Transcription profiling of rat calyceal vestibular primary afferent neurons (calretinin positive) and dimorphic-bouton vestibular primary afferent neurons (calretinin negative)
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ABSTRACT: Pooled Immunoflourescent guided laser capture microdisection of calyceal vestibular primary afferent neurons (calretinin positive) and dimorphic-bouton vestibular primary afferent neurons (calretinin negative) were used for microarray expression profiling. Transcription analysis of each of these biologically diverse pools was completed.
Project description:Using laser capture microdisection, the more superficial layer of the cristae ampullaris sensory epithelium encompassing types I and II hair cells, and the supporting cell cytoplasm interspersed between them was captured in the first pass, and the underlying supporting cell bodies resting on the basal lamina were captured in a second pass. Individual hair cells were identified under light microscopy at 200X magnification by their stereociliary tuft and the calyceal nerve ending surrounding them and the presence of their nucleus in the outermost portion of the epithelia.
Project description:The functional diversity of neuropeptides and/or their receptors may provide a complex means to modulate vestibular primary afferent neuronal function. The precise role of these neuropeptides in the physiology of the vestibular neuroepithelium is poorly understood. The vestibular caliceal afferent neurons when compared to the dimorphic and bouton afferent neurons have different functional properties. Immunofluorescent laser capture microdissection utilizing calretinin (calbindin 2, Calb2) antibodies allowed selective acquisition of these two primary afferent neuronal populations from the rat (Rattus norvegicus). After capturing the caliceal afferent neurons, as well as the dimorphic and bouton afferent neurons, microarray expression profiling was completed. Analysis of the resulting data revealed that there were 732 genes involved in synaptic/signaling, calcium binding/solute transport, and other. Of those genes, 52 were related to afferent modulation, including 21 genes representing neuropeptides or their receptors. The observed expression of 52 genes related to afferent modulation identified using microarray were confirmed using PCR from the Wackym-Soares normalized rat vestibular periphery cDNA library or by RT-PCR from fresh ganglia tissue. The majority of the neuropeptides and/or receptors were found to be expressed in both groups, although there were neuropeptides and/or receptors that were unique to the dimorphic and bouton afferent neuron pool. The expression of selected neuropeptides or their receptors was confirmed using immunohistochemistry in the crista ampullares. Our results suggest that the unexpected neuropeptide diversity and their differences in expression pattern could serve unique roles in the physiology of the vestibular neuroepithelium. Keywords: expression profiling, neuropeptides, vestibular neuroepithelium, calretinin
Project description:The spontaneous mutant Bronx waltzer (bv) mouse line is characterized by deafness and balance defect. We located the bv mutation to the Srrm4 gene which encodes a regulator of alternative pre-mRNA splicing. We found that Srrm4 is expressed in balance and hearing organs (i.e. in the vestibular maculas and the cochlea). Srrm4 is also expressed in the central nervous system including the cerebellum. To identify potential splicing defects in bv/bv mice, we analyzed RNA samples from the vestibular maculas and cerebellums of bv/bv mice and control (bv/+) littermates, using mouse exon junction microarrays (MJAY). In this dataset, we include probe-set level data obtained from vestibular macula samples. The processed data represent probe-set intensities that have been normalized to gene expression levels (Inorm). Inorm was calculated using batch-corrected data as well as data that were not corrected for a batch effect. 7 total samples were analyzed: vestibular maculas from 4 heterozygous (bv/+) and 3 homozygous (bv/bv) mouse embryos at E16.5.
