DEVELOPMENT AND POTENTIAL BEHAVIORAL SIGNIFICANCE OF PRECISE TONOTOPY IN AN INHIBITORY CIRCUIT OF THE AUDITORY BRAINSTEM

Precise neuronal connections are crucial for normal brain function. Often this is accomplished during development, as initially imprecise connections are refined in a manner that depends on neural activity, both spontaneous and sensory-evoked. In the auditory system, many connections are topographically organized according to frequency, or tonotopically, an organizational scheme important for processing information about sound. In this thesis, I investigated the development of precise tonotopy in the inhibitory connections between the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO), a pathway in the auditory brainstem involved in sound localization. Although MNTB-LSO connections exhibit tonotopy from the outset, tonotopic precision increases during development through a process of silencing imprecise inputs and strengthening maintained connections before hearing onset, followed by anatomical pruning after hearing. I teased apart the relationship between functional and anatomical refinement, as well as the degree to which spontaneous and sound-evoked activity play a role in each. Finally, I attempted to link the tonotopic specificity of this circuit to a specific aspect of auditory perception, frequency discrimination.In Chapter 2, I mapped the tonotopic precision of individual MNTB axons in the LSO over the first three weeks of postnatal development and showed that pruning does not take place before hearing onset, indicating that functional and anatomical refinement take place during distinct developmental periods. In Chapter 3, I showed that anatomical refinement after hearing onset depends on efferent cholinergic transmission in the cochlea, most likely due to its role in patterning pre-hearing spontaneous activity and the functional refinement of connections. In Chapter 4, I showed that eliminating the normal spectrotemporal structure of sound-evoked activity by rearing animals in pulsed white noise does not disrupt pruning. Finally, in Chapter 5, I showed that the loss of tonotopic precision that results from the elimination of cochlear cholinergic transmission is also accompanied by impaired frequency discrimination, providing a link between tonotopic refinement, the efferent system, and auditory perception. I discuss the results in the context of a model of tonotopic refinement and a new role of the efferent system during development

Mice Lacking the Alpha9 Subunit of the Nicotinic Acetylcholine Receptor Exhibit Deficits in Frequency Difference Limens and Sound Localization

Sound processing in the cochlea is modulated by cholinergic efferent axons arising from medial olivocochlear neurons in the brainstem. These axons contact outer hair cells in the mature cochlea and inner hair cells during development and activate nicotinic acetylcholine receptors composed of α9 and α10 subunits. The α9 subunit is necessary for mediating the effects of acetylcholine on hair cells as genetic deletion of the α9 subunit results in functional cholinergic de-efferentation of the cochlea. Cholinergic modulation of spontaneous cochlear activity before hearing onset is important for the maturation of central auditory circuits. In α9KO mice, the developmental refinement of inhibitory afferents to the lateral superior olive is disturbed, resulting in decreased tonotopic organization of this sound localization nucleus. In this study, we used behavioral tests to investigate whether the circuit anomalies in α9KO mice correlate with sound localization or sound frequency processing. Using a conditioned lick suppression task to measure sound localization, we found that three out of four α9KO mice showed impaired minimum audible angles. Using a prepulse inhibition of the acoustic startle response paradigm, we found that the ability of α9KO mice to detect sound frequency changes was impaired, whereas their ability to detect sound intensity changes was not. These results demonstrate that cholinergic, nicotinic α9 subunit mediated transmission in the developing cochlear plays an important role in the maturation of hearing

Expert Panel Curation of 113 Primary Mitochondrial Disease Genes for the Leigh Syndrome Spectrum

