Mice ; Animals ; Spiral Ganglion/physiology* ; Cochlea/physiology* ; Neurons

OBJECTIVETo establishing the cochlea slice technique and infrared visual slice patch clamp method in order to observe the electrophysiological characteristics of rat spiral ganglion neurons (SGN) METHODS: SD rats were divided into three groups according to postnatal days old (0-2 d, 3-6 d and 7-14 d). Making slice of SD rat cochlear quickly, using infrared differential interference contrast (IR-DIC) technique, together with slice patch clamp, the electrophysiological characteristics of rat spiral ganglion neurons were observed, and factors which affected the quality of cochlear slice and recording of patch clamp were analyzed.

RESULTSThe success rate of 3-6 days SD was the highest, and 2-4 pieces of slice could be made from each cochlea. Cochlea connecting with partial skull and integrity of cochlear hull were the key for making slice, and the angle of modiolus axis should be adjusted to be parallel to the knife and the preparing time should be shorter. The SGN cell of good condition could be easily found and the seal test became easier with the help of infrared visual slice patch clamp method. The rest membrane potential was (-45.6 +/- 5.3) mV (x +/- s, n=52) and the current of Na+ and K+ could be activated.

CONCLUSIONSCochlear slice technique can retain structural integrity, cell viability and their association in cochlea, which suggest that this technique provides carrier for electrophysiological study of rat spiral ganglion neurons, and patch clamp with infrared videomicroscopy method can be used to make direct real-time observation in electrophysiological experiments of SGN, which can provide important technique support and reference for deep study of electrophysiological characteristics of SGN and auditory neurotransmission in cochlea.

Animals ; Cochlea ; physiology ; Microtomy ; Neurons ; physiology ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley ; Spiral Ganglion ; physiology

Animals ; Cells, Cultured ; Electrophysiology ; Mice ; Mice, Inbred Strains ; Neurons ; physiology ; Potassium Channels ; physiology ; Spiral Ganglion ; physiology

OBJECTIVE: To compare the impedance between the modiolus and the inner wall of scala tympani with that between the modiolus and the outer wall of scala tympani. METHOD: The impedances between the modiolus and the inner wall of scala tympani and the impedance between the modiolus and the outer wall of scala tympani were measured, calculated and compared under different stimulating rates 0.1, 1.0, 10.0 kHz. RESULT: The impedance between the modiolus and the inner wall of scala tympani is less than that between the modiolus and the outer wall of scala tympani (P < 0.05). CONCLUSION To effectively stimulate the residual neurons in the spiral ganglion, the electrodes should be kept close to the inner wall of scale tympani.
Adult ; Cochlea ; physiology ; Cochlear Implants ; Electric Impedance ; Electrodes ; Humans ; Scala Tympani ; physiology ; Spiral Ganglion ; Temporal Bone ; physiology

OBJECTIVE: To explore the nature of voltage dependent ion channels and basic electrophysiological characteristics of cochlear spiral ganglion neurons of apical and basal turn by patch clamp techniques of whole cell configure on murine spiral ganglion neurons. METHOD: Different voltage dependent ionic currents were recorded with patch clamp techniques of whole cell configure on the condition of different internal electrode solution, blockers and stimulus protocol. RESULT: Inward sodium channel current (I(Na)), hyperpolarization-activated inward cationic current (Ih), outward delay rectification potassium current (I(K)) and outward transient potassium current (I(A)) were recorded ,significant difference of electrophysiological characteristics of I(A) and I(K) was found between apical and basal turns (P < 0.05). CONCLUSION Various ionic currents are recorded, which shows that spiral ganglion neurons have the base of ionic channels to complete formation,conduction and modulation of action potential for auditory information transduction, the difference of electrophysiological characteristics between apical and basal turns contributes to the course of hearing formation.
Animals ; Cells, Cultured ; Ion Channels ; physiology ; Membrane Potentials ; physiology ; Mice ; Mice, Inbred Strains ; Neurons ; physiology ; Patch-Clamp Techniques ; Spiral Ganglion ; physiology

OBJECTIVEIn this study, we investigated the potential of mouse induced pluripotent stem cells (iPSC) for use as a source of transplants for the restoration of auditory hair cells and spiral ganglion neurons.

