Project description:Sound localization requires extremely precise development of auditory brainstem circuits, the molecular mechanisms of which are largely unknown. We previously demonstrated a novel requirement for non-apoptotic activity of the protease caspase-3 in chick auditory brainstem development. Here, we used mass spectrometry to identify proteolytic substrates of caspase-3 during chick auditory brainstem development. Functional annotation analysis revealed that our caspase-3 substrates were enriched more than two-fold for proteins associated with extracellular vesicles (EVs), membrane-bound nanoparticles that function in intercellular communication. The proteome of EVs isolated from the auditory brainstem contained caspase-3 and was highly enriched for the caspase-3 substrates identified here. Additionally, we identified two caspase-3 substrates with known functions in axon guidance, namely Neural Cell Adhesion Molecule (NCAM) and Neuronal-glial Cell Adhesion Molecule (Ng-CAM), that were found in auditory brainstem EVs and expressed in the auditory pathway alongside cleaved caspase-3. Taken together, these data suggest a novel developmental mechanism whereby caspase-3 influences auditory brainstem circuit formation through the proteolytic cleavage of EV proteins.
Project description:Genome-wide gene expression was obtained in three auditory brainstem nuclei (defined below), at two different ages in mice, postnatal day (P)3 and P14. The primary aim was to identify genes which are differentially expressed between the medial nucleus of the trapezoid body (MNTB) and the superior olive (LSO), at both age groups. Tissue samples from the ventral cochlear nucleus (VCN), the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO) were collected from 200 Î¼m thick fresh transverse mouse brainstem slices prepared with a vibratome tissue slicer. Individual brainstem nuclei were dissected out with fine forceps under optical control with a binocular. For each nucleus (VCN, MNTB, and LSO), the tissue from six male C57Bl6 mice at postnatal day (P) 3, and from three male C57Bl6 mice at P14 was pooled. The RNA was isolated with the RNeasy Micro kit (Qiagen); RNA samples were amplified using a WT ovation Pico RNA amplification (Nugen, CA, USA). The cDNA was hybridized onto mouse Affymetrix microarrays 430 2.0 (Affymetrix, Santa Clara, USA).
Project description:N-methyl-D-aspartate (NMDA) receptor subunit-specific probes were used to characterize developmental changes in the distribution of excitatory amino acid receptors in the chicken's auditory brainstem nuclei. Although NR1 subunit expression does not change greatly during the development of the cochlear nuclei in the chicken (Tang and Carr  Hear. Res 191:79-89), there are significant developmental changes in NR2 subunit expression. We used in situ hybridization against NR1, NR2A, NR2B, NR2C, and NR2D to compare NR1 and NR2 expression during development. All five NMDA subunits were expressed in the auditory brainstem before embryonic day (E) 10, when electrical activity and synaptic responses appear in the nucleus magnocellularis (NM) and the nucleus laminaris (NL). At this time, the dominant form of the receptor appeared to contain NR1 and NR2B. NR2A appeared to replace NR2B by E14, a time that coincides with synaptic refinement and evoked auditory responses. NR2C did not change greatly during auditory development, whereas NR2D increased from E10 and remained at fairly high levels into adulthood. Thus changes in NMDA NR2 receptor subunits may contribute to the development of auditory brainstem responses in the chick.
Project description:Genome-wide gene expression was obtained in three auditory brainstem nuclei (defined below), at two different ages in mice, postnatal day (P)3 and P14. The primary aim was to identify genes which are differentially expressed between the medial nucleus of the trapezoid body (MNTB) and the superior olive (LSO), at both age groups. Tissue samples from the ventral cochlear nucleus (VCN), the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO) were collected from 200 μm thick fresh transverse mouse brainstem slices prepared with a vibratome tissue slicer. Individual brainstem nuclei were dissected out with fine forceps under optical control with a binocular. For each nucleus (VCN, MNTB, and LSO), the tissue from six male C57Bl6 mice at postnatal day (P) 3, and from three male C57Bl6 mice at P14 was pooled. The RNA was isolated with the RNeasy Micro kit (Qiagen); RNA samples were amplified using a WT ovation Pico RNA amplification (Nugen, CA, USA). The cDNA was hybridized onto mouse Affymetrix microarrays 430 2.0 (Affymetrix, Santa Clara, USA).
Project description:Lysosome-associated membrane protein 5 (LAMP5) is a mammalian ortholog of the Caenorhabditis elegans protein, UNC-46, which functions as a sorting factor to localize the vesicular GABA transporter UNC-47 to synaptic vesicles. In the mouse forebrain, LAMP5 is expressed in a subpopulation of GABAergic neurons in the olfactory bulb and the striato-nigral system, where it is required for fine-tuning of GABAergic synaptic transmission. Here we focus on the prominent expression of LAMP5 in the brainstem and spinal cord and suggest a role for LAMP5 in these brain regions. LAMP5 was highly expressed in several brainstem nuclei involved with auditory processing including the cochlear nuclei, the superior olivary complex, nuclei of the lateral lemniscus and grey matter in the spinal cord. It was localized exclusively in inhibitory synaptic terminals, as has been reported in the forebrain. In the absence of LAMP5, localization of the vesicular inhibitory amino acid transporter (VIAAT) was unaltered in the lateral superior olive and the ventral cochlear nuclei, arguing against a conserved role for LAMP5 in trafficking VIAAT. Lamp5 knockout mice showed no overt behavioral abnormality but an increased startle response to auditory and tactile stimuli. In addition, LAMP5 deficiency led to a larger intensity-dependent increase of wave I, II and V peak amplitude of auditory brainstem response. Our results indicate that LAMP5 plays a pivotal role in sensorimotor processing in the brainstem and spinal cord.
