Experimental febrile seizures are precipitated by a hyperthermia-induced respiratory alkalosis

Abstract

Febrile seizures are frequent during early childhood, and prolonged (complex) febrile seizures are associated with an increased susceptibility to temporal lobe epilepsy. The pathophysiological consequences of febrile seizures have been extensively studied in rat pups exposed to hyperthermia. The mechanisms that trigger these seizures are unknown, however. A rise in brain pH is known to enhance neuronal excitability. Here we show that hyperthermia causes respiratory alkalosis in the immature brain, with a threshold of 0.2-0.3 pH units for seizure induction. Suppressing alkalosis with 5% ambient CO2 abolished seizures within 20 s. CO2 also prevented two long-term effects of hyperthermic seizures in the hippocampus: the upregulation of the I(h) current and the upregulation of CB1 receptor expression. The effects of hyperthermia were closely mimicked by intraperitoneal injection of bicarbonate. Our work indicates a mechanism for triggering hyperthermic seizures and suggests new strategies in the research and therapy of fever-related epileptic syndromes.

Figure 1

Hyperthermia-induced behavioral seizures are associated with brain alkalosis. (a) Hyperthermia (HT)-induced changes in body temperature (lower panels) and breathing rate (upper panels) in P8–P11 (n = 21; left panels) and P22–P23 (right panels) rat pups. The green bar marks the onset and end of the hyperthermia-induced seizures; the red bar indicates exposure to hyperthermia. Data here and below are mean ± (or +) s.d. Seizures did not occur in P22–P23 rats. (b) Simultaneous recording of hyperthermia-induced changes in breathing rate and intracortical pH in a P9 (left) and a P23 (right) rat. The green bar indicates hyperthermia-induced seizures. (c) Intraperitoneal application (arrow) of 1 mmol/kg bicarbonate induces a small (~0.1 pH units) alkaline shift in pH in the brain of a P9 rat, but no seizure activity. (d) Application of 5 mmol/kg bicarbonate in a P9 rat induces an intracortical alkalosis of 0.24 pH units. The green bar indicates seizure activity. (e) Summary of cortical pH changes in P8–P11 rat pups at threshold for seizure initiation during hyperthermia (n = 9) and upon injection of 5 mmol/kg bicarbonate (n = 7). The two sets of data are not statistically different (P = 0.424).

Figure 2

Exposure of rat pups to 5% ambient CO₂ blocks hyperthermia- and bicarbonate-induced brain alkalosis and associated ictal activity. (a) Recording in a P9 rat showing simultaneous hyperthermia (HT)-induced ictal activity in the hippocampus and in the temporal cortex, and the fast antiepileptic action of ambient 5% CO₂. (b) Time delay between exposure of the pups to 5% CO₂ to the block of ongoing ictal activity in the cortex and hippocampus in the P8–P11 rats (n = 29). (c) Cortical recording during a continuous 25-min hyperthermia-induced seizure (upper panel) in a P9 rat. Under identical hyperthermic conditions, but in the presence of 5% CO₂, no ictal activity is observed (lower panel). The insets show simultaneous cortical and hippocampal recordings at an expanded scale. (d) Cortical recording immediately after the onset and before the end of ictal activity evoked by intraperitoneal application of 5 mmol/kg bicarbonate (upper panel) in a P10 rat. The bicarbonate-induced ictal activity is completely suppressed by exposure to 5% CO₂ (lower panel). The insets show simultaneous cortical and hippocampal recordings at an expanded scale. (e) The alkalosis induced by hyperthermia or intraperitoneal injection of 5 mmol/kg bicarbonate (P9 rats) is completely abolished in the presence of 5% CO₂. During prolonged exposure to 5% CO₂, a slow acid shift is seen.

Figure 3

Ambient 5% CO₂ applied during hyperthermia blocks the long-term upregulation of the I_h current. (a) The amplitudes of the I_h-generated voltage sag and rebound depolarization evoked by hyperpolarizing current pulses in CA1 pyramidal neurons in hippocampal slices are enhanced in slices from pups previously exposed to hyperthermia compared to control slices, but there are no significant differences between neurons from control and hyperthermia + 5% CO₂ rats (control, n (slices) = 6; hyperthermia, n = 7; hyperthermia + 5% CO₂, n = 5). The inset shows recordings performed 10 d after the induction of hyperthermia-induced seizures at P9. The number of action potentials evoked by the rebound depolarization after hyperpolarizing currents ≥ 0.3 nA is increased in slices from pups previously exposed to hyperthermia. No significant differences were found between neurons from hyperthermia + 5% CO₂ and control rats (bottom left panel; control, n (slices) = 5; hyperthermia, n = 6; hyperthermia + 5% CO₂, n = 4). Five to ten current pulses of each amplitude shown on the x-axis were applied. Specimen traces are shown on the bottom right. (b) After seizures caused by one or two bicarbonate injections, the I_h-generated sag (top left panel), the rebound depolarization (top right panel) and the number of action potentials evoked by the rebound depolarization after hyperpolarizing currents ≥ 0.3 nA (bottom panel) are enhanced (control, n (slices) = 8; one bicarbonate injection, n = 8; two bicarbonate injections, n = 6). *P < 0.05, **P < 0.01, ***P < 0.001 for test versus control (ANOVA).

Figure 4

Ambient 5% CO₂ applied during hyperthermia blocks the long-term upregulation of the CB1 receptors. (a) Western blots of CB1 receptor protein from hippocampi 1, 2, 3, 5, 10 and 120 d after exposing P10 rats to hyperthermia (red) or to the hyperthermia + 5% CO₂ treatment (blue), and from control littermates (white). β-tubulin was used as a reference protein for the quantification shown in b. (b) Quantitative analysis of western blots. An upregulation of the CB1 receptor protein is evident after 2 d following hyperthermia, and a maximum is seen after 5 d. No significant change in CB1 receptor protein expression was found in the hyperthermia + 5% CO2 rats compared to controls (all values represent percentage changes normalized to β-tubulin levels). The value at each time point is based on data from 12 hippocampi. (c) After bicarbonate injection, an upregulation in CB1 receptor is seen at 5 d after seizures (n = 6). This effect is fully blocked in rats in which the seizures were suppressed by 5% CO₂. ***P < 0.001, control versus hyperthermia, or control versus bicarbonate injection.