METHODSWe co-cultured the mouse iPSC with the cells of the cochlear organ of Corti or the modiolus in vitro. The cochlear organ of Corti (which contains cochlear hair cells) and the modiolus (which contains auditory spiral ganglion neurons) were obtained from postnatal day 3 (P3) CD-1 ICR mice. After 18 days of coculture with the cells of newborn mouse cochleae. The expressions of hair cell markers (Myosin VIIa, Math1, Calretinin, Espin) and Spiral ganglion neuron markers [Nestin, Neurofilament-M, β-III Tubulin, Vesicular glutamate transporter 1(VGluT1)] were detected by immunocytochemical analysis.

RESULTSImmunocytochemical analysis results indicated that the differentiated iPSC expressed auditory hair cell markers (MyosinVIIa,Math1, Calretinin, Espin ) and spiral ganglion markers (Nestin, Neurofilament-M,β-III Tubulin,VGluT1).

CONCLUSIONMouse iPSC in virto cultured could successfully be induced to differentiate into hair cell-like cells and spiral ganglion-like cells with hair cell and spiral ganglion molecular markers.

Animals ; Cell Differentiation ; Cochlea ; physiology ; Coculture Techniques ; Hair ; Hair Cells, Auditory ; In Vitro Techniques ; Induced Pluripotent Stem Cells ; Mice ; Mice, Inbred ICR ; Neurons ; Spiral Ganglion ; physiology

OBJECTIVETo study the differences of regulation of sodium salicylate on the auditory brain stem response (ABR) threshold and expression of glutamic acid decarboxylase (GAD) protein in spiral ganglion of juvenile and adult guinea pigs.

METHODSFourty juvenile guinea pigs which were born just four days and fourty adult guinea pigs which were born thirty days were selected. They were divided four groups (group A; group B; group C; group D). ABR threshold was detected before administration, after administration for 15 days and after administration stopped for 30 days. The protein expression of GAD were measured after administration for 15 days and after administration stopped for 30 days by the method of immunohistochemistry.

RESULTSABR threshold of juvenile sodium salicylate groups (group C) was increased remarkably than that of before administration and the control after administration for 15 days (P < 0.001). ABR threshold of group C was returned to the level of that of before administration and after administration stopped for 30 days. ABR threshold of adult sodium salicylate groups (group D) was increased remarkably than that of before administration and the control after administration for 15 days (P < 0.001). ABR threshold of group D was kept the high level after administration stopped for 30 days. The protein expression of GAD of sodium salicylate groups (group C and D) was decreased than that of the control after administration for 15 days. The protein expression of group C was more visible regression than that of group D (t = 4.7, P < 0.001). The protein expression of group C was returned the level of before administration after administration stopped for 30 days, but the protein expression of group D was kept the high level.

CONCLUSIONSThe results suggest that sodium salicylate can regulate differently ABR threshold and expression of GAD protein in spiral ganglion of juvenile and adult guinea pigs. The effects of sodium salicylate on ABR threshold and expression of GAD protein in spiral ganglion of juvenile pigs are more noticeable than that of adult guinea pigs, but these changes are easier to return the normal than that of adult guinea pigs.

Animals ; Auditory Threshold ; drug effects ; Evoked Potentials, Auditory, Brain Stem ; drug effects ; physiology ; Glutamate Decarboxylase ; metabolism ; Guinea Pigs ; Sodium Salicylate ; pharmacology ; Spiral Ganglion ; drug effects ; enzymology

BACKGROUNDOuabain, a cardiac glycoside that specifically binds to Na/K-ATPase and inhibits its activity, was applied to gerbils to develop a method for studying auditory neuropathy.

METHODSOuabain was applied to the round window of the cochlea in each gerbil by using a piece of gelfoam with 3 microl or 24 microl (1 mmol/L) ouabain solution. The changes of the threshold of auditory brainstem response, cochlear function round window electrocochleography, as well as the morphological changes of the spiral ganglion cells of the cochlea were observed after application of ouabain for 24 hours or 96 hours.

