Tau reduction affects excitatory and inhibitory neurons differently, reduces excitation/inhibition ratios, and counteracts network hypersynchrony

Abstract

The protein tau has been implicated in many brain disorders. In animal models, tau reduction suppresses epileptogenesis of diverse causes and ameliorates synaptic and behavioral abnormalities in various conditions associated with excessive excitation-inhibition (E/I) ratios. However, the underlying mechanisms are unknown. Global genetic ablation of tau in mice reduces the action potential (AP) firing and E/I ratio of pyramidal cells in acute cortical slices without affecting the excitability of these cells. Tau ablation reduces the excitatory inputs to inhibitory neurons, increases the excitability of these cells, and structurally alters their axon initial segments (AISs). In primary neuronal cultures subjected to prolonged overstimulation, tau ablation diminishes the homeostatic response of AISs in inhibitory neurons, promotes inhibition, and suppresses hypersynchrony. Together, these differential alterations in excitatory and inhibitory neurons help explain how tau reduction prevents network hypersynchrony and counteracts brain disorders causing abnormally increased E/I ratios.

Keywords: Alzheimer’s disease; axon initial segment; epilepsy; excitation-inhibition balance; hypersynchrony; interneurons; intrinsic excitability; neuronal plasticity; pyramidal cells; tau.

Figure 1.. Tau ablation reduces sAP firing and E/I ratio of PCs but does not affect their excitability

sAP firing, AP thresholds, input-output responses, and spontaneous (s) and miniature (m) EPSCs and IPSCs were recorded in PCs in acute slices of somatosensory cortex from 24- to 28-day-old WT and Mapt^−/− mice. See Table S1 for additional measures. (A) Representative traces of sAPs recorded by cell-attached patch-clamp. (B) Quantitation of sAP frequencies. The graph on the right shows cumulative probability curves of sAP frequencies binned at 2 Hz. (C) Representative APs induced by minimal current injection. (D and E) Quantitations of AP rheobase currents (D) and thresholds (E). (F) Representative AP firing patterns induced by a 200-pA current injection. (G) AP frequency in response to increasing current injections (F-I curves). (H) Representative PSC traces recorded from WT (top) and Mapt^−/− (bottom) cells. (I–L) Quantitations of sIPSC (I), mIPSC (J), sEPSC (K), and mEPSC (L) frequencies. (M) Ratios of (sEPSC frequency × amplitude) over (sIPSC frequency × amplitude). n = 43–48 cells (from 4 mice) per genotype for sAPs; 2–4 cells per slice and 3–4 slices per mouse (B) were analyzed. n = 16–17 cells (from 3 mice) per genotype for APs; 1 cell per slice and 5–6 slices per mouse (D, E, and G) were analyzed. n = 14–17 cells (from 3 mice) per genotype for PSCs; 1 cell per slice and 4–6 slices per mouse (I–M) were analyzed. *p < 0.05, **p < 0.01 by unpaired two-tailed Student’s t test (I) or Mann-Whitney test (B and M). In all figures, differences without asterisk(s) were not statistically significant. See Table S4 for all other p values and STAR methods for an explanation of box-and-whisker plots (B, D, E, and I–M). Values in (G) are means ± SEM.

Figure 2.. Tau ablation increases the excitability of interneurons but decreases the excitatory inputs they receive

AP thresholds, input-output responses, and EPSCswere recorded from fast-spiking (FS) neurons in acute slices of somatosensory cortex from 24- to 28-day-old WT and Mapt^−/− mice. See Table S2 for additional measures. (A) Representative individual APs induced by minimal current injection. (B and C) Quantitations of AP rheobase currents (B) and thresholds (C). (D) Representative AP firing patterns induced by a 200-pA current injection. (E) AP frequency in response to increasing current injections (F-I curves). (F) Representative traces of sEPSCs and mEPSCs in FS cells. (G and H) Quantitations of sEPSC (G) and mEPSC (H) frequencies. n = 25–28 cells (from 6–7 mice) per genotype for APs; 1 cell per slice and 4–6 slices per mouse (B, C, and E) were analyzed. n = 20–21 cells (from 5–6 mice) per genotype for EPSCs; 1 cell per slice and 3–5 slices per mouse (G and H) were analyzed. ^#p = 0.09, *p < 0.05, ***p < 0.001 by unpaired, two-tailed t test (B, C, and G), unpaired, two-tailed t test for areas under the curves (E), or Mann-Whitney test (H). Values in (E) are means ± SEM.

