Immunohistochemical demonstration of neuroactive substances in the inner ear of rat and guinea pig

The presence and localization of different neuropeptides and other putative neurotransmitters or -modulators were examined by immunohistochemistry in the cochleovestibular end organs and in neurons innervating them in rats and guinea pigs. In the organ of Corti neural elements beneath inner hair cells showed immunoreactivity for enkephalin (ENK), calcitonin gene-related peptide (CGRP), L-glutamate decarboxylase (GAD), substance P (SP) and tyrosine hydroxylase (TH). Nerve chalices of type I vestibular hair cells contained SP and GAD, but not consistently. SP was only occasionally observed in neuronal cell bodies of the 8th cranial nerve but fine fibers with different neuroactive substances were seen in the nerve trunk in the following relative numbers: TH greater than SP greater than CGRP greater than ENK. The present data demonstrate the presence of several different neuroactive substances in the rat and guinea pig inner ear suggesting a multiplicity of neurotransmitters or -modulators in this system.     

Nitric oxide synthase in identified olivocochlear projection neurons in rat and guinea pig.

Nitric oxide (NO) is thought to be involved in the effects of amino acids at the level of cochlear hair cell afferents. Recently, the isoform of the NO-producing enzyme, neuronal NO synthase (nNOS), has been demonstrated in neuronal structures of the cochlea in rats and guinea pigs histochemically and immunohistochemically. To investigate the sources of cochlear NO, we injected Fluoro-Gold (FG) into the cochlea of rats and guinea pigs. Upon terminal uptake of the tracer and neuronal transport we observed FG in terminals at the base of inner (IHC) and outer hair cells (OHC) and in neurons of the spiral ganglion. Ganglion cells and terminals at the IHC were clearly nNOS-positive, while terminals at the OHC exhibited nNOS-immunoreactivity to a minor degree. The immunohistochemical investigation of the auditory brainstem showed that about one-fourth of the neurons of the superior olivary complex contained nNOS. The comparison with retrogradely labeled neurons showed that perikarya in the lateral superior olivary nucleus and, in particular, the medial nucleus of the trapezoid body were double-labeled. These results were similar in both, rat and guinea pig. Our data reveal that neurons of the superior olivary complex are likely to be additional sources of neuronal NOS in the cochlea.     

Input from the inferior colliculus to medial olivocochlear neurons in the rat: A double label study with PHA-L and cholera toxin

The inferior colliculus provides a strong descending influence capable of modulating the excitability levels of olivocochlear neurons (Rajan, 1990). In an attempt to anatomically demonstrate this pathway in rats, an experimental paradigm was designed by which anterogradely transported Phaseolus vulgaris-leucoagglutinin (PHA-L), which delineates axonal arbors, and retrogradely transported cholera toxin B subunit alone (CT-B) or conjugated to horseradish peroxidase (CT-HRP), which delineate dendritic arbors, are visualized in the same brainstem sections. PHA-L was injected unilaterally into the central nucleus of the inferior colliculus of adult rats 5–9 days prior to injection of CT-B or CT-HRP into either the contralateral or the ipsilateral cochlea. Descending collicular axons labeled with PHA-L densely innervate the ventral nucleus of the trapezoid body (VNTB), which contains neurons of the medial olivocochlear system (MOCS), but do not enter the lateral superior olive, where the neurons of the lateral olivocochlear system (LOCS) are found. The collicular projection to VNTB is largely ipsilateral and supplies mostly the ventral half of the nucleus. Within VNTB, the collicular fibers intermingle with dendrites and, to a lesser extent, cell bodies of MOCS. Collicular boutons, predominantly of the en passant type, are often observed in close apposition to dendrites and, less frequently, cell bodies of both crossed and uncrossed MOCS. These light microscopic results suggest the existence of direct, synaptic contacts between descending collicular axons and ipsilateral crossed and uncrossed MOCS. Numerous collicular boutons were also seen at a distance from MOCS, suggesting that they establish synapses with other neuron types of the VNTB that do not send their axons to the cochlea.     

