Downregulation of tonic GABAergic inhibition in a mouse model of fragile X syndrome

Abstract

The absence of fragile X mental retardation protein results in the fragile X syndrome (FXS), a common form of mental retardation associated with attention deficit, autistic behavior, and epileptic seizures. The phenotype of FXS is reproduced in fragile X mental retardation 1 (fmr1) knockout (KO) mice that have region-specific altered expression of some gamma-aminobutyric acid (GABA(A)) receptor subunits. However, little is known about the characteristics of GABAergic inhibition in the subiculum of these animals. We employed patch-clamp recordings from subicular pyramidal cells in an in vitro slice preparation. In addition, semiquantitative polymerase chain reaction and western blot experiments were performed on subiculum obtained from wild-type (WT) and KO mice. We found that tonic GABA(A) currents were downregulated in fmr1 KO compared with WT neurons, whereas no significant differences were observed in phasic GABA(A) currents. Molecular biology analysis revealed that the tonic GABA(A) receptor subunits alpha5 and delta were underexpressed in the fmr1 KO mouse subiculum compared with WT. Because the subiculum plays a role in both cognitive functions and epileptic disorders, we propose that altered tonic inhibition in this structure contributes to the behavioral deficits and epileptic activity seen in FXS patients. This conclusion is in line with evidence implicating tonic GABA(A) inhibition in learning and memory.

Figure 1

Phasic component of GABAergic current is not altered in subiculum of fmr1 KO mice. (A) The sIPSC events recorded from WT (left) and fmr1 KO mice (right) during voltage-clamp experiments in presence of blockers for the glutamatergic and GABA_B receptors. Traces with higher frequency of events than mean values were chosen for figure purpose. Holding potential was −70 mV. In the inset single events from WT (black trace) and fmr1 KO (gray trace) are overlapped to show that time constants are not changed in the 2 groups. (B) Histograms reveal no statistical difference in current density, charge transferred, peak time, half decay time, and interevent interval between WT (black bars) and fmr1 KO (gray bars) tissues.

Figure 2

The tonic component of GABAergic current is downregulated in fmr1 KO subicular neurons. (A) Traces recorded in voltage-clamp from WT (left) and fmr1 KO (right) subicular neurons (holding potential −70 mV; glutamatergic and GABA_B blockers in the bath). Application of PTX induces the disappearance of synaptic events and the shift of the holding current presumably due to the block of phasic and tonic GABAergic components, respectively. Note that the shift of the holding current is more pronounced in WT compared with fmr1 KO tissue. Dash lines indicate zero-current level. (B) Histogram shows significant difference of current density for the tonic component between WT (black bar) and fmr1 KO (gray bar) mice.

Figure 3

The mRNA of α5 and δ subunits is underexpressed in subiculum of fmr1 KO mice. R Pfaffl values (Pfaffl 2001) indicating underexpression of the levels of expression of mRNA for α5 (light gray bars) and δ (dark gray bars) subunits in fmr1 KO relative to WT. Data obtained using GAPDH and TBP as housekeeping genes were not significantly different (n.s.).

Figure 4

The α5 and δ subunits are underexpressed at protein level in subiculum of fmr1 KO mice. Western blots performed in WT (left) and fmr1 KO (right) tissue for both GABA_A subunits in subiculum. On the right, DRs obtained from western blot experiments reveal underexpression of α5 (light gray bar) and δ (dark gray bar) GABA_A receptor subunits.