Dennis G. Drescher

Effect of sound stimulation at several levels on concentrations of primary amines, including neurotransmitter candidates, in perilymph of the guinea pig inner ear

Abstract: Perilymph, which bathes the sensory cells of the cochlea, was collected from guinea pigs exposed to noise and analyzed via two cation‐exchange HPLC procedures with fluorescence detection, resolving 51 and 81 primary‐amine compounds, respectively, at a sensitivity limit of 0.1 pmol relative to leucine. During a first period, each animal was either exposed to noise at 80, 90, or 115 decibels sound‐pressure level or maintained in silence (controls), and during a second period, the same animal was maintained in silence. Perilymph was collected during both periods, and perilymphatic components were compared, within animals and across animals, for several levels of sound stimulation. A 7‐aminobutyric acid‐like component was elevated in the first period in proportion to stimulus intensity by the various methods of comparison, suggesting an auditory‐neurotransmitter role for this component. Aspartic acid was elevated in the second period, 2–3.5 h after onset of sound stimulation, compatible with the release of aspartic acid from central auditory synapses. In addition, a methionine‐enkephalin‐like component, distinct from leucine‐enkephalin, was detected in perilymph from control animals and was elevated in response to noise at 115 decibels. Regression coefficients, determined for the relation between sound intensity and first‐period concentrations or the difference between first and second‐period concentrations, indicated zero linear regression at p = 0.05 for glutamic acid, aspartic acid, glycine, taurine, and 39 other perilymphatic components, consistent with the hypothesis that these compounds are unlikely to be peripheral auditory neurotransmitters.

Direct Interaction of Otoferlin with Syntaxin 1A, SNAP-25, and the L-type Voltage-gated Calcium Channel CaV1.3

The molecular mechanisms underlying synaptic exocytosis in the hair cell, the auditory and vestibular receptor cell, are not well understood. Otoferlin, a C2 domain-containing Ca2+-binding protein, has been implicated as having a role in vesicular release. Mutations in the OTOF gene cause nonsyndromic deafness in humans, and OTOF knock-out mice are deaf. In the present study, we generated otoferlin fusion proteins containing two of the same amino acid substitutions detected in DFNB9 patients (P1825A in C2F and L1011P in C2D). The native otoferlin C2F domain bound syntaxin 1A and SNAP-25 in a Ca2+-dependent manner (with optimal 61 μm free Ca2+ required for binding). These interactions were greatly diminished for C2F with the P1825A mutation, possibly because of a reduction in tertiary structural change, induced by Ca2+, for the mutated C2F compared with the native C2F. The otoferlin C2D domain also bound syntaxin 1A, but with weaker affinity (Kd = 1.7 × 10–5 m) than for the C2F interaction (Kd = 2.6 × 10–9 m). In contrast, it was the otoferlin C2D domain that bound the Cav1.3 II-III loop, in a Ca2+-dependent manner. The L1011P mutation in C2D rendered this binding insensitive to Ca2+ and considerably diminished. Overall, we demonstrated that otoferlin interacts with two main target-SNARE proteins of the hair-cell synaptic complex, syntaxin 1A and SNAP-25, as well as the calcium channel, with the otoferlin C2F and C2D domains of central importance for binding. Because mutations in the otoferlin C2 domains that cause deafness in humans impair the ability of otoferlin to bind syntaxin, SNAP-25, and the Cav1.3 calcium channel, it is these interactions that may mediate regulation by otoferlin of hair cell synaptic exocytosis critical to inner ear hair cell function.

Identification of the subunits of the nicotinic cholinergic receptors in the rat cochlea using RT-PCR and in situ hybridization.

