Harvest of Vestibular End-Organs under Physiologic Conditions during Labyrinthectomy.

The living human inner ear is challenging to study because it is encased within dense otic capsule bone that limits access to biological tissue. Traditional temporal bone histopathology methods rely on lengthy, expensive decalcification protocols that take 9-10 months and reduce the types of tissue analysis possible due to RNA degradation. There is a critical need to develop methods to access fresh human inner ear tissue to better understand otologic diseases, such as Ménière's disease, at the cellular and molecular level.

A Benchtop Round Window Model for Studying Magnetic Nanoparticle Transport to the Inner Ear.

Introduction: The round window membrane (RWM) presents a significant barrier to the local application of therapeutics to the inner ear. We demonstrate a benchtop preclinical RWM model and evaluate superparamagnetic iron oxide nanoparticles (SPIONs) as vehicles for magnetically assisted drug delivery.

Methods: Guinea pig RWM explants were inset into a 3D-printed dual chamber benchtop device.

Engineering olivocochlear inhibition to reduce acoustic trauma.

Efferent brain-stem neurons release acetylcholine to desensitize cochlear hair cells and can protect the inner ear from acoustic trauma. That protection is absent from knockout mice lacking efferent inhibition and is stronger in mice with a gain-of-function point mutation of the hair cell-specific nicotinic acetylcholine receptor. The present work uses viral transduction of gain-of-function receptors to restore acoustic prophylaxis to the knockout mice.

Characterization of HA-tagged α9 and α10 nAChRs in the mouse cochlea.

Neurons of the medial olivary complex inhibit cochlear hair cells through the activation of α9α10-containing nicotinic acetylcholine receptors (nAChRs). Efforts to study the localization of these proteins have been hampered by the absence of reliable antibodies. To overcome this obstacle, CRISPR-Cas9 gene editing was used to generate mice in which a hemagglutinin tag (HA) was attached to the C-terminus of either α9 or α10 proteins.

Transplantation of human oligodendrocyte progenitor cells in an animal model of diffuse traumatic axonal injury: survival and differentiation.

Introduction: Diffuse axonal injury is an extremely common type of traumatic brain injury encountered in motor vehicle crashes, sports injuries, and in combat. Although many cases of diffuse axonal injury result in chronic disability, there are no current treatments for this condition. Its basic lesion, traumatic axonal injury, has been aggressively modeled in primate and rodent animal models.

Synaptic calcium regulation in hair cells of the chicken basilar papilla.

Cholinergic inhibition of hair cells occurs by activation of calcium-dependent potassium channels. A near-membrane postsynaptic cistern has been proposed to serve as a store from which calcium is released to supplement influx through the ionotropic ACh receptor. However, the time and voltage dependence of acetylcholine (ACh)-evoked potassium currents reveal a more complex relationship between calcium entry and release from stores.

Ultrastructure of cisternal synapses on outer hair cells of the mouse cochlea.

C (cisternal) synapses with a near membrane postsynaptic cistern are found on motor neurons and other central neurons, where their functional role is unknown. Similarly structured cisternal synapses mediate cholinergic inhibition of cochlear hair cells via α9α10-containing ionotropic receptors and associated calcium-activated (SK2) potassium channels, providing the opportunity to examine the ultrastructure of genetically altered cisternal synapses. Serial section electron microscopy was used to examine efferent synapses of outer hair cells (OHCs) in mice with diminished or enhanced cholinergic inhibition.

Synaptic transfer from outer hair cells to type II afferent fibers in the rat cochlea.

Type II cochlear afferents receive glutamatergic synaptic excitation from outer hair cells (OHCs) in the rat cochlea. However, it remains uncertain whether this connection is capable of providing auditory information to the brain. The functional efficacy of this connection depends in part on the number of presynaptic OHCs, their probability of transmitter release, and the effective electrical distance for spatial summation in the type II fiber.

Constitutive expression of the alpha10 nicotinic acetylcholine receptor subunit fails to maintain cholinergic responses in inner hair cells after the onset of hearing.

Efferent inhibition of cochlear hair cells is mediated by alpha9alpha10 nicotinic cholinergic receptors (nAChRs) functionally coupled to calcium-activated, small conductance (SK2) potassium channels. Before the onset of hearing, efferent fibers transiently make functional cholinergic synapses with inner hair cells (IHCs). The retraction of these fibers after the onset of hearing correlates with the cessation of transcription of the Chrna10 (but not the Chrna9) gene in IHCs.

In situ hybridization approach to study mRNA expression and distribution in cochlear frozen sections.

In situ hybridization is well suited to obtaining specific topological information on gene transcripts and thereby to relating such observations to a particular function. In spite of the technical and practical difficulties, the application of molecular biological techniques such as in situ hybridization to the cochlea can provide important insights. However, the rarity of gene products (mRNA and proteins) in the cochlea and its fragile structure require the refinement and adaptation of in situ hybridization methods.

Calcium-dependent inactivation of calcium channels in cochlear hair cells of the chicken.