Synaptic connections and putative functions of the dopaminergic innervation of the guinea pig cochlea

We report our findings in the guinea pig involving dopamine in postsynaptic regulation of the activity of glutamatergic inner hair cells (IHCs) and in protection of primary auditory neurons during transient ischemia. Seven days after intracochlear perfusion of 6-hydroxydopamine, no immunoreactivity to tyrosine hydroxylase (TH) was demonstrable within the organ of Corti. TH and aromatic amino acid decarboxylase were immunolocalized at an ultrastructural level within lateral olivocochlear varicosities synapsing with radial auditory dendrites postsynaptic to the IHCs. The D₂ agonist piribedil induced a dose-dependent decrease in the amplitude of the compound action potential of the auditory nerve. Piribedil also prevented appearance of ischemia-induced swelling of the radial dendrites.     

三种神经活性物质对离体豚鼠耳蜗外毛细胞内游离钙浓度的影响

目的研究神经活性物质对耳蜗外毛细胞内游离钙离子浓度（［Ｃａ２＋］ｉ）的影响。方法应用显微荧光测钙技术检测豚鼠耳蜗离体外毛细胞［Ｃａ２＋］ｉ在加入乙酰胆碱、ＡＴＰ、碳酰胆碱之后的变化。结果在含钙的细胞外液中，乙酰胆碱、ＡＴＰ和碳酰胆碱均可引起［Ｃａ２＋］ｉ升高，幅度分别为（０．７４±０．１２９）μｍｏｌ／Ｌ（乙酰胆碱），（０．６５±０．１１）μｍｏｌ／Ｌ（ＡＴＰ），（１．１６±０．２７）μｍｏｌ／Ｌ（碳酰胆碱）；而细胞外液中无钙时，ＡＴＰ仅可引起缓慢而小幅度（０．１８±０．０５）μｍｏｌ／Ｌ的［Ｃａ２＋］ｉ升高。结论同属胆碱能毒蕈碱受体激动剂的乙酰胆碱和碳酰胆碱可作用于受体，打开离子通道，使细胞外Ｃａ２＋进入细胞内，导致外毛细胞［Ｃａ２＋］ｉ升高；细胞外液有Ｃａ２＋时，ＡＴＰ引致的［Ｃａ２＋］ｉ升高，可能是因为引发了内向的Ｃａ２＋流，细胞外液无Ｃａ２＋时，［Ｃａ２＋］ｉ升高则可能源于细胞内钙库的释放。     

Decreased Immunoreactivities of the Chloride Transporters, KCC2 and NKCC1, in the Lateral Superior Olive Neurons of Kanamycin-treated Rats

Objectives

From our previous study about the weak expressions of potassium-chloride (KCC2) and sodium-potassium-2 chloride (NKCC1) co-transporters in the lateral superior olive (LSO) in circling mice, we hypothesized that partially damaged cochlea of circling mice might be a cause of the weak expressions of KCC2 or NKCC1. To test this possibility, we reproduced the altered expressions of KCC2 and NKCC1 in the LSO of rats, whose cochleae were partially destroyed with kanamycin.

Methods

Rat pups were treated with kanamycin from postnatal (P)3 to P8 (700 mg/kg, subcutaneous injection, twice a day) and sacrificed for immunohistochemical analysis, scanning electron microscope (SEM) and auditory brain stem response.

Results

The SEM study revealed partially missing hair cells in P9 rats treated with kanamycin, and the hearing threshold was elevated to 63.8±2.5 dB SPL (4 ears) at P16. Both KCC2 and NKCC1 immunoreactivities were more prominent in control rats on P16. On 9 paired slices, the mean densities of NKCC1 immunoreactivities were 118.0±1.0 (control) and 112.2±1.2 (kanamycin treated), whereas those of KCC2 were 115.7±1.5 (control) and 112.0±0.8 (kanamycin treated).

