Genetic and pharmacological modulation of giant depolarizing potentials in the neonatal hippocampus associates with increased seizure susceptibility

Abstract

The expression of Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) is responsible for high intracellular Cl(-) resulting in the excitatory action of GABA(A) receptor activation in the developing brain. Giant depolarizing potentials (GDPs) are spontaneous network oscillations that involve GABA(A) receptors and are thought to be important in establishing neuronal circuit wiring. Earlier work established that seizure susceptibility in the GABA(A) γ2(R43Q) epilepsy mouse is impacted by developmental consequences of impaired GABA(A) receptor function. We investigated the potential mechanism of the developmental influence by recording GDPs in the CA3 pyramidal neurons from brain slices of the neonatal GABA(A) γ2(R43Q) mouse. Interestingly, the number of GPDs was significantly lower in slices from mutant mouse compared with wild-type control, suggesting an involvement in setting seizure susceptibility. To test this idea we blocked NKCC1 with bumetanide in neonatal mice and reduced the number of GDPs to a level similar to that seen in the mutant mice. We found that neonatal treatment with bumetanide resulted in a similar level of susceptibility to thermally induced seizures as described for the GABA(A) γ2(R43Q) mouse. These results provide evidence that a human GABA(A) receptor epilepsy mutation exerts a developmental influence by modulating the number of GDPs. It also draws attention to the potential risk of early treatment with bumetanide.

Figure 1. A decrease in giant depolarizing potentials (GDPs) frequency is evident in the GABA_Aγ2_R43Q epilepsy mouse

Representative whole-cell recordings from CA3 pyramidal neurons in acute neonatal hippocampal slices from A wild-type (WT; RR) and B GABA_Aγ2_R43Q (RQ) mice. Stars represent GDP events detected using the automatic algorithm. The expanded time scale of CA3 pyramidal recordings is shown below. C, mean pooled data of GDP frequency. *P < 0.05. D, example recording indicating the effect of bicuculline (10 μm; Bic) in a GABA_Aγ2_R43Q hippocampal slice.

Figure 2. Spontaneous sPSCs are reduced in the GABA_Aγ2_R43Q epilepsy mouse

A, representative whole-cell recording from CA3 pyramidal neurons in neonatal hippocampal slices from wild-type (WT) and mutant GABA_Aγ2_R43Q mice. B, average postsynaptic events recorded from a single CA3 pyramidal neuron from a WT and mutant mouse. C, mean pooled data of frequency, amplitude, and rise and decay times of sPSC from WT and GABA_Aγ2_R43Q mice. *P < 0.05.

Figure 3. Bumetanide reduces giant depolarizing potentials (GDPs) frequency in WT mice

Representative whole-cell recordings from CA3 pyramidal neurons in acute neonatal hippocampal slices perfused with: A, normal artificial cerebrospinal fluid (ACSF); and B, 10 μm bumetanide. Stars represent GDP events detected using the automatic algorithm. Expanded time scale of CA3 pyramidal recordings is shown below. C, mean pooled data of GDP frequency. *P < 0.05.

Figure 4. Bumetanide reduces spontaneous sPSCs

A, representative whole-cell recording from CA3 pyramidal neurons in neonatal hippocampal slices in normal artificial cerebrospinal fluid (ACSF) or in bumetanide (10 μm). B, average postsynaptic events recorded from CA3 pyramidal neurons in ASCF and bumetanide. C, mean pooled data of frequency, amplitude, and rise and decay times. *P < 0.05.

Figure 5. Neonatal exposure to bumetanide increases susceptibility to thermogenic seizures

Survival curve of the latency to first tonic-clonic seizure of mice injected with saline or bumetanide (2 mg kg⁻¹). P < 0.05, Mantel–Cox test.