Critical period plasticity is disrupted in the barrel cortex of FMR1 knockout mice

Abstract

Alterations in sensory processing constitute prominent symptoms of fragile X syndrome; however, little is known about how disrupted synaptic and circuit development in sensory cortex contributes to these deficits. To investigate how the loss of fragile X mental retardation protein (FMRP) impacts the development of cortical synapses, we examined excitatory thalamocortical synapses in somatosensory cortex during the perinatal critical period in Fmr1 knockout mice. FMRP ablation resulted in dysregulation of glutamatergic signaling maturation. The fraction of silent synapses persisting to later developmental times was increased; there was a temporal delay in the window for synaptic plasticity, while other forms of developmental plasticity were not altered in Fmr1 knockout mice. Our results indicate that FMRP is required for the normal developmental progression of synaptic maturation, and loss of this important RNA binding protein impacts the timing of the critical period for layer IV synaptic plasticity.

Figure 1. FMRP expression levels parallel the critical period for plasticity in layer IV of the somatosensory cortex

(Ai, Bi, Ci, Di) Coronal sections containing the primary somatosensory cortex stained with α-FMRP antibody (rAM1) from P4, P7,P14, and P21 wildtype mice. Calibration 250μm (Aii, Bii, Cii, Dii) Tangential sections through layer IV of the PMBSF at each developmental age. The expression of FMRP is highest during the critical period, peaking at P7. Expression in layer IV of older mice is reduced. Calibration 100μm. (Ei-iii) Examples of immunoelectron micrographs from layer IV somatosensory cortex containing labeled asymmetric spines. No FMRP labeling was observed in presynaptic elements at P7. (F) Immunoelectron micrograph of a layer IV neuron from P7 mouse. DAB immunoreactivity is observed in the cytoplasm and primary dendrite. (NU: nucleus. Cyt: cytoplasm)

Figure 2. Critical period maturation of glutamate receptor signaling at thalamocortical synapses is altered in FMRP knockout mice

(Ai) Cartoon representation of para-coronal thalamocortical slice depicting placement of the recording electrode in the layer IV barrels and the extracellular stimulating electrode in the ventrobasal thalamus (VB) (H: hippocampus; IC: internal capsule; C: caudate). (Aii) Bright field image of the thalamocortical slice and (Aiii) image of the barrels in layer IV. Calibration: 250μm (Aiv) Higher magnification image of a spiny stellate neuron in layer IV (SS: spiny stellate; IN: interneuron. Calibration: 25μm (Bi) Time course of development in NMDA/AMPA ratio at thalamocortical synapses in wildtype mice (Fmr1^+/y). Relatively high NMDA/AMPA ratio is observed at P4 when synaptic contribution of NMDA receptors is large. Over the course of the first week NMDA/AMPA ratio declines in wildtype mice and then stabilizes at the close of the critical period (depicted by shaded area). (Bii) Representative EPSC recordings from wildtype Fmr1^+/y mice at P4 and P7. AMPA receptor mediated EPSCs were measured as the peak current at −70mV and the NMDA component was measured by depolarizing the cell to +40mV and measuring the mean current over a 2.5ms window, 60ms after the onset of the outward current. At this time point the contribution of the outward AMPA component is negligible and the measured current is mediated solely by NMDA receptors (Marie et al., 2005). Calibration: 20pA, 100ms (Ci) Time course of development of NA ratio in Fmr1^−/y mice. NA ratio at P4 is significantly lower than in Fmr1^+/y mice (0.5 ± 0.1, n = 13, p = 0.0003). Over the critical period NMDA/AMPA ratio increases to maximal level at P7 (1.5 ± 0.2, n = 18, p = 0.0007) and then reverts to similar levels as wildtype recordings beyond this developmental timepoint. (Cii) Representative EPSC recordings from Fmr1^−/y mice at P4 and P7 recorded from spiny stellate neurons voltage clamped at −70mV and +40mV. Calibration: 10pA, 100ms.

Figure 3. Quantal glutamate receptor content is not altered in thalamocortical synapses in Fmr1 knockout mice during the critical period

(A) Western blot analysis of synaptoneurosome fraction from layer IV S1 tissue during critical period development. Expression of NR1 protein was significantly elevated in the synaptic membrane fraction. (B) Analysis of protein expression in layer IV after closure of the critical period. No significant differences were observed between Fmr1^+/y and Fmr1^−/y mice (Ci) Cumulative distribution of amplitude of all quantal events measured in Sr²⁺ from Fmr1^+/y (black) and Fmr1^−/y mice (grey). (Cii) Mean amplitude of events from all recordings. No difference is observed between Fmr1^+/y and Fmr1^−/y mice (p>0.05, KS test) (Ciii & iv) Representative traces EPSCs recorded in Ca²⁺ (upper) and desynchronized events recorded in 6mM Sr²⁺. Calibration: 10pA, 100ms (Di) Cumulative distribution of quantal NMDA mediated events and (Dii) mean amplitudes from all recordings. No significant difference in the size of quantal NMDA events was observed between Fmr1^+/y and Fmr1^−/y mice. (Diii & iv) Representative recordings of desynchronized NMDA mediated Sr²⁺-mEPSCs. Calibration: 5pA, 250ms.