Conclusion

We concluded that weak expressions of KCC2 and NKCC1 in circling mice were due to partial destruction of cochleae.     

Decreased Immunoreactivities and Functions of the Chloride Transporters, KCC2 and NKCC1, in the Lateral Superior Olive Neurons of Circling Mice

Objectives

We tested the possibility of differential expression and function of the potassium-chloride (KCC2) and sodium-potassium-2 chloride (NKCC1) co-transporters in the lateral superior olive (LSO) of heterozygous (+/cir) or homozygous (cir/cir) mice.

Methods

Mice pups aged from postnatal (P) day 9 to 16 were used. Tails from mice were cut for DNA typing. For Immunohistochemical analysis, rabbit polyclonal anti-KCC2 or rabbit polyclonal anti-NKCC1 was used and the density of immunolabelings was evaluated using the NIH image program. For functional analysis, whole cell voltage clamp technique was used in brain stem slices and the changes of reversal potentials were evaluated at various membrane potentials.

Results

Immunohistochemical analysis revealed both KCC2 and NKCC1 immunoreactivities were more prominent in heterozygous (+/cir) than homozygous (cir/cir) mice on P day 16. In P9-P12 heterozygous (+/cir) mice, the reversal potential (E_gly) of glycine-induced currents was shifted to a more negative potential by 50 µM bumetanide, a known NKCC1 blocker, and the negatively shifted E_gly was restored by additional application of 1 mM furosemide, a KCC2 blocker (-58.9±2.6 mV to -66.0±1.5 mV [bumetanide], -66.0±1.5 mV to -59.8±2.8 mV [furosemide+bumetanide], n=11). However, only bumetanide was weakly, but significantly effective (-60.1±2.9 mV to -62.7±2.6 mV [bumetanide], -62.7±2.6 mV to -62.1±2.5 mV [furosemide+bumetanide], n=7) in P9-P12 homozygous (cir/cir) mice.

Conclusion

The less prominent immunoreactivities and weak or absent responses to bumetanide or furosemide suggest impaired function or delayed development of both transporters in homozygous (cir/cir) mice.     

Low frequency neurons in the lateral central nucleus of the cat inferior colliculus receive their input predominantly from the medial superior olive

When iontophoretic injections of horseradish peroxidase were made in the vicinity of inferior colliculus units in the cat responding to low sound frequencies, retrograde labelling occurred in the ipsilateral medial superior olive. Both bipolar and multipolar cells of the medial superior olive participated in this projection, and the focus of labelling shifted topographically within the olive as the injection site best frequency varied. These observations confirm previous anatomical findings and link them to the tonotopic organization of the central nucleus of the inferior colliculus. However, the fact that the medial superior olive alone provides between 50 and 98% of labelled cells in the brain stem projecting to this region of the central nucleus is an unexpected observation. This study gives further support to an hypothesis of 'core zones' within the central nucleus that receive preferentially input from specific brain stem auditory nuclei.     

三种鼠对脉冲噪音损伤敏感性的研究

目的 研究豚鼠、大鼠和小鼠对脉冲噪音暴露的敏感性。方法 共分6组,第1组豚鼠(n=5)给予160 dB SPL50次脉冲噪音暴露;第2组豚鼠(n=5)予160 dB SPL 100次脉冲噪音暴露;第3组豚鼠(n=5)给予160 dB SPL 200次脉冲噪音暴露;第4组豚鼠(n=6)给予160 dB SPL 400次脉冲噪音暴露。第5组10只Sprague-Dawley大鼠给予160 dB SPL 50次脉冲噪音暴露。第6组10只小鼠(Bagg Albino雌鼠与DBA雄鼠杂交)予160 dB SPL 50次脉冲噪音暴露。脑干诱发电位检测在脉冲噪音前,噪音后立刻、1 d、1、2、4周。结果 给予160 dB SPL50次脉冲噪音暴露后,大鼠和小鼠都显示暂时和永久的阈值漂移,豚鼠无暂时和永久的阈值漂移,给予160 dB SPL 400次脉冲噪音暴露后显示暂时和永久的阈值漂移。结论 豚鼠、大鼠和小鼠对脉冲噪音敏感性不同,豚鼠对脉冲噪音不如大鼠小鼠敏感,而大鼠比小鼠更敏感。     