Project description:Atoh1 is a basic helix-loop-helix transcription factor necessary for the specification of inner ear hair cells and central auditory system neurons derived from the rhombic lip. We used the Cre-loxP system and two Cre-driver lines (Egr2(Cre) and Hoxb1(Cre)) to delete Atoh1 from different regions of the cochlear nucleus (CN) and accessory auditory nuclei (AAN). Adult Atoh1-conditional knock-out mice (Atoh1(CKO)) are behaviorally deaf, have diminished auditory brainstem evoked responses, and have disrupted CN and AAN morphology and connectivity. In addition, Egr2; Atoh1(CKO) mice lose spiral ganglion neurons in the cochlea and AAN neurons during the first 3 d of life, revealing a novel critical period in the development of these neurons. These new mouse models of predominantly central deafness illuminate the importance of the CN for support of a subset of peripheral and central auditory neurons.
Project description:During development of the CNS, secreted morphogens of the fibroblast growth factor (FGF) family have multiple effects on cell division, migration, and survival depending on where, when, and how much FGF signal is received. The consequences of misregulating the FGF pathway were studied in a mouse with decreased levels of the FGF antagonist Sef. To uncover effects in the nervous system, we focused on the auditory system, which is accessible to physiological analysis. We found that the mitogen-activated protein kinase pathway is active in the rhombic lip, a germinal zone that generates diverse types of neurons, including the cochlear nucleus complex of the auditory system. Sef is expressed immediately adjacent to the rhombic lip, overlapping with FGF15 and FGFR1, which is also present in the lip itself. This pattern suggests that Sef may normally function in non-rhombic lip cells and prevent them from responding to FGF ligand in the vicinity. Consistent with this idea, overexpression of Sef in chicks decreased the size of the auditory nuclei. Cochlear nucleus defects were also apparent in mice with reduced levels of Sef, with 13% exhibiting grossly dysmorphic cochlear nuclei and 26% showing decreased amounts of GFAP in the cochlear nucleus. Additional evidence for cochlear nucleus defects was obtained by electrophysiological analysis of Sef mutant mice, which have normal auditory thresholds but abnormal auditory brainstem responses. These results show both increases and decreases in Sef levels affect the assembly and function of the auditory brainstem.
Project description:Glutamate is the main excitatory neurotransmitter in the auditory system, but associations between glutamatergic neuronal populations and the distribution of their synaptic terminations have been difficult. Different subsets of glutamatergic terminals employ one of three vesicular glutamate transporters (VGLUT) to load synaptic vesicles. Recently, VGLUT1 and VGLUT2 terminals were found to have different patterns of organization in the inferior colliculus, suggesting that there are different types of glutamatergic neurons in the brainstem auditory system with projections to the colliculus. To positively identify VGLUT-expressing neurons as well as inhibitory neurons in the auditory brainstem, we used in situ hybridization to identify the mRNA for VGLUT1, VGLUT2, and VIAAT (the vesicular inhibitory amino acid transporter used by GABAergic and glycinergic terminals). Similar expression patterns were found in subsets of glutamatergic and inhibitory neurons in the auditory brainstem and thalamus of adult rats and mice. Four patterns of gene expression were seen in individual neurons. 1) VGLUT2 expressed alone was the prevalent pattern. 2) VGLUT1 coexpressed with VGLUT2 was seen in scattered neurons in most nuclei but was common in the medial geniculate body and ventral cochlear nucleus. 3) VGLUT1 expressed alone was found only in granule cells. 4) VIAAT expression was common in most nuclei but dominated in some. These data show that the expression of the VGLUT1/2 and VIAAT genes can identify different subsets of auditory neurons. This may facilitate the identification of different components in auditory circuits.
Project description:Rhombomeres (r) contribute to brainstem auditory nuclei during development. Hox genes are determinants of rhombomere-derived fate and neuronal connectivity. Little is known about the contribution of individual rhombomeres and their associated Hox codes to auditory sensorimotor circuitry. Here, we show that r4 contributes to functionally linked sensory and motor components, including the ventral nucleus of lateral lemniscus, posterior ventral cochlear nuclei (VCN), and motor olivocochlear neurons. Assembly of the r4-derived auditory components is involved in sound perception and depends on regulatory interactions between Hoxb1 and Hoxb2. Indeed, in Hoxb1 and Hoxb2 mutant mice the transmission of low-level auditory stimuli is lost, resulting in hearing impairments. On the other hand, Hoxa2 regulates the Rig1 axon guidance receptor and controls contralateral projections from the anterior VCN to the medial nucleus of the trapezoid body, a circuit involved in sound localization. Thus, individual rhombomeres and their associated Hox codes control the assembly of distinct functionally segregated sub-circuits in the developing auditory brainstem.
Project description: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.