Figure 4. Silent synapses are prevalent at the close of the critical period in Fmr1 knockout mice

(Ai) Representative traces and (Aii) timecourse from a single silent synapse experiment in Fmr1^+/y mice. Minimal stimulation was used such that approximately 50% failures were observed when recording with the membrane potential clamped at −70mV. After 100 consecutive trials, the membrane potential was depolarized to +40mV and NMDA EPSCs measured (60ms latency from current onset, see methods). After a further 100 trials, D-APV was applied to block the NMDA component. (Bi & Bii) Representative silent synapse experiment and traces from a Fmr1^−/y mouse. Calibration: 20pA, 50ms (C) Percentage of failures for each recording at −70mV and +40mV in Fmr1^+/y mice. There is no difference in failure rate of AMPA and NMDA components suggesting that there are few NMDA-only synapses present at P7 in Fmr1^+/y mice. (D) Percent failures at −70mV and +40mV in Fmr1^−/y mice. A significantly reduced number of failures at +40mV demonstrates that a fraction of NMDA-only synapses remain at P7 in Fmr1^−/y mice (p = 0.01). (E) The failure ratio is larger than 1.0 in Fmr1^−/y underlining that there is a fraction of silent synapses remaining at P7 in Fmr1 knockout animals.

Figure 5. NMDA receptor stoichiometry and spine density in layer IV stellate neurons

(Ai & Aii) Representative NMDA receptor mediated responses at P4 and P10 before and after application of the NR2B specific antagonist ifenprodil (IFN, 3μM) in Fmr1^+/y and (Bi & Bii) Fmr1^−/y mice Calibration: 5pA, 200ms (P4) and 5pA, 100ms (P10). (Aiii & Biii) Grouped data from all recordings demonstrating a developmental reduction in the sensitivity of thalamocortical NMDA receptors to ifenprodil between P4 and P10 that is not significantly different between Fmr1^+/y and Fmr1^−/y mice. (Ci & Cii) Representative pictures of Golgi stained neurons and enlarged dendritic regions from layer IV sections taken from P7 Fmr1 knockout animals and littermate controls. Calibration: 25μm (Ciii) Analysis of spine protrusion density in P7 and (Civ) P14 mice.

Figure 6. Temporal window for LTP at thalamocortical synapses is shifted in Fmr1 knockout mice

(Ai) Grouped data from all wildtype recordings at each postnatal day between P3 and P10. Robust potentiation is observed in all recordings at P3 whereas no LTP is observed after closure of the critical period (P7). (Aii) Grouped data from all Fmr1^−/y mice thalamocortical LTP recordings. No LTP is observed at P3 whereas increasing amounts of potentiation are observed on subsequent postnatal days with maximal potentiation observed at P6 & P7. In some recordings LTP is observed even beyond closure of the critical period. (Bi-iii) Grouped time-courses of thalamocortical LTP experiments in Fmr1^−/y and control mice at P3 & P4, P6 & P7, P9 & P10.

Figure 7. Barrel arealization and lesion induced map plasticity in Fmr1 knockout mice

(Ai & Aii) Representative cytochrome oxidase stained tangential sections through layer IV of the barrel cortex in P7 Fmr1^+/y and Fmr1^−/y mice. Calibration: 250μm (B) Barrel areas for each individual barrel in rows b-d in wildtype and knockout mice. (C) Total area occupied by the posterior medial barrel subfield (PMBSF) in Fmr1^+/y and Fmr1^−/y mice (D) Ratio of each individual barrel to the total territory occupied by the PMBSF. (Ei & Eii) Representative cytochrome oxidase stained sections of mice with unilateral c-row whisker lesioning performed at P0. White dashed regions highlight the c-row barrels. (Eiii) Map Plasticity Index of barrel areas for P0 lesioned mice of the ipsilateral and contralateral hemispheres. (Fi & Fii) Representative sections of mice subjected to c-row whisker lesioning at P5. (Fiii) Map Plasticity Index for ipsi- and contralateral barrel areas of P5 lesioned mice.