A limited role of NKCC1 in telencephalic glutamatergic neurons for developing hippocampal network dynamics and behavior

Abstract

NKCC1 is the primary transporter mediating chloride uptake in immature principal neurons, but its role in the development of in vivo network dynamics and cognitive abilities remains unknown. Here, we address the function of NKCC1 in developing mice using electrophysiological, optical, and behavioral approaches. We report that NKCC1 deletion from telencephalic glutamatergic neurons decreases in vitro excitatory actions of γ-aminobutyric acid (GABA) and impairs neuronal synchrony in neonatal hippocampal brain slices. In vivo, it has a minor impact on correlated spontaneous activity in the hippocampus and does not affect network activity in the intact visual cortex. Moreover, long-term effects of the developmental NKCC1 deletion on synaptic maturation, network dynamics, and behavioral performance are subtle. Our data reveal a neural network function of NKCC1 in hippocampal glutamatergic neurons in vivo, but challenge the hypothesis that NKCC1 is essential for major aspects of hippocampal development.

Keywords: GABA; NKCC1; development; hippocampus; in vivo.

Fig. 1.

Behavioral performance of NKCC1 KO^Emx1 mice. (A) In the targeted Slc12a2 locus, exons 8 to 10 are flanked by loxP sites. Cre-dependent recombination causes a frameshift (dotted) and introduces a stop codon in exon 12. (B) Confocal image demonstrating Cre-reporter expression (GCaMP3) in a horizontal brain slice (P4). (C) Hippocampal Slc12a2 mRNA levels compared with the geometric mean of WT, normalized to Gapdh and Hmbs. (D) Sample trajectories in the open field. (E) Total distance covered and relative time spent in the center. (F) Sample trajectories in the Y maze. (G) Spontaneous alternations (dotted, chance level). (H) Morris water maze. (I and J) Distance to platform for cued trial (visible platform) and acquisition (hidden platform). (K) Time in target quadrant during probe trial (no platform). (L–N) Freezing time during fear conditioning (L), reexposure to cue (M), and context (N). (C–K) Open circles represent single animals. (E–N) Mean ± SEM; CS, conditioned stimulus; US, unconditioned stimulus; ***P < 0.001. See SI Appendix, Fig. S1 and Table S1.

Fig. 2.

Loss of NKCC1 attenuates the depolarizing action of GABA. (A) Sample gramicidin perforated-patch current-clamp recordings in response to puff-applied isoguvacine (Iso). (B) Resting (V_rest) and peak isoguvacine-induced (V_peak) membrane potential. (C) Correlation of isoguvacine-induced membrane potential changes (ΔV_m) in two successive trials. ρ, Spearman’s rank correlation coefficient. (D) Sample cell-attached recordings in response to puff-applied isoguvacine. (E) Number of action currents (300-ms intervals) immediately before (base) and after (Iso) puff onset. (F) Reduced fraction of responsive cells (resp) in KO^Emx1 mice. Open circles represent single cells. Mean ± SEM; ns, not significant; *P < 0.05, ***P < 0.001. See SI Appendix, Fig. S2 and Table S2.

Fig. 3.

NKCC1^Emx1 deletion impairs spontaneous hippocampal activity in vitro. (A) Sample whole-cell recordings of sGPSCs isolated by reversal potential. (B) sGPSC frequency. (C) Burst index vs. sGPSC frequency. The dotted line represents a Poisson point process. (D) Burst indices. (E) GCaMP-based confocal imaging of spontaneous activity in corticohippocampal slices. Top: GCaMP3 fluorescence overlaid with ROIs for analysis (Left) and sample ΔF/F₀ images for time periods indicated below. (Scale bars, 500 µm.) Bottom: ΔF/F₀ sample traces. (F and G) Total active time (F) and mean area under the curve per event (AUC, G). Open circles represent single cells (B–D) or slices (F and G). Mean ± SEM; ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001. See SI Appendix, Figs. S3 and S4 and Table S3.

Fig. 4.

NKCC1 effects on hippocampal network dynamics are not accounted for by alterations in intrinsic excitability or basic synaptic properties. (A) Sample cell-attached recordings in the presence of ionotropic glutamate and GABA receptor antagonists. (B) Mean spontaneous AP frequency. (C) Sample current-clamp recordings in response to current injections. (D and E) Current–frequency relationship (D) and AP threshold (E) are unaltered. (F) Sample voltage-clamp measurements of mEPSCs. (G) mEPSC frequency, median mEPSC amplitude, and mean mEPSC decay-time constant. (H) Sample voltage-clamp measurements of mGPSCs. (I) mGPSC frequency, median mGPSC amplitude and mean mGPSC decay-time constant. Open circles represent single cells. Mean ± SEM; ns, not significant; ***P < 0.001. See SI Appendix, Table S4.

Fig. 5.