Effect of high-intensity sound on cochlear microphonics and activity of inferior colliculus neurons in the guinea pig

Summary The input-output functions of cochlear microphonics (recorded from the round window) in guinea pigs exposed briefly to high-intensity sound (2 kHz, 130 dB, 30 min) were shifted toward higher intensities by about 15 dB in comparison with normal animals. 2–4 h after the exposure this shift decreases to 10 dB. The response characteristics of the inferior colliculus neurons were compared under similar conditions. Thresholds at the characteristic frequencies (CF) measured 2–4 h after exposure, were raised by 30–50 dB, a higher threshold increase was observed at frequencies from 5–7 kHz. 48 h after exposure the thresholds approached those obtained in normal animals. There was no pronounced hair cell loss after exposure. It is assumed that in addition to the impairment of sound transduction in hair cells there may be a specific effect of high-intensity sound exposure on neuronal transmission in the auditory pathway.     

Effects of pentoxifylline and nitroprusside on guinea pig cochlear blood flow in relationship to various hematocrit values

To understand better the microcirculation in the inner ear in relation to blood viscosity, we examined the effects of pentoxifylline (PXF) and nitroprusside (NP) on cochlear blood flow (CBF) in healthy adult guinea pigs in relationship to various hematocrit (Ht) values. There was no correlation between Ht value and the maximum decrease of mean blood pressure after PXF infusion, whereas there was a positive correlation between Ht and the maximum increase in CBE No such relationship was found after NP infusion. A graph plot of each animal's blood oxygen transport capacity (i.e., Ht/blood viscosity) was found to have a convex form and to increase with a shift of the peak to a higher Ht area after PXF infusion. These findings indicate that a decrease in blood viscosity may improve CBF and increase oxygen transport capacity of blood.     

Differences in the distribution of responses to ATP and acetylcholine between outer hair cells of rat and guinea pig

Adenosine 5′ triphosphate (ATP) and acetylcholine (ACh) are neurotransmitters (ACh) and/or modulators (ATP) in the mammalian cochlea. In guinea pig, it appears that both neurotransmitters have a similar response distribution, with larger responses being evoked by the ligands in short hair cells compared to long hair cells (e.g., Chen et al., 1995b. Noise exposure alters the response of outer hair cells to ATP. Hear. Res. 88, 215–221.; Erostegui et al., 1994. In vitro pharmacologic characterization of a cholinergic receptor on outer hair cells. Hear. Res. 74, 135–147). The purpose of the present study was to test whether the distribution of responses to ACh and ATP in the OHCs of rat is the same as guinea pig. The ligand-induced current was monitored using the whole-cell configuration of the patch-clamp technique. Results show that in guinea pig OHCs, extracellular application of 100 μM ATP induced a current response in a majority of the same cells that responded to the application of 100 μM ACh. In contrast in rat OHCs, 100 μM ATP did not induce a current in the majority of cells that responded to the application of 100 μM ACh. N-methyl-glucamine (NMG⁺) substituted for K⁺ in the pipette solution failed to unmask an ATP-evoked inward current in rat OHCs. In addition, no response was produced in rat or guinea pig OHCs by adenosine, adenosine 5′-monophosphate (AMP) or adenosine 5′-diphosphate (ADP) at 100 μM. Results suggest that in guinea pig ACh-gated channels are present on most of the same OHCs that have ATP-gated ion channels, whereas in rat ACh-gated ion channels are present without ATP-gated channels on some OHCs.