Project description:Microarray analysis was used to examine the expression of genes upregulated or downregulated in the ipsilateral vestibular nucleus at 1 and 7 days following unilateral labyrinthectomy. Changes in gene expression during the chronic phase of vestibular compensation following unilateral labyrinthectomy in rats Three conditioned experiment: nl:control with sham operation, 1day:1day after labyrninthectomy, 7day:7day after labyrinthectomy
Project description:To investigate which miRNAs regulate the vestibular compensation after unilateral vestibular deafferentiation (UVD), we have performed microarray for miRNAs as a discovery platform. UVD induces breakdown of the activity of ipsilesional vestibular nuclei and an unbalance of activity between bilateral vestibular nuclei. Vestibular compensation is a course of rebalancing of activities of bilateral vestibular nuclei. It takes place mainly in medial vestibular nucleus. This study was performed using seven week-old-male Sprague–Dawley rats. Based on our previous experiment about vestibular compensation course, we set two time points for harvesting medial vestibular nuclei: 4hr and 4 days after unilateral vestibular deafferentiation. Twenty four animals were divided into two experimental groups: UVD group undergoing UVD at left side (n = 12); and SO group undergoing sham operation (SO) at left side (n=12). Six animals of each group were anesthetized deeply and euthanized at 4 hr or 4 days after surgery, respectively. Medial vestibular nucleus at left side was harvested. Medial vestibular nucleus from three animals became one sample for microarray. Sequentially two samples were obtained for each time point in one group. Microarray for miRNAs was performed using the Agilent Rat miRNA Microarray 8x15K platforms. Considering the fold change of normalized signal intensities between two time points in UVD group and between UVD and SO groups at the same time, miR-31a-5p, 133a-3p, 133b-3p, 204-5p, 206-3p, 218a-5p, 219a-5p, 221-3p and 497-5p were selected as the candidate miRNAs. This result was validated by quantitative reverse transcription-PCR.
Project description:Vestibular Schwannomas are benign neoplasms that arise from the vestibular nerve. The hallmark of these tumors is the biallelic inactivation of NF2. Transcriptomic alterations, such as the Nrg1/ErbB2 pathway, have been described in Schwannomas. Here, we have performed a whole transcriptomic analysis in 31 vestibular Schwannomas and 9 control nerves in the Affymetrix Gene 1.0ST platform, validated by quantitative Real-Time PCR using TaqMan Low Density Arrays. We performed a mutational analysis of NF2 by PCR/dHPLC and MLPA as well as a microsatellite marker analysis of the loss of heterozygosity of chromosome 22q. The microarray analysis showed that 1516 genes were deregulated, and 48 of the genes were validated by qRT-PCR. At least two genetic hits (allelic loss and/or gene mutation) in NF2 were found in 16 tumors, seven cases showed one hit and eight tumors showed no NF2 alteration. As conclusion, MET and associated genes such as ITGA4/B6, PLEXNB3/SEMA5 and CAV1 showed a clear deregulation in vestibular Schwannomas. In addition, androgen receptor (AR) downregulation may denote a hormonal effect or cause in this tumor. Furthermore, the osteopontin gene (SPP1), which is involved in Merlin protein degradation, was upregulated, which suggests that this mechanism may also exert a pivotal role in Schwannoma Merlin depletion. Finally, no major differences were found between tumors of different sizes, histological types or NF2 status, which suggests that at the mRNA level all Schwannomas, regardless of molecular and clinical characteristics, may share common features that can be used in the fight against them. In order to find target to fight against vestibular schwannoma, we performed an analysis of gene expression by microarrays.
Project description:Vestibular schwannoma is the most common benign neoplasm of the cerebellopontine angle. Its first symptoms include hearing loss, tinnitus, and vestibular symptoms, followed by cerebellar and brainstem symptoms, along with palsy of the adjacent cranial nerves. However, the clinical picture has unpredictable dynamics and currently, there are no reliable predictors of tumour behaviour. The major objective of this study was to verify whether a technique using in-sample specific protein digestion with trypsin would have the potential to provide proteomic characterisation of these pathological tissues.
Project description:Cerebrospinal fluid (liquor) samples (N = 44) have been derived from patients with vestibular schwannoma that comprises about 10% of all intracranial tumors. Applying high resolution tandem mass-spectrometry 525 proteins were identified with high confidence (at least 2 peptides per protein, FDR <1%) in the liquor samples. This dataset provides unique information on proteomic composition of vestibular schwannoma liquor samples.
Project description:Microarray analysis was used to examine the expression of genes upregulated or downregulated in the ipsilateral vestibular nucleus at 1 and 7 days following unilateral labyrinthectomy. Changes in gene expression during the chronic phase of vestibular compensation following unilateral labyrinthectomy in rats