Distribution of resting [Cl⁻]_i and GABA action in the intact neonatal hippocampus. (a) Two-photon fluorescence confocal scanning images of the CA3 pyramidal cell layer in the intact hippocampus in vitro of the neonatal (P7) transgenic CLM-1 mouse expressing Clomeleon. Top panel: overlay of confocal multiple planes (150 samples; 2 μm step) of the Cl⁻ sensitive YFP signals of the CA3 neurons. Inset shows the photograph of the intact hippocampus. Bottom panel: pseudo-colored regions of interest (ROI) from neurons according to [Cl⁻]_i. (b) Distribution of resting [Cl⁻]_i (bin size 2.5 mM) from 81 individual neurons shown in (a). A Gaussian fit yielded a peak at 17.4 ± 0.6 mM and width of 14.6 ± 2.3 mM. Multi-peak fit yielded three peaks at 6.5 ± 0.6 mM, 15.9 ± 1 mM and 25.3 ± 2.1 mM. (c–e) Effect of isoguvacine (ISO) on multiple-unit activity (MUA) in the intact neonatal (P5–7) hippocampal preparations. (c) Extracellular recording of MUA in the CA3 pyramidal cell layer and corresponding frequency of MUA. Bath application of isoguvacine (10 μM) caused a transient decrease in the frequency of MUA; peak function fit is indicated by red line. (d–e) Individual hippocampus responses to isoguvacine and corresponding mean frequency of MUA (*p=0.00002, two sample t-Test). Decrease in MUA frequency indicates that the net effect of GABA_A-R activation was inhibitory.

Neonatal seizure-induced chloride accumulation and facilitation of recurrent seizures. (a) Extracellular field potential recordings in the CA3 pyramidal cell layer in the intact hippocampus in vitro of the neonatal (P5) CLM-1 mouse. Recurrent seizures were induced by low-Mg²⁺ ACSF and stopped by perfusion of control ACSF. (b) Effect of recurrent seizures on [Cl⁻]_i. Example of two-photon confocal imaging of YFP (left) of CA3 neurons yielded pseudo-color map of [Cl⁻]_i before seizures, after the first, forth and eighth recurrent seizures. (c) Distribution of [Cl⁻]_i (bin size 10 mM) in 36 identified neurons before seizures (dark blue), after one (green), four (orange) and eight (red) recurrent seizures. (d) Corresponding changes of mean [Cl⁻]_i in subpopulation of neurons with low (0–10 mM), medium (10–20 mM) and high (20–30 mM) range of control resting chloride. (e – g) Seizure induced facilitation of recurrent seizures. (e) Inter-seizure intervals decreased as a function of the number of recurrent seizures. (f) Power spectra of three consecutive seizures in frequency band from 1 to 1000 Hz. (g) Corresponding power of recurrent seizures as a function of the number of seizures.

Seizure-dependent changes in the effects of the GABA_A agonist isoguvacine on MUA and epileptiform discharges. (a–b) Extracellular field potential recordings in the CA3 pyramidal cell layer in the intact hippocampus in vitro of the neonatal (P5) CLM-1 mouse. Recurrent seizures were induced by low-Mg²⁺ ACSF. Application of isoguvacine (ISO; 10 μM for 1 min) after N=1 seizure decreased neuronal firing. Similar applications of isoguvacine after N=2 and N=3 seizures transiently increased neuronal firing rate in association with a transient increase of the frequency of interictal epileptiform discharges (IEDs). (c) Corresponding normalized frequency of MUA. Excitatory action of isoguvacine increased as a function of recurrent seizures. Red lines represent adjacent averaging of 20 data points. Vertical dashed lines on (b) and (c) panels delineate isoguvacine application.

Seizure-dependent changes in the effects of phenobarbital on neonatal seizures. (a) Low- Mg²⁺ ACSF – induced recurrent seizures in the intact hippocampus in vitro. (b) Application of phenobarbital (100 μM) in low-Mg²⁺ ACSF after one ictal-like event (N=1) abolished seizures in the intact neonatal hippocampus. (c) Application of phenobarbital (100 μM) after five ictal-like events (N=5) failed to abolish recurrent ictal-like activity in low-Mg²⁺ ACSF. (a–c) Extracellular field potential recordings were performed in the CA3 pyramidal cell layer in the intact hippocampus in vitro of neonatal (P5–6) rats. (d) Frequency of recurrent seizures as a function of preceding seizures (N) in control low-Mg²⁺ ACSF and after application of phenobarbital. Phenobarbital applied after N=1 and N=3 seizures significantly reduced the mean frequency of recurrent seizures (*p < 0.05). Phenobarbital applied after more than N=5 seizures failed to reduce the frequency of recurrent seizures. (e) Fraction of hippocampi in low-Mg²⁺ ACSF in which seizures were abolished by 100 uM phenobarbital, plotted as a function of the number of seizures prior to Phenobarbital application. Efficacy of phenobarbital in neonatal seizures decreased as a function of quantity of prior seizure activity (N).

