The cation-chloride cotransporter NKCC1 promotes sharp waves in the neonatal rat hippocampus

Abstract

Earlier studies indicate a crucial role for the interconnected network of intrinsically bursting CA3 pyramidal neurons in the generation of in vivo hippocampal sharp waves (SPWs) and their proposed neonatal in vitro counterparts, the giant depolarizing potentials (GDPs). While mechanisms involving ligand- and voltage-gated channels have received lots of attention in the generation of CA3 network events in the immature hippocampus, the contribution of ion-transport mechanisms has not been extensively studied. Here, we show that bumetanide, a selective inhibitor of neuronal Cl- uptake mediated by the Na+-K+-2Cl- cotransporter isoform 1 (NKCC1), completely and reversibly blocks SPWs in the neonate (postnatal days 7-9) rat hippocampus in vivo, an action also seen on GDPs in slices (postnatal days 1-8). These findings strengthen the view that GDPs and early SPWs are homologous events. Gramicidin-perforated patch recordings indicated that NKCC1 accounts for a large ( approximately 10 mV) depolarizing driving force for the GABAA current in the immature CA3 pyramids. Consistent with a reduction in the depolarization mediated by endogenous GABAA-receptor activation, bumetanide inhibited the spontaneous bursts of individual neonatal CA3 pyramids, but it slightly increased the interneuronal activity as seen in the frequency of spontaneous GABAergic currents. An inhibitory effect of bumetanide was seen on the in vitro population events in the absence of synaptic GABAA receptor-mediated transmission, provided that a tonic GABAA receptor-mediated current was present. Our work indicates that NKCC1 expressed in CA3 pyramidal neurons promotes network activity in the developing hippocampus.

Figure 1. Inhibition of NKCC1 blocks hippocampal SPWs in neonatal rats

A, a direct current (DC) recording (left) of a typical SPW in a P8 rat pup hippocampus in vivo and the same event after high-pass filtering at 0.5 Hz (right). B, intraperitoneal application of 5 μmol kg⁻¹ bumetanide (bume) blocks early SPWs in a reversible manner (high-pass 0.5 Hz).

Figure 2. NKCC1 is required for the depolarizing action of GABA in immature CA3 pyramidal neurons

A, currents evoked by somatic GABA uncaging (indicated by the dots above the traces) at various membrane voltages (from −68 mV, 10 mV steps) in control and in bumetanide. B, peak GABA_A receptor-mediated current versus holding potential from the recordings in A. C, resting membrane potential (RMP) and reversal potential of GABA_A-mediated currents (E_GABA) from individual recordings in control (n = 10) and in bumetanide (n = 4). D, driving force of GABA_A-mediated currents (E_GABA− RMP) from individual recordings (small circles) and the mean ± s.d. (large circles with error bars). Filled and open circles indicate data in control and in bumetanide, respectively, in B–D.

Figure 3. NKCC1 facilitates spontaneous activity of individual neonatal CA3 pyramidal cells

A, field potential recordings showing unit activity of CA3 pyramidal neurons and the effect of bumetanide (upper traces). Lower traces show unit activity in the presence of picrotoxin (PiTX) in 9 mm extracellular K⁺ and the effect of bumetanide. B, time course of the effect of bumetanide on the mean frequency (+ s.e.m.) of unit activity from 4 and 7 experiments in the absence and presence of PiTX, respectively (bin 5 min). In all recordings, GDPs were blocked by NBQX and AP-5.

Figure 4. Bumetanide slightly increases the frequency of spontaneous postsynaptic GABA_A currents

A, voltage-clamp recording at 0 mV with a low-chloride pipette filling solution shows a minor increase in spontaneous GABA-PSC frequency but no effect on the baseline holding current. B, a bar graph showing mean (+ s.e.m.) spontaneous GABA-PSC frequency relative to control (n = 5 cells). Data obtained within 15–20 min after the beginning of bumetanide application were used for calculation of spontaneous GABA-PSC frequency. C, cumulative probability histogram of spontaneous GABA-PSC amplitudes from the recording in A (black line – control, grey line – bumetanide). D, averaged spontaneous GABA-PSCs from 40 single events in control and in bumetanide from the recording in A. In all recordings, GDPs were blocked by NBQX and AP-5.

Figure 5. Bumetanide blocks spontaneous network events driven by CA3 pyramidal neurons in vitro in the presence and absence of interneuronal input

Aa, a field potential (FP) recording showing a block of field giant depolarizing potentials (fGDPs) by 10 μm bumetanide (band-pass 0.2–5 Hz; inset shows a DC recording of a single fGDP). b, voltage-clamp recording showing GDPs as bursts of postsynaptic GABA_A receptor-mediated currents that are blocked by bumetanide. B, in the presence of the competitive GABA_A receptor antagonist SR 95531, application of 32 μm isoguvacine (isog) increases fGDP frequency. Further addition of bumetanide blocks fGDPs (band-pass 0.2–5 Hz; inset shows a DC recording of a single fGDP). Note that SR 95531 completely blocks synaptic GABA_A responses (interneuronal input) and a subsequent application of isoguvacine facilitates the tonic mode of GABA_A activation (see Results).