD; n_dPC = 12 CT, 9 5xFAD). I, sEPSQ = normalized spontaneous excitatory charge (integrated current per unit time). J, swrEPSQ = total excitatory charge during SWRs, integrated over a 100-ms window centered around the SWR peak. K–N, Summary plots of sIPSC frequency (K), amplitude (L), rise tau (M), and decay tau (N; n_sPC = 7 CT, 5 5xFAD; n_dPC = 14 CT, 11 5xFAD). O, P, Cumulative distribution functions, averaged over all cells for sIPSC amplitude (O) and decay tau (P), both for sPCs (top) and dPCs (bottom). Dark lines represent cell average, shaded region represents SEM. Q, sIPSQ = normalized spontaneous inhibitory charge (integrated current per unit time). R, swrIPSQ = total inhibitory charge during SWRs. S, Spontaneous synaptic E/I ratio of normalized charge for each cell (sEPSQ/sIPSQ). T, Synaptic E/I ratio for 100-ms window centered around SWR peak (swrEPSQ/swrIPSQ). U, V, During SWRs, the charge for each cell (EPSQ and IPSQ) was calculated in 2-ms bins by integrating the current in a sliding 100-ms window centered around that bin, for both sPCs (U) and dPCs (V). Summary data in J and R thus represent these curves at the y-intercept. Asterisks indicate regions that survived Šidák's multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. W, X, The ratios of curves in U, V yield the synaptic E/I ratio during SWRs. Summary data in T represent these curves at the y-intercept. For all plots, individual data points represent a cell. Closed circles represent cells from males, open circles females. Box-whisker plots represent non-normal data with median and IQRs. Bar plots represent normal data with mean ± SEM; p values of pairwise comparisons indicated above brackets.

PV cells in CA1 delineate into basket (PVBCs), bistratified (PVBSCs), and axo-axonic (PVAACs) cells. A, Diagram of LFP + PV cell recording in three-month 5xFAD/+; PV^Cre/+;tdTom/+ and PV^Cre/+;tdTom/+ littermate controls. B, Diagram of PV cell subtypes and their axonal targets in CA1 str. pyr. Examples of (C1) PVBCs, (C2) PVBSCs, and (C3) PVAACs. Green fluorescence indicates avidin-fluorescein bound to the biocyin in the filled PV cell. Red fluorescence indicates PV^Cre-dTom expression. Dashed gray lines approximately distinguish layers of hippocampus, str. l.m. = stratum lacunosum-moleculare, str. rad. = stratum radiatum, str. pyr. = stratum pyramidale, str. or. = stratum oriens. Notice that str. pyr. is dimly red due to the expression of other non-filled PVBCs targeting perisomatic regions of PCs. Scale bars = 100 μm. Bottom traces, Examples of LFP and cell-attached recording of spikes, which was used to aid in distinguishing PVBCs and PVAACs. PVAACs are unique in that they reduce their firing during SWRs. C4, Ankyrin G immunostaining showing co-localization of synaptic boutons with AIS of PCs for identified PVAAC. Scale bar = 10 μm. D, Proportions of sorted cells in each genotype.

PVBCs have selectively reduced spiking. Spiking data for PVBCs (panel subheading 1), PVBSCs (panel subheading 2), and PVAACs (panel subheading 3). A, Example LFP and cell-attached traces for CT (left) and 5xFAD (right), for a PVBC (A1), PVBSC (A2), and PVAAC (A3). Spike and SWR events are color-coded as in Figure 4B. B, Summary spike rate data for PVBCs (n_PVBC = 13 CT, 16 5xFAD, B1), PVBSCs (n_PVBSC = 5 CT, 10 5xFAD, B2), and PVAACs (n_PVAAC = 6 CT, 6 5xFAD, B3) during spontaneous and SWR periods. C, Peri-SWR spike probability. Asterisks indicate regions surviving Šidák's multiple comparisons correction. D, Burst rate, defined as 3 or more spikes each within 40 ms. E, The fraction of SWRs with one or more spike. F, The fraction of SWRs with a burst. G-H, Polar phase plots of phase-locking of spikes to SWR-nested slow γ (G) and ripple (H). The angles of individual data points represent the average phase of all spikes for a cell. Length from origin (0–1) indicates the degree of phase-locking. Lines with arrowheads represent cell average, solid colored = CT, dashed gray = 5xFAD. Asterisks indicate result of Raleigh's test for non-uniformity, p values indicate results of Watson–Williams or circular mean test; *p < 0.05, **p < 0.01, ***p < 0.001. For all plots, individual data points represent a cell. Closed circles represent cells from males, open circles females. Box-whisker plots represent non-normal data with Median and IQRs; p values indicated above brackets.

PVBCs have selective decrease in excitatory synaptic input and decreased E/I ratio. Whole-cell data for PVBC EPSCs (panel subheading 1), PVBSC EPSCs (panel subheading 2), PVAAC EPSCs (panel subheading 3), and PVBC IPSCs (panel subheading 4). A, Example recordings of LFP + whole-cell PV cell recording in three-month 5xFAD/+; PV^Cre/+;tdTom/+ and PV^Cre/+;tdTom/+ littermate controls, recording synaptic input during spontaneous and SWR period. EPSCs were recorded at −70 mV in PVBCs (A1), PVBSCs (A2), and PVAACs (A3), and IPSCs recorded at 0 mV in a subset of PVBCs (A4). B, Summary data for spontaneous PSC frequency and amplitude for PVBC sEPSCs (n_PVBC = 12 CT, 16 5xFAD, B1), PVBSC sEPSCs (n_PVBSC = 4 CT, 6 5xFAD, B2), PVAAC sEPSCs (n_PVAAC = 5 CT, 6 5xFAD, B3), and PVBC sIPSCs (n_PVBC = 8 CT, 8 5xFAD, B4). C, Spontaneous PSC kinetics: rise and decay tau. D, Cumulative distribution function of PVBC decay tau. Dark lines represent cell average, shaded region represents SEM. E, swrEPSQ and swrIPSQ = total excitatory and inhibitory charge during SWRs, integrated over a 100-ms window centered around the SWR peak. F, left, PVBC spontaneous synaptic E/I ratio of normalized charge (sEPSQ/sIPSQ). F, right, PVBC synaptic E/I ratio for 100-ms window centered around SWR peak (swrEPSQ/swrIPSQ). G, The charge for each PVBC (EPSQ and IPSQ), calculated in 2-ms bins by integrating the current in a sliding 100-ms window centered around that bin. Summary data in E1, E4 thus represent these curves at the y-intercept. Asterisks indicate regions that survived Šidák's multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. H, The ratios of curves in G yield the synaptic E/I ratio during SWRs. Summary data in F represents these curves at the y-intercept. I, Linear regression of spike rate during SWRs versus the E/I ratio. For all plots, individual data points represent a cell. Closed circles represent cells from males, open circles females. Bar plots represent normal data with Mean ± SEM; p values indicated above brackets.