Figure 3.. Tau ablation preferentially modulates AISs of inhibitory neurons

In (A)–(E), coronal sections of somatosensory cortex from 23- to 25-day-old WT and Mapt^−/− mice were co-immunostained for the AIS marker AnkG and for PV and analyzed by confocal microscopy. (A) Representative images depicting AISs (green) of PV cells (red) and PCs (not labeled). In the inset, dashed lines indicate the soma boundary, and arrowheads indicate the start and end of AISs. Scale bars: 20 μm, 10 μm (inset). (B–E) AIS length (B and D) and AIS-soma distance (C and E) in PV cells (B and C) and PCs (D and E). Cumulative probability curves binned at 1 μm for AIS length and at 0.5 μm for AIS-soma distance are shown to the right of box-and-whisker plots. (F–H) At DIV13–14, primary hippocampal neuronal cultures from P0–1 WT and Mapt^−/− mice were or were not exposed to 15 mM KCl (10 mM added plus 5 mM in medium) for 4 or 16h; fixed; immunostained for markers of the AIS (AnkG, white), excitatory cells (CaMKII, cytoplasmic, green), including granule cells (PROX1, nuclear, white), and interneurons (GAD67, cytoplasmic, red); and analyzed by confocal microscopy. (F) Representative images depicting AISs of GABAergic interneurons. Arrowheads mark the start and end of AISs and dashed lines the soma boundary. Scale bar: 20 μm. (G and H) AIS-soma distance (G) and AIS length (H) in GABAergic cells. Cumulative probability curves binned at 2 μm are shown on the right. For AIS measurements in granule cells, see Figures S2A-S2C. n = 83–92 AISs (from 4 mice) per genotype; 8–12 AISs per section and 2 sections per mouse (B and C) were analyzed. n = 68–84 AISs for AIS length and 55–56 AISs for AIS-soma distance (from 4 mice) per genotype; 7–14 AISs per section and 2 sections per mouse (D and E) were analyzed. n = 24–42 AISs (from 3 mice) per genotype and treatment; 7–14 AISs per well and 1 well per mouse (G and H) were analyzed. ^#p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired, two-tailed t test (B–D) or two-way ANOVA and Holm-Sidak test (G and H).

Figure 4.. Tau ablation supports inhibition and prevents network hypersynchrony after stimulation of neuronal cultures

In (A) and (B), primary hippocampal neuronal cultures from P0–1 WT and Mapt^−/− mice were transduced on DIV4 to express iGABASnFR. At DIV13–14, cultures were or were not exposed to 15 mM KCl (10 mM added plus 5 mM in medium) for 4 or 16 h, followed by incubation in conditioned medium (without KCl addition) for 1 h. Fluorescence changes indicating GABA release were then triggered by electrical field stimulation (1 ms, 90 mA, 100 Hz, and 5 pulses) and monitored by fluorescence microscopy. (A) Representative traces of fluorescence changes in neuronal somas triggered by electrical field stimulation. (B) GABA release (areas under signal traces). See Figure S3 for input-output curves. In (C)–(F), spontaneous activity in neuronal cultures recorded by MEA after KCl treatment and incubation in conditioned medium as in (A) and (B) is shown. (C) Representative traces from single-channel MEA recordings showing spontaneous spiking in neuronal cultures at baseline (0 h KCl). (D) Spike frequencies per channel. Individual values were calculated by averaging spike frequencies recorded from all 16 channels per MEA well. (E) Raster plots from cultures before and after 4 h KCl treatment. (F) Global synchrony index obtained from the phase synchronization matrix of all pairs of electrodes (see STAR Methods). See Table S3 for additional measures. n = 64–84 cells (from 5 mice) per genotype and treatment; 7–29 cells per coverslip and 1–2 coverslips per mouse (B) were analyzed. n = 16–18 wells (from 8–9 mice) per genotype and treatment; 2 wells were analyzed per mouse (D and F). *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA and Holm-Sidaktest (D and F) or permutation test with Holm-Sidak correction (B). Table S4 provides p values for all comparisons.