Cell-Autonomous Alterations in Dendritic Arbor Morphology and Connectivity Induced by Overexpression of MeCP2 in Xenopus Central Neurons In Vivo

Methyl CpG binding protein-2 (MeCP2) is an essential epigenetic regulator in human brain development. Mutations in the MeCP2 gene have been linked to Rett syndrome, a severe X-linked progressive neurodevelopmental disorder, and one of the most common causes of mental retardation in females. MeCP2 duplication and triplication have also been found to affect brain development, indicating that both loss of function and gain in MeCP2 dosage lead to similar neurological phenotypes. Here, we used the Xenopus laevis visual system as an in vivo model to examine the consequence of increased MeCP2 expression during the morphological maturation of individual central neurons in an otherwise intact brain. Single-cell overexpression of wild-type human MeCP2 was combined with time-lapse confocal microscopy imaging to study dynamic mechanisms by which MeCP2 influences tectal neuron dendritic arborization. Analysis of neurons co-expressing DsRed2 demonstrates that MeCP2 overexpression specifically interfered with dendritic elaboration, decreasing the rates of branch addition and elimination over a 48 hour observation period. Moreover, dynamic analysis of neurons co-expressing wt-hMeCP2 and PSD95-GFP revealed that even though neurons expressing wt-hMeCP2 possessed significantly fewer dendrites and simpler morphologies than control neurons at the same developmental stage, postsynaptic site density in wt-hMeCP2-expressing neurons was similar to controls and increased at a rate higher than controls. Together, our in vivo studies support an early, cell-autonomous role for MeCP2 during the morphological differentiation of neurons and indicate that perturbations in MeCP2 gene dosage result in deficits in dendritic arborization that can be compensated, at least in part, by synaptic connectivity changes

Deletion of Fmr1 alters function and synaptic inputs in the auditory brainstem.

Fragile X Syndrome (FXS), a neurodevelopmental disorder, is the most prevalent single-gene cause of autism spectrum disorder. Autism has been associated with impaired auditory processing, abnormalities in the auditory brainstem response (ABR), and reduced cell number and size in the auditory brainstem nuclei. FXS is characterized by elevated cortical responses to sound stimuli, with some evidence for aberrant ABRs. Here, we assessed ABRs and auditory brainstem anatomy in Fmr1-/- mice, an animal model of FXS. We found that Fmr1-/- mice showed elevated response thresholds to both click and tone stimuli. Amplitudes of ABR responses were reduced in Fmr1-/- mice for early peaks of the ABR. The growth of the peak I response with sound intensity was less steep in mutants that in wild type mice. In contrast, amplitudes and response growth in peaks IV and V did not differ between these groups. We did not observe differences in peak latencies or in interpeak latencies. Cell size was reduced in Fmr1-/- mice in the ventral cochlear nucleus (VCN) and in the medial nucleus of the trapezoid body (MNTB). We quantified levels of inhibitory and excitatory synaptic inputs in these nuclei using markers for presynaptic proteins. We measured VGAT and VGLUT immunolabeling in VCN, MNTB, and the lateral superior olive (LSO). VGAT expression in MNTB was significantly greater in the Fmr1-/- mouse than in wild type mice. Together, these observations demonstrate that FXS affects peripheral and central aspects of hearing and alters the balance of excitation and inhibition in the auditory brainstem

Deletion of <i>Fmr1</i> Alters Function and Synaptic Inputs in the Auditory Brainstem

<div><p>Fragile X Syndrome (FXS), a neurodevelopmental disorder, is the most prevalent single-gene cause of autism spectrum disorder. Autism has been associated with impaired auditory processing, abnormalities in the auditory brainstem response (ABR), and reduced cell number and size in the auditory brainstem nuclei. FXS is characterized by elevated cortical responses to sound stimuli, with some evidence for aberrant ABRs. Here, we assessed ABRs and auditory brainstem anatomy in <i>Fmr1</i>^-/- mice, an animal model of FXS. We found that <i>Fmr1</i>^-/- mice showed elevated response thresholds to both click and tone stimuli. Amplitudes of ABR responses were reduced in <i>Fmr1</i>^-/- mice for early peaks of the ABR. The growth of the peak I response with sound intensity was less steep in mutants that in wild type mice. In contrast, amplitudes and response growth in peaks IV and V did not differ between these groups. We did not observe differences in peak latencies or in interpeak latencies. Cell size was reduced in <i>Fmr1</i>^-/- mice in the ventral cochlear nucleus (VCN) and in the medial nucleus of the trapezoid body (MNTB). We quantified levels of inhibitory and excitatory synaptic inputs in these nuclei using markers for presynaptic proteins. We measured VGAT and VGLUT immunolabeling in VCN, MNTB, and the lateral superior olive (LSO). VGAT expression in MNTB was significantly greater in the <i>Fmr1</i>^-/- mouse than in wild type mice. Together, these observations demonstrate that FXS affects peripheral and central aspects of hearing and alters the balance of excitation and inhibition in the auditory brainstem.</p></div

Nissl staining revealed a significant decrease in VCN and MNTB cell area in <i>Fmr1</i>^-/- mice.

<p>Nissl stains were performed on mouse brainstem tissue and the borders of VCN (A), MNTB (D), and LSO (G) were identified in wild type and <i>Fmr1</i>^-/- mice. Cells were counted within these regions and used to calculate cell density for each nucleus. No significant differences in cell density were found in VCN (B), MNTB (E), or LSO (H). Cell area was significantly reduced in <i>Fmr1</i>^-/- mice in VCN (C) and MNTB (F), but not in LSO (I). Scale bar in A, 200 μm; applies to A, D, and G.</p

Input-output functions in response to click, 8kHz, and 16kHz stimuli for peak amplitude (A) and peak latency (B).

<p>Peak I is represented by the furthest left column, peak II by the middle left column, peak III by the middle right column, and peak IV by the furthest right column. Significant differences in peak amplitude (A) were found between wild type (black circles) and <i>Fmr1</i>^-/- (open circles) at peaks I and III. Few significant differences were found in peak latency (B).</p

<i>Fmr1</i>^-/- MNTB cells receive significantly more GABAergic input.

<p>The fractional coverage of VGLUT and VGAT was assessed in the VCN (A), MNTB (B), and LSO (C). No significant differences were found in VGLUT or VGAT fractional coverage in VCN (A). <i>Fmr1</i>^-/- (gray) MNTB had significantly more VGAT coverage than wild type MNTB (black), though there was no difference in VGLUT coverage. VGLUT and VGAT fractional coverage was significantly greater in <i>Fmr1</i>^-/- within the LSO. VGLUT and VGAT fractional coverage was compared by calculating a synaptic protein index value for each nucleus (D). No significant differences were found in VCN or LSO, but <i>Fmr1</i>^-/- MNTB had lower index values than wild type MNTB. The smaller I_SP value likely reflects the heightened VGAT staining in <i>Fmr1</i>^-/- MNTB.</p

Analysis of ABR mean thresholds.

<p>Analysis of ABR mean thresholds.</p