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. 2015 Feb 13;10(2):e0117266.
doi: 10.1371/journal.pone.0117266. eCollection 2015.

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

Sarah E Rotschafer  1 , Sonya Marshak  1 , Karina S Cramer  1
Affiliations

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

Sarah E Rotschafer et al. PLoS One. .

Abstract

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.

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Conflict of interest statement

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

Figures

Fig 1
Fig 1. Fmr1 -/- mice have higher hearing thresholds in response to click and pure tone stimuli.
(A) Representative ABR traces in response to click stimuli from wild type mice (left) and Fmr1 -/- mice (right). (B) ABR thresholds are significantly higher in Fmr1 -/- mice (gray) than in wild type mice (black) in response to clicks. (C) Fmr1 -/- mouse ABR thresholds were significantly higher in response to pure tone stimuli then those of wild type mice.
Fig 2
Fig 2. Input-output functions in response to click, 8kHz, and 16kHz stimuli for peak amplitude (A) and peak latency (B).
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 Fmr1 -/- (open circles) at peaks I and III. Few significant differences were found in peak latency (B).
Fig 3
Fig 3. Nissl staining revealed a significant decrease in VCN and MNTB cell area in Fmr1 -/- mice.
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 Fmr1 -/- 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 Fmr1 -/- mice in VCN (C) and MNTB (F), but not in LSO (I). Scale bar in A, 200 μm; applies to A, D, and G.
Fig 4
Fig 4. Wild type (left column) and Fmr1 -/- (right column) brainstem tissue was stained for markers of excitatory and inhibitory inputs.
VCN (A-B), MNTB (C-D), and LSO (E-F) were stained for VGLUT (red) and VGAT (green). Scale bar in A, 50 μm; applies to all panels.
Fig 5
Fig 5. Fmr1 -/- MNTB cells receive significantly more GABAergic input.
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). Fmr1 -/- (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 Fmr1 -/- 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 Fmr1 -/- MNTB had lower index values than wild type MNTB. The smaller ISP value likely reflects the heightened VGAT staining in Fmr1 -/- MNTB.
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