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. 2017 Sep 20;12(9):e0185067.
doi: 10.1371/journal.pone.0185067. eCollection 2017.

Excitability is increased in hippocampal CA1 pyramidal cells of Fmr1 knockout mice

M Angeles Luque  1 , Pablo Beltran-Matas  1 , M Carmen Marin  1 , Blas Torres  1 , Luis Herrero  1
Affiliations

Excitability is increased in hippocampal CA1 pyramidal cells of Fmr1 knockout mice

M Angeles Luque et al. PLoS One. .

Abstract

Fragile X syndrome (FXS) is caused by a failure of neuronal cells to express the gene encoding the fragile mental retardation protein (FMRP). Clinical features of the syndrome include intellectual disability, learning impairment, hyperactivity, seizures and anxiety. Fmr1 knockout (KO) mice do not express FMRP and, as a result, reproduce some FXS behavioral abnormalities. While intrinsic and synaptic properties of excitatory cells in various part of the brain have been studied in Fmr1 KO mice, a thorough analysis of action potential characteristics and input-output function of CA1 pyramidal cells in this model is lacking. With a view to determining the effects of the absence of FMRP on cell excitability, we studied rheobase, action potential duration, firing frequency-current intensity relationship and action potential after-hyperpolarization (AHP) in CA1 pyramidal cells of the hippocampus of wild type (WT) and Fmr1 KO male mice. Brain slices were prepared from 8- to 12-week-old mice and the electrophysiological properties of cells recorded. Cells from both groups had similar resting membrane potentials. In the absence of FMRP expression, cells had a significantly higher input resistance, while voltage threshold and depolarization voltage were similar in WT and Fmr1 KO cell groups. No changes were observed in rheobase. The action potential duration was longer in the Fmr1 KO cell group, and the action potential firing frequency evoked by current steps of the same intensity was higher. Moreover, the gain (slope) of the relationship between firing frequency and injected current was 1.25-fold higher in the Fmr1 KO cell group. Finally, AHP amplitude was significantly reduced in the Fmr1 KO cell group. According to these data, FMRP absence increases excitability in hippocampal CA1 pyramidal cells.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Rheobase is not modified in CA1 pyramidal cells of Fmr1 KO mice.
(A) Voltage responses of two representative WT and Fmr1 KO cells to depolarizing and hyperpolarizing current steps. (B) Histogram showing input resistance in WT and Fmr1 KO cell groups. (C) Representative recordings of the rheobase that was 70 pA for both WT and Fmr1 KO cells (left panel). Phase-plane plots to determine the action potential voltage threshold (right panel). (D) Histograms showing resting membrane potential, depolarization voltage, action potential voltage threshold and rheobase in WT and Fmr1 KO cell groups. Calibrations as indicated. In this and following figures, histograms show the mean value and standard error of the mean. Asterisks (*) indicate statistically significant differences (P<0.05). n.s. indicates non-significant statistical differences.
Fig 2
Fig 2. Action potential duration is increased in CA1 pyramidal cells of Fmr1 KO mice.
(A) Single action potential in a WT and a Fmr1 KO cell generated by a short pulse (100 μs) with an intensity that was adjusted to the spike voltage threshold. The recordings also depict the half-width (dashed line) of the spike for the measurement of action potential duration. St indicates the onset of the stimulus. (B) Train of action potentials (8–12 spikes) produced by applying 500 ms current injections at 200 pA in a WT cell and at 100 pA in a Fmr1 KO cell. The durations of the first spike (▼), the spike in the middle of the train (250 ms after stimulus onset; ■) and the last spike of the train (●) were measured in both cell groups. (C) Histogram showing action potential duration evoked in a single action potential and in trains of action potentials in WT and Fmr1 KO cell groups. Calibrations as indicated. Asterisks (*) indicate statistically significant differences (P<0.05).
Fig 3
Fig 3. Action potential firing frequency is increased in CA1 pyramidal cells of Fmr1 KO mice.
(A) Firing frequency depended on injected current in both WT and Fmr1 KO cells, but in response to the same current intensity the number of spikes was higher in Fmr1 KO cells. (B) Histogram showing firing frequency (spikes s-1) evoked by current steps of 100, 200, 300 and 400 pA in WT and Fmr1 KO cell groups. (C) Plot of the firing frequency versus injected current relationship for representative WT and Fmr1 KO cells. The gain corresponds to the slope of the linear relationships. (D, E) Linear relationships between injected current and firing frequency for WT and Fmr1 KO cells are represented in grey color. The black lines correspond to the mean fits for each group: WT, firing frequency = 0.12 ∙ injected current—2.7; Fmr1 KO, firing frequency = 0.15∙ injected current—3.7. Calibrations as indicated. Asterisks (*) indicate statistically significant differences (P<0.05).
Fig 4
Fig 4. Amplitude of the action potential fast and medium after-hyperpolarization (AHP) diminished in CA1 pyramidal cells of Fmr1 KO mice.
(A) Action potential after-depolarization in response to a short stimulus (100 μs, current intensity adjusted to spike voltage threshold). (B) Action potential AHP in response to depolarizing current steps of 500 ms that shifted the membrane potential to the spike voltage threshold and produced action potentials at low firing frequencies (≤ 2 spikes s-1). It should be noted that fast (▲), but not medium (∆), AHPs were observable in Fmr1 KO cells. (C) Amplitude and duration of the medium AHP during a repetitive firing, measured in the spike closest to 250 ms after stimulus onset. Current intensity was adjusted to evoke similar number of spikes (8–12 action potentials for 500 ms pulse durations) in both cell groups. (D) Medium AHP after a train of action potentials (area indicated by square) in WT and Fmr1 KO cells. Note the absence of a medium AHP in the Fmr1 KO cell. (E, F) Histograms show the amplitude and duration of medium AHPs in WT and Fmr1 KO cell groups. Calibrations as indicated. Asterisks (*) indicate statistically significant differences (P<0.05). n.s. indicates non-significant statistical differences.
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The authors received no specific funding for this work.