The role of NKCC1 in seizure-induced chloride accumulation and facilitation of recurrent seizures. (a) Top: low-Mg²⁺ ACSF induced recurrent seizures in control and in the presence of NKCC1 blocker bumetanide (10 μM). Extracellular field potential recordings in the CA3 pyramidal cell layer in the intact hippocampus in vitro of neonatal (P5) CLM-1 mouse. Bottom: example of two-photon confocal imaging of YFP (left) of CA3 neurons yielded pseudo-color map of [Cl⁻]_i before seizures, after two seizures, and after nine seizures in presence of bumetanide. (b) Distribution of [Cl⁻]_i (bin size 10 mM) in 48 identified neurons before seizures (black), after two seizures in control (low Mg) solution (red), and after nine seizures in the presence of bumetanide (green). Gauss fits yielded mean ± width values of 22.1 ± 19 mM in control, 29.8 ± 18.4 mM after N=2 seizures, and 44.4 ± 24.1 mM after N=9 seizures. (c) Seizure-induced changes of mean [Cl⁻]_i in subpopulation of neurons with low (0–10 mM), medium (10–20 mM) and high (20–30 mM) range of control resting chloride. Bumetanide prevented seizure-induced neuronal chloride accumulation. (d) Effects of recurrent seizures on intracellular chloride accumulation in control low-Mg²⁺ ACSF and in the presence of bumetanide. Solid lines represent linear regression (Control, low-Mg²⁺ ACSF [black]: r = 0.97 ± 1.66; p = 0.03; Bumetanide [green]: r = − 0.43 ± 2.6; p = 0.57). In low-Mg²⁺ ACSF, [Cl⁻]_i progressively increases as a function of recurrent seizures. Bumetanide prevents progressive chloride accumulation induced by recurrent seizures. (e–f) Mean inter-seizure intervals (e) and seizure power (f) as a function of the number of recurrent seizures. (e) Solid lines represent linier regression fit of the mean inter-seizure intervals (ISIs) in control (black; r = − 0.82 ± 1.37; p = 0.02) and in the presence of bumetanide (green: r = −0.55 ± 0.7; p = 0.2). (f) Corresponding linear regression fit of normalized mean power in control (black: r = 0.95 ± 0.07; p = 0.0003; n = 8) and in the presence of bumetanide (green: r = 0.08 ± 0.08; p = 0.85; n = 8). Bumetanide prevents a progressive increase of seizure power and frequency.

The role of NKCC1 in seizure-induced changes in the action of GABA. (a–b) Extracellular field potential recordings in the CA3 pyramidal cell layer in the intact hippocampus in vitro of the neonatal (P5) mouse. Recurrent seizures were induced by low-Mg²⁺ ACSF in the presence of NKCC1 blocker bumetanide (10 μM). Applications of isoguvacine (ISO; 10 μM for 1 min) after seizure and recovery from post-ictal depression transiently decreased neuronal firing rate. (c) Corresponding frequency of MUA. Vertical dashed lines delineate isoguvacine application. Red lines represent adjacent averaging of 20 data points. Bumetanide prevented seizure-induced changes in the effects of the GABA_A-R agonist isoguvacine on MUA and epileptiform discharges (Fig. 3).

Concentration gradient of potassium regulates NKCC1 activity. (a–e) Changes in intracellular chloride distribution as a function of extracellular concentration of potassium ([K⁺]_o) in the presence of sodium channel blocker TTX (1 μM). (a, c) Examples of two-photon confocal imaging of YFP (left) of CA3 neurons yielded pseudo-color map of [Cl⁻]_i at different [K⁺]_o (3.5 mM, 8.5 mM and 13.5 mM) in control (a) and in presence of bumetanide (c). (b, d) Corresponding distributions of [Cl⁻]_i (bin size 5 mM) in control (b; n = 30 identified neurons) and in the presence of bumetanide (d; n = 40 identified neurons). Data were fitted with a Gaussian function. (e) Mean [Cl⁻]_i as a function of [K⁺]_o in control and in the presence of bumetanide.