Hippocampal sharp waves in vivo at P4 persist in NKCC1 KO^Emx1 mice. (A) Example SPW (red arrow) and beta-frequency (∼10- to 30-Hz range) hippocampal network oscillation (HNO, blue line) recorded by linear silicon probes. LFP traces (black) are superimposed over the current source density (CSD). 0 µm represents the field reversal of s.p. (B) Example wavelet spectrograms of CSD in s.l.m. in median absolute deviation (MAD) units per frequency. Events marked as in A and B. Note that some HNOs identified in the LFP may be of too small amplitude to be clearly visible in the wavelet of the CSD. (C) Median LFP and CSD depicted from SPWs with sinks either below (“rad/lm,” Left) or above (“oriens,” Right) s.p. Left two panels are from a WT, and Right two are from a KO^Emx1 mouse. Lower panels display the SPW trough-aligned average MUA for all sets with five or more events. (D) Frequency, LFP amplitude, and MUA spike count of rad/lm and oriens SPWs. Displayed MUA counts are from 50-ms bins centered around the peak at 0 [−25, 25] ms for oriens SPWs, and after the peak from 0 [0, 50] ms for rad/lm SPWs. (E, Left) HNO start-aligned average MUA. Note the −200-ms peak in MUA due to (the ∼30 to 60% of) HNO events having a preceding SPW (c.f. A and B). Note also that the MUA decay results from averaging HNOs of varying length. Right three panels, HNO occurrence rate, duration and MUA count in the [0, 500]-ms bin. Open circles represent single animals. Mean ± SEM. See SI Appendix, Fig. S5 and Table S5.

Fig. 6.

NKCC1 promotes correlated network activity in an event type- and region-specific manner in vivo. (A) Experimental design. EC, extracellular electrode. (B) GCaMP fluorescence and ROIs for analysis. (C) Time-aligned GCaMP fluorescence (ROIs indicated in B), LFP (0.5 to 100 Hz) and respiration/movement (resp) signal. Ca²⁺ clusters are indicated by gray, SPWs by red arrows. Note CaTs with or without a concurrent SPW. (D) Boxed regions in C at higher magnification. +SPWs CaTs display faster rise kinetics and higher amplitudes. (E) Raster plots indicating CaT times per ROI. Insets: GCaMP fluorescence (F₀) and binary area plots of Ca²⁺ clusters indicated on the Left. (F) Reduced CaT frequencies per ROI. (G) NKCC1^Emx1 deletion reduced the frequency of –SPW Ca²⁺ clusters. (H–J) Rise kinetics (H), cluster area (I), and amplitude (J). SI, sampling interval. (K) Sample ΔF/F₀ traces (mean of all analyzed ROIs), LFP (0.5 to 100 Hz), and raster plots before/after superfusion of gabazine (40 µM). Red arrows indicate detected SPWs (control) and SPW-like events (gabazine). (L) +SPW and –SPW Ca²⁺ cluster frequencies. (M) Sample raster plots demonstrating cluster activity in the visual neocortex. Insets: Transcranial GCaMP fluorescence overlaid with binary area plots of three events indicated on the Left. (Scale bars, 200 µm.) (N–P) Average CaT frequency per ROI (N), mean cluster area (O), and cluster frequency (P). Open circles represent single animals. Mean ± SEM; ns, not significant; *P < 0.05, ***P < 0.001. See SI Appendix, Figs. S6–S8 and Table S6.

Fig. 7.

Long-term alterations of in vivo hippocampal network dynamics upon NKCC1^Emx1 deletion. (A) Top: Representative example of REM- and SWS-like activity classification of the hippocampal LFP in adult urethane-anesthetized mice. SWS- and REM-like activity in s.l.m. showed characteristic inverted peaks in the delta band (blue) and theta/delta ratio (red), respectively. Bottom: Z-score normalized wavelet spectrogram showing differences in theta and delta power between REM- and SWS-like oscillations. (B) Band-limited power analysis during REM-like activity. Theta amplitude (Left) in s.l.m. and s.m. was higher in KO^Emx1 compared with WT mice. Low-gamma (Middle) and MUA (0.7 to 3 kHz, Right) frequency bands were unaffected, as were the remaining frequency bands analyzed (see SI Appendix). Shaded areas represent ± SEM across animals. (C) Power spectra from s.p. and s.l.m. (D) SPW-R events occurring during SWS-like activity showed lower spectral frequency (Left) and incidence (Right) in KO^Emx1 mice. Ripple-associated SPWs showed no significant amplitude difference between the genotypes (Middle). (E) Experimental arrangement and sample OGB1 fluorescence images. (F, Left) Two-photon fluorescence images; (F, Middle) sample ΔF/F₀ traces; (F, Right) Pearson correlation matrices for the same FOVs. (G) Single-cell CaT frequencies (averaged across animals). (H) Mean CaT frequencies. (I) Mean pairwise Pearson correlation coefficients for measured and randomly shuffled CaT trains. (J) Distributions of Pearson correlation coefficients. (K) Mean spike-time tiling coefficients (STTC) for measured data and randomly shuffled CaT trains. Open circles represent single animals. Mean ± SEM; ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001. See SI Appendix, Fig. S9 and Table S7.