The developmental switch in GABA polarity is delayed in fragile X mice

Abstract

Delays in synaptic and neuronal development in the cortex are key hallmarks of fragile X syndrome, a prevalent neurodevelopmental disorder that causes intellectual disability and sensory deficits and is the most common known cause of autism. Previous studies have demonstrated that the normal progression of plasticity and synaptic refinement during the critical period is altered in the cortex of fragile X mice. Although the disruptions in excitatory synapses are well documented in fragile X, there is less known about inhibitory neurotransmission during the critical period. GABAergic transmission plays a crucial trophic role in cortical development through its early depolarizing action. At the end of cortical critical period, response properties of GABA transform into their mature hyperpolarizing type due to developmental changes in intracellular chloride homeostasis. We found that the timing of the switch from depolarizing to hyperpolarizing GABA is delayed in the cortex of fragile X mice and there is a concurrent alteration in the expression of the neuronal chloride cotransporter NKCC1 that promotes the accumulation of intracellular chloride. Disruption of the trophic effects of GABA during cortical development could contribute to the altered trajectory of synaptic maturation in fragile X syndrome.

Figure 1.

E_Cl- remains depolarized in Fmr1 ko mice during cortical development. A, Representative example of a perforated patch-clamp recording from a layer IV neuron in the somatosensory cortex of a P10 Fmr1 wt mouse. Recordings were made at several hold potentials and the E_Cl- calculated from the linear fit of the current–voltage relationship. GABA responses shown at −80, 0, and +40 mV were evoked by extracellular stimulation in the presence of glutamate blockers d-APV (50 μm) and CNQX (10 μm). Calibration for current traces: 50 ms, 200 pA. B, Representative recording from Fmr1 ko at P10 and current–voltage relationship of GABA-mediated currents. E_Cl- is significantly more depolarized at this age in recordings from Fmr1 ko mice. Calibration for current traces: 50 ms, 50 pA. C, Grouped data from all recordings. The average E_Cl- calculated from each individual recording is plotted against the age of the mouse (postnatal day). The RMP measured at P10 is denoted by the dashed line and shaded area represents points at which GABA would have a mature hyperpolarizing response. *p < 0.05 (P5: wt, n = 13; ko n = 4. P6: wt, n = 6; ko, n = 11. P7: wt, n = 6; ko, n = 4. P8: wt, n = 10: ko, n = 8. P9: wt, n = 8; ko, n = 11.P10: wt, n = 8; ko, n = 5. P11: wt, n = 16; ko, n = 14. P12: wt, n = 17; ko, n = 6. P13: wt, n = 12: ko, n = 5. P14: wt, n = 11: ko, n = 10. P15: wt, n = 12; ko, n = 7).

Figure 2.

Expression of the juvenile chloride cotransporter NKCC1. A, The expression of NKCC1 is elevated in the cortex of Fmr1 ko mice at the close of the critical period. Representative gel run with cortical homogenates from Fmr1 wt and Fmr1 ko mice and probed with anti-NKCC1 and anti-β-tubulin (β-tub). Western blots were used to measure NKCC1 protein levels at three developmental time points: P5, P10, and P15. B, Grouped data from all experiments. The average intensity of NKCC1 was normalized to β-tubulin in Fmr1 wt and Fmr1 ko in each sample at each age (P5: wt, n = 8; ko, n = 8. P10: wt, n = 11; ko, n = 11. P15: wt, n = 8; ko, n = 7). A significant elevation of NKCC1 protein levels was observed at P10 in samples from the Fmr1 ko mice. *p < 0.05. C, Representative Western blots for cortical homogenates probed with anti-KCC2 antibodies from Fmr1 wt and Fmr1 ko mice at P5, P10, and P15. D, Grouped data for all KCC2 Western blots (P5: wt, n = 8; ko, n = 8. P10: wt, n = 11; ko, n = 11; P15: wt, n = 8; ko, n = 7). No difference in relative protein levels was observed at any age between the two genotypes (p > 0.05). All values are means ± SEM.

Figure 3.

Chloride cotransporter transcript expression is not altered in cortex of Fmr1 ko mice. A, Relative transcript abundance of NKCC1 in cortex of Fmr1 wt and Fmr1 ko at P5, P10, and P15. B, Relative RNA expression of KCC2 in Fmr1 wt and Fmr1 ko at P5, P10, and P15. R_q was calculated as the ratio of the target gene to GAPDH as the reference gene (p > 0.05 for all age groups for both NKCC1 and KCC2).