Voltage-gated calcium channels support both spontaneous and sound-evoked neurotransmitter release from ribbon synapses of cochlear hair cells. A variety of regulatory mechanisms must cooperate to ensure the appropriate level of activity in the restricted pool of synaptic calcium channels ( approximately 100) available to each synaptic ribbon. One potential feedback mechanism, calcium-dependent inactivation (CDI) of voltage-gated, L-type calcium channels, can be modulated by calmodulin-like calcium-binding proteins.

Progressive deafness and altered cochlear innervation in knock-out mice lacking prosaposin.

After a yeast two-hybrid screen identified prosaposin as a potential interacting protein with the nicotinic acetylcholine receptor (nAChR) subunit alpha10, studies were performed to characterize prosaposin in the normal rodent inner ear. Prosaposin demonstrates diffuse organ of Corti expression at birth, with gradual localization to the inner hair cells (IHCs) and its supporting cells, inner pillar cells, and synaptic region of the outer hair cells (OHCs) and Deiters' cells (DCs) by postnatal day 21 (P21). Microdissected OHC and DC quantitative reverse transcriptase-PCR and immunohistology localizes prosaposin mRNA to DCs and OHCs, and protein predominantly to the apex of the DCs.

Alternative splicing of the Ca(v)1.3 channel IQ domain, a molecular switch for Ca2+-dependent inactivation within auditory hair cells.

Native Ca(V)1.3 channels within cochlear hair cells exhibit a surprising lack of Ca2+-dependent inactivation (CDI), given that heterologously expressed Ca(V)1.3 channels show marked CDI.

Switching of Ca2+-dependent inactivation of Ca(v)1.3 channels by calcium binding proteins of auditory hair cells.

Ca(V)1.3 channels comprise a vital subdivision of L-type Ca2+ channels: Ca(V)1.3 channels mediate neurotransmitter release from auditory inner hair cells (IHCs), pancreatic insulin secretion, and cardiac pacemaking.

The glutamate-aspartate transporter GLAST mediates glutamate uptake at inner hair cell afferent synapses in the mammalian cochlea.

Ribbon synapses formed between inner hair cells (IHCs) and afferent dendrites in the mammalian cochlea can sustain high rates of release, placing strong demands on glutamate clearance mechanisms. To investigate the role of transporters in glutamate removal at these synapses, we made whole-cell recordings from IHCs, afferent dendrites, and glial cells adjacent to IHCs [inner phalangeal cells (IPCs)] in whole-mount preparations of rat organ of Corti. Focal application of the transporter substrate D-aspartate elicited inward currents in IPCs, which were larger in the presence of anions that permeate the transporter-associated anion channel and blocked by the transporter antagonist D,L-threo-beta-benzyloxyaspartate.

A "synaptoplasmic cistern" mediates rapid inhibition of cochlear hair cells.

Cochlear hair cells are inhibited by cholinergic efferent neurons. The acetylcholine (ACh) receptor of the hair cell is a ligand-gated cation channel through which calcium enters to activate potassium channels and hyperpolarize the cell. It has been proposed that calcium-induced calcium release (CICR) from a near-membrane postsynaptic store supplements this process.

Characterization of the human nicotinic acetylcholine receptor subunit alpha (alpha) 9 (CHRNA9) and alpha (alpha) 10 (CHRNA10) in lymphocytes.

Though the nicotinic acetylcholine receptor (nAChR) subunits alpha9 and alpha 10 have been thoroughly characterized within hair cells of the organ of Corti in the inner ear, prior studies have shown that they are also expressed in lymphocytes. In this report, we sought to more definitively characterize the nAChR subunits alpha9 and alpha10 within various populations of human lymphocytes. Using a combination of techniques, including RT-PCR, single-cell RT-PCR, Northern and western blot analysis, and immunofluorescence, expression of both alpha9 and alpha 10 was demonstrated in purified populations of T-cells (CD3+, CD4+, CD8+ and the Jurkat, MT2 and CEM T-cell lines) and B-cells (CD19+, CD80+ and EBV-immortalized B-cells).

PHR1, a PH domain-containing protein expressed in primary sensory neurons.

Previously, we identified PHR1 as an abundantly expressed gene in photoreceptors and showed that it encodes four isoforms, each with N-terminal pleckstrin homology (PH) and C-terminal transmembrane domains. To better understand PHR1 function and expression, we made a Phr1 null mouse by inserting a beta-galactosidase/neor cassette into exon 3. In addition to photoreceptors, we found abundant expression of specific Phr1 splice forms in olfactory receptor neurons and vestibular and cochlear hair cells.

Topological and developmental gradients of calbindin expression in the chick's inner ear.

Mobile intracellular calcium buffers play an important role in regulating calcium flux into mechanosensory hair cells and calbindin D-28k is expressed at high levels in the chick's basilar papilla. We have used RT-PCR, in situ hybridization, and immunohistology to demonstrate that calbindin expression varies systematically according to hair cell position and developmental age. RT-PCR using microdissected quarters of the posthatch basilar papilla showed that mRNA levels were lowest in the (low frequency) apex and higher in basal quadrants.