Aa, configuration of dual recording electrodes (whole-cell (WC), normal chloride, field (extracellular)) placed at the CA1 region in the middle septotemporal level. Infra-red image of the CA1 pyramidal cell layer as observed from a top view of the hippocampus (bottom inset). Ab, field recording of recurrent seizure-like events with three states: interictal, preictal and ictal, with a distinction between the preictal and ictal states by a clearly denoted 20 Hz oscillation at ictal onset (♦, bottom inset). Ba, whole-cell recording from pyramidal cell (PC) with field activity during the preictal state exhibits hyperpolarizing (preictal_h), mixed hyper-/depolarizing (preictal_m), and depolarizing (preictal_d) activity prior to ictal onset. Bb, representative intracellular activity during the three preictal substates. The preictal_m state is composed of hyperpolarizing (h arrow) and depolarizing (d arrow) activity. C, prior to ictal onset (t = 0 s), hyperpolarizing activity (t =−20 s to −10 s) decreases in amplitude towards a reversal in polarity with a gradually increase in the depolarizing peak amplitudes (measured by the arrows), with an average rate of approximately 2.0 mV s⁻¹. The initial resting membrane potential was −40 mV. Coupled with the phasic changes during this state, there is also a slow depolarization in the membrane potential prior to ictal onset.

Aa, intracellular recordings of the preictal_h state in a pyramidal cell using whole-cell (WC), perforated patch, and whole-cell technique with high internal chloride (whole-cell high Cl_i) to measure the reversal potential of the spontaneous preictal_h activity by manually injecting current to slowly depolarize and hyperpolarize the cell. In a sequence of intracellular events, the region of reversal (ROR) was defined as the region between the last positivity (+) and the first negativity (−) when depolarizing the cell, or the region between the last negativity and first positivity when hyperpolarizing the cell. The peak amplitude (arrows) was measured at the respective membrane potential (V_m). Ab, the reversal potential is sensitive to the intracellular chloride concentration, as whole-cell high Cl_i had a more depolarized reversal potential than standard WC and perforated patch. B, average reversal potentials (E_rev) of the preictal_h state in pyramidal cells, FS and non-FS interneurons with the three intracellular recording methodologies. Perforated patch reveals the most hyperpolarized E_rev, while a more depolarized E_rev is observed with whole-cell high internal chloride recordings, as suggested by the Nernst E_Cl. C, sample intracellular responses to electrical stimulation at varying membrane potentials in the presence of glutamatergic blockers (60 μm APV, 10 μm CNQX) and somatic pressure-injection of muscimol in two interneurons (perforated patch recording). The ROR for the evoked responses is between −70 and −55 mV. Da, dual whole-cell (normal chloride) and field recordings from a non-fast-spiking interneuron during BMI (10 μm) application created more recurrent and fragmented ictal episodes. Db, interneuronal preictal activity in low-Mg²⁺ (•, expanded from Da) is completely abolished with the application of BMI (♦, expanded from Da).

Aa, IR image from the CA1 stratum pyramidale (SP) cell layer in a hippocampal slice (400 μm), with a patched CA1 pyramidal cell (PC). Membrane voltage responses of the pyramidal cell to positive and negative current injections prior to seizure induction showing spike frequency adaptation that is common to these cell types (bottom inset). Ab, whole-cell recording (normal chloride) with field activity during the preictal state exhibits the three preictal substates observed in the intact hippocampus prior to ictal onset (♦, bottom inset) with the representative intracellular activity as observed during the three substates. Ba, IR image of the stratum pyramidale (SP) and stratum radiatum (SR) of the hippocampal slice with an intracellular recording electrode and the pressure-injector (PI) positioned in the dendritic region. Bb, dendritic pressure-injection of the GABA_A agonist muscimol (•, 500 μm, 10 ms, 10 p.s.i.) onto the pyramidal cell evokes hyperpolarizing GABAergic responses during the preictal_m and preictal_d substates, as shown by two recordings. C, sample of intracellular responses during the interictal state, at varying membrane potentials, to dendritic muscimol pressure-injection with a reversal potential of −55.0 mV.

A, configuration of dual recording electrodes (PC, whole-cell, field (extracellular)) placed at the CA3 and CA1 region in the middle septotemporal level. B, whole-cell pyramidal cell recording with field activity during the preictal and ictal states. C, the preictal state (♦, expanded from B) exhibits a mixture of synaptic activity and action potentials, which later develop into EPSPs prior to ictal onset, similar to the preictal_d state observed in CA1 neurons. D, intracellular CA3 activity leads the CA1 field (•, expanded from C), while IPSPs follow field activity. Vertical lines indicate onset of intracellular CA3 activity.

Aa, morphological reconstruction of a FS interneuron (basket cell). The basket cell interneuron has an extensive axonal arbor in the CA1 pyramidal layer and dendrites extending in the stratum oriens and radiatum, with an average peak frequency of 67.0 ± 5.0 Hz. o, stratum oriens; p, stratum pyramidale; r, stratum radiatum; l, stratum lacunosum-moleculare. Ab, whole-cell recording from a FS interneuron demonstrates similar intracellular dynamics during the preictal state recordings to those observed in pyramidal cells, with all preictal h, m and d states preserved (Ac) prior to ictal onset. Ba, morphological reconstruction of a non-FS interneuron (O-LM). The O-LM interneuron (dendrites, black; axons, grey) has a characteristic axonal arbor in the stratum lacunosum-moleculare and a dendritic tree in the stratum oriens, with an average peak frequency of 35.0 ± 4.0 Hz. Bb and c, similar to pyramidal cells and FS interneurons, whole-cell recording from a non-FS interneuron in the CA1 stratum oriens region demonstrates three distinct preictal substates prior to ictal onset.

Aa, whole-cell recording from a pyramidal cell reveals that application of the carbonic anhydrase inhibitor ETZ (100 μm) does not attenuate spontaneous activity and maintains the interictal, preictal and ictal states. Ab, the three preictal substates are maintained, and the cell reveals an ROR similar to that of a pyramidal cell in low-Mg²⁺ ACSF. Dashed line indicates intracellular activity at the same membrane potential, with reversed polarities, just before and after the administration of the slow hyperpolarizing and depolarizing current steps. B, somatic pressure-injection of the GABA_A agonist muscimol (•, 500 μm, 10 ms, 10 p.s.i.) to pyramidal cells. Hyperpolarizing GABAergic responses are maintained throughout the preictal state as pharmacologically evoked by somatic activation of the GABA_A receptors. These responses are also evoked in FS interneurons (C) and non-FS interneurons (D) by somatic application of muscimol (•) throughout the preictal substates. All whole-cell recordings were performed using normal chloride intracellular solution.

A, independent whole-cell (normal chloride) recordings from two interneurons in different hippocampi. The normal action potentials (upper trace) are attenuated with QX222 (middle trace). Electrically evoked responses were recorded at −50, −60 and −70 mV and demonstrate the attenuation of EPSPs at these resting membrane potentials (boxed region, stimulus artifact omitted for clarity, represented by •). B, IPSPs are unmasked with intracellular application of QX222, which attenuates EPSPs during the preictal and ictal states, recorded in an interneuron (IN) with correlated field activity. C, IPSPs are maintained throughout the entire preictal state (♦, expanded from B). D, intracellular activity from the early preictal (•) and late preictal (^) reveals that application of QX222 does not completely attenuate all excitatory depolarization (d arrow), but weakens the effect of compound EPSPs that mask the underlying IPSPs.