Persistently modified h-channels after complex febrile seizures convert the seizure-induced enhancement of inhibition to hyperexcitability

Abstract

Febrile seizures are the most common type of developmental seizures, affecting up to 5% of children. Experimental complex febrile seizures involving the immature rat hippocampus led to a persistent lowering of seizure threshold despite an upregulation of inhibition. Here we provide a mechanistic resolution to this paradox by showing that, in the hippocampus of rats that had febrile seizures, the long-lasting enhancement of the widely expressed intrinsic membrane conductance Ih converts the potentiated synaptic inhibition to hyperexcitability in a frequency-dependent manner. The altered gain of this molecular inhibition-excitation converter reveals a new mechanism for controlling the balance of excitation-inhibition in the limbic system. In addition, here we show for the first time that h-channels are modified in a human neurological disease paradigm.

Fig 1

Long-lasting shift in h-current activation following hyperthermia-induced seizures. a, I_h activation in CA1 cells from animals one week after seizures (●) and sham-treated littermate controls (○). The fitted curve is the Boltzmann function. The data points are normalized per cell and averaged. R_S: control, 8.5 ± 1.1 MΩ; HT, 10.1 ± 1.7 MΩ; no difference. The maximal tail current did not increase; at −120 mV: control, 127.6 ± 8.6 pA; HT, 119.1 ± 8.7 pA. The tail current tended to saturate at negative voltages; currents at −120 mV and −110 mV were not statistically different in either group. Right inset, Raw traces; voltage stepped from −40 mV up to −120 mV, then to −60 mV. Left inset, Gramicidin recordings (1 wk post-seizure; R_S: control, 40.8 ± 4.6 MΩ; HT, 41.0 ± 3.4 MΩ; no difference). b, I_h activation 9 wk after seizures (●) and in controls (○). R_S: control, 9.6 ± 1.0 MΩ; HT, 9.0 ± 1.0 MΩ; no difference). Inset, Steady-state current (following subtraction of instantaneous current; 9 wk after seizures).

Fig. 2

Kinetic changes in I_h following seizures and the effect of modulation of intracellular cAMP. a, ‘SSE Improvement’ shows that double exponentials provided better fits to I_h at most voltages. Inset, Traces evoked at −110mV; single exponentials (arrows) provided poorer fits than double exponentials. b, Voltage versus fast activation time constants (1 wk after seizures; in b–f, control, ○; HT, ●). Inset, Activation kinetics 9 wk following seizures. c, Voltage versus slow activation time constants. Inset, Weights of fast and slow exponential components (w_fast and w_slow, respectively). d, Fast activation versus fast de-activation rates (data points are mean values from cells at −110 mV). Inset, Slow activation and de-activation time constants. e, I_h activation curves without (continuous lines) and with 1 mM cAMP (dashed lines) in the pipette. f, I_h activation curves in Rp-cAMPS. Inset, Activation curves in propranolol.

Fig. 3

Enhanced activation of altered I_h by hyperpolarizing inputs and post-inhibitory rebound firing following hyperthermia-induced seizures. a, Intracellularly injected hyperpolarizing current amplitude versus the depolarizing ‘sag’ (left y-axis; continuous lines), and the rebound depolarization ‘dep’; right y-axis; dotted lines). In control ACSF, cells held at −60 mV to avoid rebound firing. b, Rebound firing. Membrane potential: control, −54.1 ± 1.6 mV; HT, −53.3 ± 2.0 mV. In b–f, APV, CNQX and SCH50911 were present. c, Block of rebound firing and depolarization by ZD-7288 in cells from HT animals (Pre-ZD, ●; ZD-7288, ○). d, IPSPs (6 stimuli at 50Hz) and rebound firing in HT animals. Inset, Single IPSP. e, Summary data for post-inhibitory firing from experiments similar to those in d (membrane potential: control, −53.3 ± 2.4 mV; HT, −53.4 ± 1.3 mV; the duration of the post-IPSP depolarization was also longer by 321.0 ± 67% in HT. f, Block of post-inhibitory rebound firing by ZD-7288 in HT cells. Throughout this figure (except for c and f): control, ○; HT, ●.

Fig. 4

Mechanism of post-inhibitory rebound firing, specificity of the changes affecting I_h, and pattern of spontaneous GABA-receptor activation after febrile seizures. a, Block of the sag response and the post-IPSP firing following intracellular application of ZD-7288 (stimulation: as in Fig. 3; hyperpolarization of V_m by extracellular ZD-7288 with intracellular ZD-7288 present, 0.5 ± 0.5 mV, n = 4; without intracellular ZD-7288, 7.3 ± 0.7, n = 8). b, Evoked IPSPs with Cl⁻ filled pipettes (E_Cl = 0 mV). c and d, Rebound firing in HEPES/O₂ buffer (c) and in acetazolamide (d). Control, 40 stimuli at 100 Hz; HT, 6 stimuli at 50 Hz. e and f, Lack of a change in I_D (e, control, ○; HT, ●) and I_AHP (f) after seizures. g and h, Spontaneous IPSCs after experimental febrile seizures (intra-burst IPSC frequency: 95.1 ± 10.6 Hz).

Fig. 5

Multicompartmental modeling shows importance of altered h-channels in limiting inhibitory inputs and generating post-inhibitory rebound firing. a, Single IPSPs, of smaller (upper panel) or larger amplitude (lower panel), did not result in rebound firing. b, Dependence of rebound firing on shift in I_h and on the strength of inhibition (g_syn). c and d, Dependence of post-inhibitory rebound firing on the position of inhibitory inputs along the somatodendritic axis (c, ‘perisomatic’ inputs; d, ‘somatic’ inputs; upper panels, inhibitory synaptic inputs; lower panels, hyperpolarizing current steps). e, Lack of significant rebound firing following placement of all dendritic h-channels into the most proximal dendritic compartment; upper panel, small IPSPs; lower panel, larger IPSPs. f, Shifted V₅₀ for I_h, but I_h kinetics were kept control-like. g, No shift in V₅₀, but HT-like kinetics. h, Lack of rebound firing when input resistance was decreased (as found experimentally after seizures) in a control model cell.