There are two tissues in the adult mammalian cochlea that are post-synaptic to cholinergic efferent fibers: The outer hair cells (OHCs) and the dendrites of the afferent fibers of the type I spiral ganglion cells. The unusual nicotinic-like pharmacology of cochlear cholinergic responses and the unique embryonic development of cochlear tissues suggest that the inner-ear nicotinic cholinergic receptor (nAChR) may be different from nAChRs described previously at synapses in the mammalian brain, autonomic ganglia, or skeletal muscle. In this study, we determined the mRNA expression of the alpha2-7, alpha9, and beta2-4 subunits of the nicotinic acetylcholine receptor (nAChR) family in the rat cochlea. In micro-dissected tissue from the organ of Corti, spiral ganglion, and the membranous lateral wall, we found mRNA expression of the alpha7 and alpha9 subunits in the organ of Corti and alpha5-7, and beta2 and beta3 in the spiral ganglion using RT-PCR. Employing in situ hybridization with 35S-riboprobes, we localized alpha9 in hair cells regions and alpha6, alpha7 and beta2 in the type I cells of the spiral ganglion. No evidence of nAChR subunit mRNA expression was found in supporting cells, but beta2 was expressed in type II spiral ganglion cells, which are neither cholinergic nor cholinoceptive.

Expression of nicotinic acetylcholine receptor mRNA in the adult rat peripheral vestibular system

The mRNA expression of the neuronal nicotinic acetylcholine receptor subunits was determined in adult rat vestibular end-organs and in Scarpas ganglion (SCG) by in situ hybridization with [35S] riboprobes. Neurons in the SCG expressed the alpha 4-7 and beta 2-3 mRNAs, but not alpha 3 or beta 4 mRNAs. Not all SCG neurons expressed every mRNA found in SCG. The alpha 6 and beta 2-3 riboprobes labeled all neurons, but alpha 4, alpha 5, and alpha 7 mRNAs were selectively expressed in one or more subpopulations of SCG neurons. Vestibular sensory hair cells, in contrast, expressed only alpha 9 mRNA.

Aldosterone mediates an increase in [3H]ouabain binding at Na+,K+-ATPase sites in the mammalian inner ear

Na+,K(+)-ATPase has been implicated in the maintenance of high [K+], low [Na+] in endolymph of the inner ear, ionic properties considered to support transduction by the receptor cells. In exocrine ion-transporting epithelia, Na+,K(+)-ATPase activity is modulated by aldosterone, a mineralocorticoid hormone. In the present study, the effect of alteration of serum aldosterone levels on Na+,K(+)-ATPase in ion-transporting regions of the mammalian inner ear was investigated. A high Na+/low K+ diet offered ad libitum for 5 days was utilized to significantly decrease serum aldosterone in male Hartley guinea pigs compared to controls. An injection of aldosterone (10 micrograms/100 g b.wt.) 21 h prior to sacrifice resulted in significant elevation of serum aldosterone over that obtained with the high Na+/low K+ diet. Binding of [3H]ouabain, a specific inhibitor of Na+,K(+)-ATPase, was significantly elevated in microdissected lateral wall of the basal turn of the cochlea and in the ampulla of the semicircular canal, for aldosterone-injected vs. vehicle-injected animals. Serum [Na+] and [Cl-] were elevated in animals on the high Na+/low K+ diet and unaltered by administration of exogenous aldosterone. The enhancement of ouabain binding in inner ear tissues observed in aldosterone-injected animals, therefore, did not appear to reflect an alteration of serum electrolytes per se. The results of these experiments are consistent with the hypothesis that aldosterone increases the number of Na+,K(+)-ATPase sites in ion-transporting epithelia of the mammalian cochlea and semicircular canal.

Calcium channel subunits in the mouse cochlea.

Abstract: Messages for subunits of voltage‐gated calcium channels were examined in the cochlea of the CBAJ mouse by PCR analysis. Total RNA was extracted from the auditory organs of 16–18‐day‐old animals. After reverse transcription, resulting cDNA was amplified by PCR with primers targeted to nucleotide sequences corresponding to 12 different calcium channel subunits. PCR products representing subunit gene expression were strongly and consistently amplified for α1C, α1D, α1E, α2δ, β1, β3, and β4 but not for α1A, α1B, α1S, β2, or γ. The chosen primers amplified cochlear cDNA to yield an overall pattern of bands different from that of any tissue studied thus far, in particular with respect to the α2δ and β1 subunits; the α2δ product was found to be significantly shorter than the corresponding brain and skeletal muscle isoforms. Nucleotide sequencing confirmed the identity of mouse cochlear subunit cDNAs. The results suggest that L‐type and presumptive R‐type calcium channels are expressed in the mammalian cochlea and that the α2δ subunits may be coded by a characteristic splice‐variant mRNA.