OBJECTIVE: Primary mitochondrial diseases (PMDs) are heterogeneous disorders caused by inherited mitochondrial dysfunction. Classically defined neuropathologically as subacute necrotizing encephalomyelopathy, Leigh syndrome spectrum (LSS) is the most frequent manifestation of PMD in children, but may also present in adults. A major challenge for accurate diagnosis of LSS in the genomic medicine era is establishing gene-disease relationships (GDRs) for this syndrome with >100 monogenic causes across both nuclear and mitochondrial genomes. METHODS: The Clinical Genome Resource (ClinGen) Mitochondrial Disease Gene Curation Expert Panel (GCEP), comprising 40 international PMD experts, met monthly for 4 years to review GDRs for LSS. The GCEP standardized gene curation for LSS by refining the phenotypic definition, modifying the ClinGen Gene-Disease Clinical Validity Curation Framework to improve interpretation for LSS, and establishing a scoring rubric for LSS. RESULTS: The GDR with LSS across the nuclear and mitochondrial genomes was classified as definitive for 31/114 gene-disease relationships curated (27%); moderate for 38 (33%); limited for 43 (38%); and 2 as disputed (2%). Ninety genes were associated with autosomal recessive inheritance, 16 were maternally inherited, 5 autosomal dominant, and 3 X-linked. INTERPRETATION: GDRs for LSS were established for genes across both nuclear and mitochondrial genomes. Establishing these GDRs will allow accurate variant interpretation, expedite genetic diagnosis of LSS, and facilitate precision medicine, multi-system organ surveillance, recurrence risk counselling, reproductive choice, natural history studies and eligibility for interventional clinical trials. This article is protected by copyright. All rights reserved

Clinical validity assessment of genes frequently tested on intellectual disability/autism sequencing panels.

[en] PURPOSE: Neurodevelopmental disorders (NDDs), such as intellectual disability (ID) and autism spectrum disorder (ASD), exhibit genetic and phenotypic heterogeneity, making them difficult to differentiate without a molecular diagnosis. The Clinical Genome Resource Intellectual Disability/Autism Gene Curation Expert Panel (GCEP) uses systematic curation to distinguish ID/ASD genes that are appropriate for clinical testing (ie, with substantial evidence supporting their relationship to disease) from those that are not. METHODS: Using the Clinical Genome Resource gene-disease validity curation framework, the ID/Autism GCEP classified genes frequently included on clinical ID/ASD testing panels as Definitive, Strong, Moderate, Limited, Disputed, Refuted, or No Known Disease Relationship. RESULTS: As of September 2021, 156 gene-disease pairs have been evaluated. Although most (75%) were determined to have definitive roles in NDDs, 22 (14%) genes evaluated had either Limited or Disputed evidence. Such genes are currently not recommended for use in clinical testing owing to the limited ability to assess the effect of identified variants. CONCLUSION: Our understanding of gene-disease relationships evolves over time; new relationships are discovered and previously-held conclusions may be questioned. Without periodic re-examination, inaccurate gene-disease claims may be perpetuated. The ID/Autism GCEP will continue to evaluate these claims to improve diagnosis and clinical care for NDDs