RESULTSIn ouabain treated gerbils, auditory brainstem response and compound action potential thresholds showed either elevation or no response at all. However, the thresholds of cochlear microphonic and distortion product otoacoustic emissions were not affected. Degeneration and necrosis of some spiral ganglion cells in ears with applications of ouabain (24 hours, 3 microl, 1 mmol/L; 96 hours, 24 microl, 1 mmol/L ouabain). The number of spiral ganglion cells was decreased (24 hours, 3 microl, 1 mmol/L ouabain) or near to a total loss (96 hours, 24 microl, 1 mmol/L ouabain).

CONCLUSIONSThese results indicate a high degree of independence between the spiral ganglion cells and the outer hair cell systems in the cochlear transduction mechanism. The method used in this study would provide a valuable tool for studying auditory neuropathy.

Action Potentials ; drug effects ; Animals ; Cochlea ; drug effects ; physiology ; Evoked Potentials, Auditory, Brain Stem ; drug effects ; Gerbillinae ; Ouabain ; toxicity ; Spiral Ganglion ; drug effects

The aim of the present study was to investigate the effect of precursor brain-derived neurotrophic factor (proBDNF) on survival and neurite outgrowth of cultured rat spiral ganglion neurons (SGNs). Spiral ganglions (SG) were collected from postnatal day 5 Sprague Dawley (SD) rats, then enzymatically digested and suspended. Dissociated SGNs were plated on poly-D-lysine/laminin coated eight-well chamber plates and maintained at 37 °C for 4 h to promote the attachment of the neurons. Cultured SGNs were randomly divided into five groups: control group, BDNF group (BDNF 10 ng/mL), C10 group (proBDNF 10 ng/mL), C50 group (proBDNF 50 ng/mL), and C100 group (proBDNF 100 ng/mL). All groups were incubated in a serum-free medium. 48 h after incubation, SGNs were fixed and stained for βIII tubulin. Immunostaining of the cultured SGNs showed that, compared with the control group, the cellular survival of C50 group and C100 group were significantly reduced (P < 0.001). Furthermore, surviving numbers of the three proBDNF-treated groups were all lower than the BDNF group. In order to assess the effect of proBDNF on cell morphology, SGNs were divided into two categories: SGNs with or without neurites. The results demonstrated that proBDNF significantly increased the proportions of SGNs without neurites in C10, C50 and C100 groups compared with that in control group (P < 0.001). In addition, c-Jun N-terminal kinase (JNK) inhibitor, SP600125 (20 μmol/L) significantly increased the surviving number of SGNs in C50 group. These results suggest that proBDNF reduces the survival rate of cultured SGNs and inhibits the sprouting of neurites. Furthermore, the inhibition of JNK signaling attenuates the effect of proBDNF on SGNs survival.
Animals ; Axons ; physiology ; Brain-Derived Neurotrophic Factor ; pharmacology ; Cell Survival ; Cells, Cultured ; JNK Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; MAP Kinase Signaling System ; Neurites ; physiology ; Neurons ; cytology ; Protein Precursors ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Spiral Ganglion ; cytology

The aim of the present study was to assess the ototoxicity of kanamycin sulfate (KM) in adult rats and its underlying mechanism. Forty male Sprague-Dawley rats (6-7 weeks old) were randomly divided into the experimental group and the control group. The animals in the experimental group were injected subcutaneously with KM (500 mg/kg per day) for two weeks, and the control group received equal volume of normal saline. To assess the ototoxicity of KM, the auditory brainstem response (ABR) was recorded to monitor the changes in hearing thresholds, and the density of spiral ganglion cells (SGCs) and morphology of cochlea were observed using surface preparations and frozen sections of cochlea. The results showed that the hearing threshold of rats in the experimental group was elevated by more than 60 dB across all the frequencies two weeks after the first administration of KM. And in the experimental group, the density of SGCs became lower, and organ of Corti suffered loss of hair cells. The loss of outer hair cells (OHCs) was more severe than that of inner hair cells (IHCs), correlated with the density decrease of SGCs. We conclude that the ototoxicity of KM in the adult rats was apparent and the underlying mechanism is associated with the loss of SGCs and hair cells.
Animals ; Cochlea ; drug effects ; pathology ; Evoked Potentials, Auditory, Brain Stem ; drug effects ; Hair Cells, Auditory, Outer ; cytology ; drug effects ; pathology ; Hearing Loss ; chemically induced ; physiopathology ; Kanamycin ; toxicity ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spiral Ganglion ; pathology ; physiology ; ultrastructure