Seizure-induced changes in the action of GABA are NMDA receptor independent. (a) Repetitive applications of kainic acid (KA; 1 uM for 5 min; opened bars) induced ictal-like epileptiform discharges (seizures) followed by spontaneous interictal epileptiform discharges (IEDs). Brief applications of isoguvacine (ISO; 10 uM for 1 min; solid bars) transiently increased the frequency of IEDs. (b) Perfusion of the non-competitive NMDA receptor antagonist MK801 (20 uM) depressed KA- induced seizures. Applications of isoguvacine increased the frequency of spontaneous IEDs. (c) Simultaneous perfusion of MK801 and bumetanide (10 uM) strongly depressed KA-induced seizures and prevented excitatory shift in the action of GABA. Applications of isoguvacine reduced the frequency of MUA and/or IEDs. (a–c) Extracellular field potential recordings in the CA3 pyramidal cell layer in the intact hippocampus of neonatal (P5–6) mice. (d) Normalized power of KA-induced seizures (mean±se) in control (squares), in the presence of MK801 (circles), and both MK801 and bumetanide (triangles). (e) Effects of isoguvacine on the frequency of spontaneous IEDs.

Recurrent seizures invert the effects of GABA_A receptors via NKCC1-dependent mechanism. (a) Spontaneous seizures (Status epilepticus) were induced by brief bath application of the GABA_A receptor antagonist SR95531 (10 μM). In the lower panel, spontaneous epileptiform discharges are shown on an expanded time scale. (b–c) Effects of the GABA_A-R agonist isoguvacine on MUA and spontaneous epileptiform activity. In control ACSF, application isoguvacine (10 μM) decreased the frequency of MUA. 30–40 min after onset of spontaneous seizures isoguvacine increased the frequency of MUA and interictal epileptiform discharges. Excitatory effects of isoguvacine on population activity persisted at least 1–2 hours after termination of seizures. (c) Bumetanide (10 μM) prevented the excitatory action of isoguvacine on the MUA and interictal epileptiform discharges. (a–c) Extracellular recordings of MUA and population activity were performed in the CA3 pyramidal cell layer of intact neonatal (P4–5) hippocampal preparations in vitro. (d, e) Mean frequency of MUA in control ACSF and in the presence of bumetanide (n=6) before, during and after spontaneous seizures. (d) Time course of isoguvacine action on MUA frequency. Bumetanide (10 μM) prevented the seizure-induced reversal of the effects of GABA_A-R activation.

Neonatal seizures alter the effect of synaptic activation of GABA_A receptors. (a–c) Effects on spontaneous neuronal firing rate of suppressing synaptic excitation and inhibition in control and during seizures. In control (a) Kynurenic acid (KYNA; 2 mM) co-applied with CGP55845 (1 μM) reduced the mean frequency of MUA in the CA3 pyramidal cell layer in the intact neonatal hippocampi at P4–5. Subsequent application of the GABA_A-R antagonist SR95531, in the presence of KYNA and CGP, increased MUA frequency, demonstrating the net inhibitory effect of GABA_A-R when activated by endogenously released GABA. (b) Spontaneous seizures were induced by brief (2–3 min) application of SR95531. After 30 min of spontaneous seizure activity following washout of SR95531, application of KYNA and CGP stopped recurrent seizures and reduced MUA frequency. Subsequent application of SR95531 did not change the mean frequency of MUA. Red plots represent the averaged mean frequency of MUA. (c) Mean ratio of MUA frequency (SR/(KYNA+CGP)) reversed as a function of the number of seizures. Solid line represents corresponding linear regression fit (r = −0.98; p = 0.02). (d, e) Synchronous extracellular field potentials and two-photon fluorescence [Ca²⁺]_i imaging in the CA3 pyramidal cell layer at P5 in the intact hippocampal preparation. Neurons were labeled by the calcium indicator dye Oregon Green BAPTA-1 AM (OGB). Field excitatory postsynaptic potentials and intracellular calcium signals were induced by electrical stimulation in the stratum radiatum proximal of the CA3 area. In control (d), bath application of KYNA (2 mM) and GGP (1 μM) depressed calcium signals (ΔF/F) in the majority of CA3 neurons. Subsequent application of SR 95531 (10 μM), in the presence of KYNA and CGP 55845, increased ΔF/F in the majority of the cells. The increase in Ca²⁺ in the presence of ionotropic glutamate and GABA receptor antagonists likely represents metabotropic receptor activation and direct electrical stimulation. (e) After spontaneous seizures, the effect of SR 95531 on ΔF/F was reversed in ~50% of neurons. (f, g) Mean population response (ΔF/F) and fraction of cells with a stimulus-induced increase in calcium responses during applications of drugs in control and after spontaneous seizures. (h) After recurrent seizure activity nearly half of neurons lose their inhibitory response to endogenous GABA or switch their inhibitory response from inhibition to excitation.