Transcriptional maturation of the mouse auditory forebrain

Background: The maturation of the brain involves the coordinated expression of thousands of genes, proteins and regulatory elements over time. In sensory pathways, gene expression profiles are modified by age and sensory experience in a manner that differs between brain regions and cell types. In the auditory system of altricial animals, neuronal activity increases markedly after the opening of the ear canals, initiating events that culminate in the maturation of auditory circuitry in the brain. This window provides a unique opportunity to study how gene expression patterns are modified by the onset of sensory experience through maturity. As a tool for capturing these features, next-generation sequencing of total RNA (RNAseq) has tremendous utility, because the entire transcriptome can be screened to index expression of any gene. To date, whole transcriptome profiles have not been generated for any central auditory structure in any species at any age. In the present study, RNAseq was used to profile two regions of the mouse auditory forebrain (A1, primary auditory cortex; MG, medial geniculate) at key stages of postnatal development (P7, P14, P21, adult) before and after the onset of hearing (~P12). Hierarchical clustering, differential expression, and functional geneset enrichment analyses (GSEA) were used to profile the expression patterns of all genes. Selected genesets related to neurotransmission, developmental plasticity, critical periods and brain structure were highlighted. An accessible repository of the entire dataset was also constructed that permits extraction and screening of all data from the global through single-gene levels. To our knowledge, this is the first whole transcriptome sequencing study of the forebrain of any mammalian sensory system. Although the data are most relevant for the auditory system, they are generally applicable to forebrain structures in the visual and somatosensory systems, as well. Results: The main findings were: (1) Global gene expression patterns were tightly clustered by postnatal age and brain region; (2) comparing A1 and MG, the total numbers of differentially expressed genes were comparable from P7 to P21, then dropped to nearly half by adulthood; (3) comparing successive age groups, the greatest numbers of differentially expressed genes were found between P7 and P14 in both regions, followed by a steady decline in numbers with age; (4) maturational trajectories in expression levels varied at the single gene level (increasing, decreasing, static, other); (5) between regions, the profiles of single genes were often asymmetric; (6) GSEA revealed that genesets related to neural activity and plasticity were typically upregulated from P7 to adult, while those related to structure tended to be downregulated; (7) GSEA and pathways analysis of selected functional networks were not predictive of expression patterns in the auditory forebrain for all genes, reflecting regional specificity at the single gene level. Conclusions: Gene expression in the auditory forebrain during postnatal development is in constant flux and becomes increasingly stable with age. Maturational changes are evident at the global through single gene levels. Transcriptome profiles in A1 and MG are distinct at all ages, and differ from other brain regions. The database generated by this study provides a rich foundation for the identification of novel developmental biomarkers, functional gene pathways, and targeted studies of postnatal maturation in the auditory forebrain. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1709-8) contains supplementary material, which is available to authorized users

Additional file 1: Figure S1. of Transcriptional maturation of the mouse auditory forebrain

Sample harvesting. Low magnification coronal images at the level of A1 and MG. (A) Gapdh in situ hybridization; (B) photograph of a frozen brain during harvesting of samples from A1 and MG for sequencing. The location of A1 within the auditory cortex (AC) is shown, along with a sketch of the 0.5 mm punch used to obtain samples. Note that the size and shape of the punch compresses tissue outside of the punched volume. The left MG has been circumscribed prior to extraction. Scale bars, 1 mm all panels

Additional file 3: of Transcriptional maturation of the mouse auditory forebrain

Differential expression comparing A1 and MG at at P7, P14, P21, and Adult. Table S5. Differential expression analyses comparing MG with A1 at P7 for all genes using DESeq2, EdgeR, and BaySeq. For each gene, the log2 fold-change, p-values, and rank are listed. The final column (All Rank) is the sum of rankings obtained by all three methods. Gene listing is given in. Table S6. Differential expression analyses comparing MG with A1 at P14 for all genes using DESeq2, EdgeR, and BaySeq. For each gene, the log2 fold-change, p-values, and rank are listed. The final column (All Rank) is the sum of rankings obtained by all three methods (lowest number = highest rank). Gene listing is ordered from highest to lowest rank. Table S7. Differential expression analyses comparing MG with A1 at P21 for all genes using DESeq2, EdgeR, and BaySeq. For each gene, the log2 fold-change, p-values, and rank are listed. The final column (All Rank) is the sum of rankings obtained by all three methods (lowest number = highest rank). Gene listing is ordered from highest to lowest rank. Table S8. Differential expression analyses comparing MG with A1 in adult animals for all genes using DESeq2, EdgeR, and BaySeq. For each gene, the log2 fold-change, p-values, and rank are listed. The final column (All Rank) is the sum of rankings obtained by all three methods (lowest number = highest rank). Gene listing is ordered from highest to lowest rank

Additional file 2: Table S1. of Transcriptional maturation of the mouse auditory forebrain

Population and quality control data for all A1 and MG samples obtained for total RNA sequencing. For each sample, the following information is provided: region of interest (ROI), age, sex, RNA integrity number (RIN), 28 s:18 s ratio, 260/280 ratio. Samples are arranged sequentially by an identifier that is used in all other tables. Table S2. Raw data quality control matrix. Contains information about the sequencing platform, total reads, and sample quality measures. Table S3. Quality control of alignments. For each sample, information about the sequencing platform and mapping details are listed. Table S4. Raw counts of all genes for the 48 samples sequenced