Compensatory enhancement of intrinsic spiking upon NKCC1 disruption in neonatal hippocampus

Abstract

Depolarizing and excitatory GABA actions are thought to be important in cortical development. We show here that GABA has no excitatory action on CA3 pyramidal neurons in hippocampal slices from neonatal NKCC1(-/-) mice that lack the Na-K-2Cl cotransporter isoform 1. Strikingly, NKCC1(-/-) slices generated endogenous network events similar to giant depolarizing potentials (GDPs), but, unlike in wild-type slices, the GDPs were not facilitated by the GABA(A) agonist isoguvacine or blocked by the NKCC1 inhibitor bumetanide. The developmental upregulation of the K-Cl cotransporter 2 (KCC2) was unperturbed, whereas the pharmacologically isolated glutamatergic network activity and the intrinsic excitability of CA3 pyramidal neurons were enhanced in the NKCC1(-/-) hippocampus. Hence, developmental expression of KCC2, unsilencing of AMPA-type synapses, and early network events can take place in the absence of excitatory GABAergic signaling in the neonatal hippocampus. Furthermore, we show that genetic as well as pharmacologically induced loss of NKCC1-dependent excitatory actions of GABA results in a dramatic compensatory increase in the intrinsic excitability of glutamatergic neurons, pointing to powerful homeostatic regulation of neuronal activity in the developing hippocampal circuitry.

Figure 1.

Developmental upregulation of KCC2 in the NKCC1^−/− mouse hippocampus takes place in the absence of depolarizing GABA action. A, Left, Gramicidin perforated-patch recordings showing currents evoked by somatic GABA uncaging (horizontal short bars) in WT and NKCC1^−/− neurons. Right, Current–voltage relationships obtained using the average current signal between the time points indicated by vertical lines on the left. B, Quantified and normalized KCC2 immunoreactivity in protein samples of hippocampi from WT and NKCC1^−/− mice (P3/P4: WT, n = 4; NKCC1^−/−, n = 6; P6/P7: WT, n = 4; NKCC1^−/−, n = 3; P20: WT, n = 6; NKCC1^−/−, n = 5).

Figure 2.

Network events similar to GDPs are generated by neonatal NKCC1^−/− hippocampal slices. A, Whole-cell voltage-clamp recordings with the low-chloride (4 mm) pipette filling solution at 0 mV showing spontaneous unitary postsynaptic currents and GDPs seen as bursts of GABAergic currents (insets) in WT (top trace and insets) and NKCC1^−/− (bottom trace and insets) hippocampal slices. B, fp recordings demonstrating that the variability of fGDP amplitude and duration is higher in NKCC1^−/− (bottom traces) than in WT (top traces) slices. Continuous fp recordings and sample fGDPs are shown on the left and right, respectively. C, Left, Simultaneous fp recording (top trace) and whole-cell recording with the high-chloride (49 mm) pipette filling solution at −60 mV (bottom trace) showing a GDP in an NKCC1^−/− slice. Right, Simultaneous fp (top trace) and cell-attached (bottom trace) recordings showing a GDP from an NKCC1^−/− slice.

Figure 3.

Effects of pharmacological manipulation of GABAergic transmission on fGDPs in WT and NKCC1^−/− slices. A, Isoguvacine (isog) at 5 μm induces a pronounced transient increase in fGDP frequency in P0–P1 WT but not in P0–P1 NKCC1^−/− slices. Mean ± SEM of fGDP frequency in seven WT and six NKCC1^−/− slices during isoguvacine application. B, Left, fGDPs are blocked by bumetanide (bume) in WT but not in NKCC1^−/− slices (bandpass, 0.3–20 Hz). Right, Mean ± SEM of normalized fGDP frequency obtained from six WT and six NKCC1^−/− slices and the effect of 10 μm bumetanide. C, fGDPs in WT slices reappear during a prolonged application of bumetanide.

Figure 4.

Enhanced AMPA receptor-mediated network events and intrinsic firing in neonatal NKCC1^−/− CA3 pyramidal neurons in the absence of GABA_A signaling. A, Top traces, The effect of the AMPA/kainate antagonist NBQX (50 μm) on fGDPs in a WT slice. Bottom traces, Simultaneous fp and voltage-clamp (V_h of −60 mV; [Cl⁻]_p = 49 mm) recordings from an NKCC1^−/− slice showing the effect of 50 μm NBQX. B, The effect of 100 μm picrotoxin on fGDPs in a WT and an NKCC1^−/− slice and a further addition of 20 μm GYKI53655. C, Left, mEPSCs recorded in WT and NKCC1^−/− CA3 pyramidal neurons. Right, Normalized cumulative peak mEPSC amplitude histograms obtained from 25 WT and 20 NKCC1^−/− CA3 pyramidal neurons. D, Cell-attached recordings from WT and NKCC1^−/− CA3 pyramidal neurons in the presence of NBQX, AP-5, and picrotoxin.