KCC2-Mediated Cl- Extrusion Modulates Spontaneous Hippocampal Network Events in Perinatal Rats and Mice

Abstract

It is generally thought that hippocampal neurons of perinatal rats and mice lack transport-functional K-Cl cotransporter KCC2, and that Cl^- regulation is dominated by Cl^- uptake via the Na-K-2Cl cotransporter NKCC1. Here, we demonstrate a robust enhancement of spontaneous hippocampal network events (giant depolarizing potentials [GDPs]) by the KCC2 inhibitor VU0463271 in neonatal rats and late-gestation, wild-type mouse embryos, but not in their KCC2-null littermates. VU0463271 increased the depolarizing GABAergic synaptic drive onto neonatal CA3 pyramidal neurons, increasing their spiking probability and synchrony during the rising phase of a GDP. Our data indicate that Cl^- extrusion by KCC2 is involved in modulation of GDPs already at their developmental onset during the perinatal period in mice and rats.

Keywords: KCC2 knockout; bumetanide; cation-chloride cotransporter; chloride; correlated activity; critical window; pacemaker.

Graphical abstract

Figure 1

KCC2 Modulates GDPs in the Perinatal Rodent Hippocampus (A) Effect of the specific KCC2 inhibitor VU0463271 (10 μM) on GDPs in the perinatal rat (P0–P2) and mouse (E17.5–E18.5) hippocampal CA3 area. From left to right: magnified example events from rat, KCC2^+/+, and KCC2^−/− mouse local field potential (LFP) recordings (filtered 1–10 Hz); sample LFP traces (filtered 1–10 Hz); and quantification of normalized network activity after application of VU0463271 are shown. Data are presented as median and IQR; Wilcoxon signed-rank test was used for statistical analysis; ^∗∗p < 0.01; N.S., non-significant; n values are indicated in the bar diagram. (B) KCC2 expression in E18.5 mouse hippocampal CA3 area of KCC2^+/+ and KCC2^−/− mice. In both panels, the areas indicated with rectangles (left) are shown as magnified insets (right). Examples of KCC2-expressing cells are indicated with stars. See also Figures S1, S2, and S3.

Figure 2

Inhibition of KCC2 Increases GABA-Driven Spontaneous Spiking in CA3 Pyramidal Neurons, but Not in Interneurons (A) KCC2 expression in E18.5 GAD67-GFP mouse hippocampal CA3 area. The areas indicated with rectangles (left) are shown in more detail on the right. Examples of KCC2-expressing interneurons and pyramidal neurons are indicated with arrowheads and stars, respectively. (B) Scheme of experimental paradigm to record interneuronal activity from a pyramidal neuron (left). Sample traces of a whole-cell voltage-clamp recording (middle) and median of the normalized frequency values (right) of sIPSCs recorded from pyramidal neurons before and after the application of VU0463271 (10 μM) are shown. (C) Scheme of experimental paradigm to record pyramidal neuron activity in loose cell-attached configuration (left). Sample traces of a loose cell-attached recording of CA3 pyramidal neuron spiking (middle) and median of the normalized spike frequency (right) in the presence and absence of VU0463271 and picrotoxin (100 μM) are shown. Recordings in (B) and (C) were done in the presence of CNQX (10 μM) and d-AP5 (20 μM), indicated in the figure as iGluR block. Data are presented as median and IQR; Wilcoxon signed-rank test was used for statistical analysis; ^∗p < 0.05; ^∗∗p < 0.01; n values are indicated in the bar diagrams.

Figure 3

VU0463271-Sensitive Chloride Extrusion in Neonatal CA3 Pyramidal Neurons (A) An Alexa Fluor 488-filled CA3 pyramidal neuron from a P1 rat representing the position of the whole-cell patch pipette imposing a 19 mM Cl⁻ load onto the neuron and the sites of UV photolysis of caged GABA. (B) Chloride extrusion efficacy in P1 rat CA3 pyramidal neurons determined using local UV photolysis of caged GABA at the soma (n = 4) and apical dendrite at 50 μm (n = 4) and 200 μm (n = 5) distance from the somatic Cl⁻ load, under control conditions (black) and in the presence of VU0463271 (10 μM, gray). Stippled line delimits the approximate residual negative deflection in E_GABA from E_GABA-GHK at 50 μm that is insensitive to VU0463271 (see Spoljaric et al., 2017) and present in KCC2^−/− neurons (see Li et al., 2007). (C) Sample E_GABA recordings and their corresponding I-V plots in the presence of iGluR block and bumetanide (2.5 μM). (D) Sample traces of gramicidin-perforated patch recordings (left) and median of the normalized amplitude of miniature inhibitory postsynaptic currents (mIPSCs; right) recorded from pyramidal neurons before and after the application of VU0463271 or DMSO. Recordings were done in the presence of iGluR block and TTX (0.5 μM). Data in (B) and (D) are presented as median and IQR; Wilcoxon signed-rank test were used for statistical analysis; ^∗p < 0.05; n values are indicated in the bar diagram.

Figure 4

Inhibition of KCC2 Shifts the Spike Distribution of CA3 Pyramidal Neurons toward the Rising Phase of GDPs (A) Sample traces from simultaneous loose cell-attached (top) and local field potential (LFP) recordings (middle: multi-unit activity [MUA], bandpass 400-2000 Hz; bottom: GDP, bandpass 1-10 Hz) of CA3 pyramidal neuron spiking in the absence and presence of VU0463271 (10 μM). The colored boxes over the sample traces highlight the rising phase (green) and falling phase (red) of the GDP. (B and C) Example raster plots of CA3 pyramidal neuron spiking during 10 control GDPs and 10 GDPs during VU0463271 application from a simultaneous loose cell-attached recording of spiking (B) and LFP recording of MUA (C). The peak of the GDPs is set to time point 0 s. The mock GDP shape in the background of the raster plots portrays the rising and falling phases of the GDPs. (D) Median values of normalized GDP-nested spikes in both loose cell-attached and MUA recordings during VU0463271 (left). Number of spikes observed in loose cell-attached and MUA recordings during the rising phase of GDPs, in relation to the total number of GDP-nested spikes, in the absence and presence of VU0463171 (right) is shown. Data are presented as median and IQR; Wilcoxon signed-rank test was used for statistical analysis; ^∗p < 0.05; n values are indicated in the bar diagrams.