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

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

The aim of the present study was to investigate the role of glutamate receptors in the damage of spiral ganglion neurons (SGNs) induced by acute acoustic noise. This investigation included in vivo and in vitro studies. In vivo, kynurenic acid (KYNA), a broad-spectrum antagonist of glutamate receptors, was applied to the round window of guinea pigs, and its protective effect was observed. The animals were divided into three groups: control (saline, 0.9%, 10 microL), saline (0.9%, 10 microL) + noise and KYNA (5 mmol/L, 10 microL) + noise. Saline and KYNA were applied to the round window membrane with a microsyringe. The animals were exposed to 110 dB SPL of white noise for 1 h. Hearing thresholds for auditory brainstem responses (ABRs) and compound action potentials (CAPs) in all animals were measured before and after treatment. The amplitudes of III waveform of ABR and N1 waveform of CAP and the latency of N1 waveform at different stimulation levels (intensity-amplitude and intensity-latency functions) were also measured. The cochleas were then dissected for transmission electron microscopy (TEM) after final electrophysiological measurement. In vitro, the SGNs of the normal guinea pigs were isolated and glutamate (100 micromol/L or 1 000 micromol/L) was added into the medium. The morphology of the SGNs was examined by light microscopy. In vivo results showed that the hearing function and morphology of the inner ear including hair cells and SGNs in the control group were normal. Compared with that in the control group the thresholds for ABR and CAP (click and tone burst) in saline + noise group were elevated significantly. The input-output functions showed that the amplitudes of III waveform of ABR and N1 waveform of CAP decreased and the latency of N1 waveform increased obviously. There was significant difference in the amplitude and latency between saline + noise group and KYNA + noise group (P<0.05). TEM indicated obvious swelling and vacuoles at the terminate of dendrites of SGNs in NS + noise group. On the contrary, the afferent dendrites in KYNA + noise group showed normal appearance without swelling and vacuoles. In vitro experiment showed that the isolated SGNs of guinea pigs obviously swelled and even died after application of 100 micromol/L or 1 000 micromol/L glutamate. These results suggest that noise exposure causes hearing impairment, damage of hair cells and hair cell/afferent synapse and death of SGNs. The antagonist of glutamate receptors provides protective effects against hearing loss and SGN damage. It is inferred that excessive release of glutamate from the inner hair cells induced by noise may be responsible for these damages. Glutamate receptors are involved in the degeneration and death of SGNs.
Action Potentials ; physiology ; Animals ; Evoked Potentials, Auditory, Brain Stem ; physiology ; Excitatory Amino Acid Antagonists ; pharmacology ; Guinea Pigs ; Hearing Loss, Noise-Induced ; metabolism ; pathology ; physiopathology ; Kynurenic Acid ; pharmacology ; Male ; Neurons ; pathology ; Noise ; adverse effects ; Random Allocation ; Receptors, Glutamate ; metabolism ; Spiral Ganglion ; pathology