Analysis of muscarinic receptor subtypes in the mouse cochlea by means of the polymerase chain reaction.

Abstract: Total RNA was extracted with guanidine thiocyanate from the cochleas of 16‐day‐old CBAJ mice. The mRNA was purified from the total RNA using oligo‐dT cellulose, and the mRNA was treated with DNase to degrade genomic DNA. After reverse transcription, resulting cDNA was amplified by polymerase chain reaction (PCR), using primers specific for the nucleotide sequences m1‐m5, representing subtypes of muscarinic acetylcholine receptors. PCR products corresponding to subtypes m1, m3, and m5, but not to m2 and m4, were amplified. These results suggest that muscarinic acetylcholine receptors of these odd‐numbered subtypes are expressed in the mammalian cochlea.

The SNARE complex in neuronal and sensory cells.

Transmitter release at synapses ensures faithful chemical coding of information that is transmitted in the sub-second time frame. The brain, the central unit of information processing, depends upon fast communication for decision making. Neuronal and neurosensory cells are equipped with the molecular machinery that responds reliably, and with high fidelity, to external stimuli. However, neuronal cells differ markedly from neurosensory cells in their signal transmission at synapses. The main difference rests in how the synaptic complex is organized, with active zones in neuronal cells and ribbon synapses in sensory cells (such as photoreceptors and hair cells). In exocytosis/neurosecretion, SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) and associated proteins play a critical role in vesicle docking, priming, fusion and synchronization of neurotransmitter release. Recent studies suggest differences between neuronal and sensory cells with respect to the molecular components of their synaptic complexes. In this review, we will cover current findings on neuronal and sensory-cell SNARE proteins and their modulators. We will also briefly discuss recent investigations on how deficits in the expression of SNARE proteins in humans impair function in brain and sense organs.

Surface plasmon resonance (SPR) analysis of binding interactions of proteins in inner-ear sensory epithelia.

Surface plasmon resonance is an optical technique utilized for detecting molecular interactions. Binding of a mobile molecule (analyte) to a molecule immobilized on a thin metal film (ligand) changes the refractive index of the film. The angle of extinction of light, reflected after polarized light impinges upon the film, is altered, monitored as a change in detector position for the dip in reflected intensity (the surface plasmon resonance phenomenon). Because the method strictly detects mass, there is no need to label the interacting components, thus eliminating possible changes of their molecular properties. We have utilized surface plasmon resonance to study the interaction of proteins of hair cells.

Glucocorticoid receptors in the mammalian inner ear: RU 28362 binding sites.

The effects of glucocorticoid hormones are thought to be initiated by binding of the steroid to stereospecific intracellular receptor proteins in target tissues. The synthetic glucocorticoid [3H]-RU 28362, which demonstrates negligible affinity for mineralocorticoid (Type I) receptors [Philibert et al., (1983) Endocrine Soc. Abstr. 65, 335], was employed to identify the high-affinity glucocorticoid (Type II) receptors in the inner ear. By Scatchard analysis, the Kd of the [3H]-RU 28362-cytoplasmic receptor complex was 11.4 x 10(-9) M for the lateral wall of the basal turn of the cochlea and 12.7 x 10(-9) M for the ampullae of the semicircular canals. The concentration of binding sites, Bmax, was 240 fmol/mg dry tissue for the cochlear specimen and 89 fmol/mg dry tissue for the ampullae. Time course studies indicated that the binding of [3H]-RU 28362 by inner ear tissues reached equilibrium within 30 min of incubation at 25 degrees C. Based on the total specific binding measured with [3H]-RU 28362, the glucocorticoid receptor concentration in the lateral wall of the basal turn of the cochlea appears to exceed that in the ampullae of the semicircular canal by a factor of 2.7. Substantial specific [3H]-RU 28362 binding to the cochlear lateral wall and ampullar tissue suggests the presence of glucocorticoid receptors and sites of glucocorticoid action in the inner ear.