Rat Cochlear Nucleus Research Articles

Optogenetically modified human embryonic stem cell-derived otic neurons establish functional synaptic connection with cochlear nuclei

Spiral ganglia neurons (SGNs) impairment can cause deafness. One important therapeutic approach involves utilizing stem cells to restore impaired auditory circuitry. Nevertheless, the inadequate implementation of research methodologies poses a challenge in accurately assessing the functionality of derived cells within the circuit. Here, we describe a novel method for converting human embryonic stem cells (hESCs) into otic neurons (ONs) and assess their functional connectivity using an optogenetic approach with cells or an organotypic slice of rat cochlear nucleus (CN) in coculture. Embryonic stem cell-derived otic neurons (eONs) exhibited SGN marker expression and generated functional synaptic connection when cocultured with cochlear nucleus neurons (CNNs). Synapsin 1 and VGLUT expression are found in the cochlear nucleus of brain slices, where eONs projected processes during the coculture of eONs and CN brain slices. Action potential spikes and INa+/IK+ of CNNs increased in tandem with light stimulations to eONs. These findings provide further evidence that eONs may be a candidate source to treat SGN-deafness.

Hydralazine alleviates noise-induced hearing loss by scavenging acrolein

Noise-induced hearing loss (NIHL) is the most common cause of hearing loss. This study investigates the therapeutic efficacy of the acrolein scavenger hydralazine for NIHL in rats. NIHL was induced by exposure to a continuous pure tone of 10 kHz. Auditory function was evaluated by auditory brainstem response (ABR) testing and scanning electron microscopy. The expression of acrolein and glial cell markers GFAP and OX42 was assessed by immunofluorescence staining. The protein and mRNA expression of GFAP, OX42, interleukin-18 (IL-18), IL-1β, and fractalkine (FTK) was measured by qRT-PCR and western blotting. A rat model of NIHL was successfully developed, as evidenced by increased ABR thresholds. The results showed that noise exposure increased the expression of acrolein, GFAP, OX42, FTK, IL-1β, and IL-18 in the rat cochlear nucleus. Furthermore, hydralazine alleviates NIHL by reversing the effects of acrolein. These results demonstrate that acrolein is involved in glial cell activation and NIHL, it’s also a therapeutic potential target for NIHL.

MRNA Abundance of Neurogenic Factors Correlates with Hearing Capacity in Auditory Brainstem Nuclei of the Rat.

Neural stem cells (NSCs) have previously been described up to the adult stage in the rat cochlear nucleus (CN). A decreasing neurogenic potential was observed with critical changes around hearing onset. A better understanding of molecular factors affecting NSCs and neurogenesis is of interest as they represent potential targets to treat the cause of neurologically based hearing disorders. The role of genes affecting NSC development and neurogenesis in CN over time on hearing capacity has remained unclear. This study investigated the mRNA abundance of genes influencing NSCs and neurogenesis in rats' CN over time. The CN of rats on postnatal days 6, 12, and 24 were examined. Real-time quantitative polymerase chain reaction arrays were used to compare mRNA levels of 84 genes relevant to NSCs and neurogenesis. Age- and hearing-specific patterns of changes in mRNA abundance of neurogenically relevant genes were detected in the rat CN. Additionally, crucial neurogenic factors with significant and relevant influence on neurogenesis were identified. The results of this work should contribute to a better understanding of the molecular mechanisms underlying the neurogenesis of the auditory pathway.

A liquid chromatography-mass spectroscopy-based untargeted metabolomic study of the rat cochlear nucleus at various stages of maturity

The cochlear nucleus receives numerous inputs from auditory and nonauditory systems. This extensive innervation of the cochlear nucleus is involved in sound source localization and the integration of auditory signals with other sensory modalities. The dorsal cochlear nucleus may also have an important role in tinnitus. Although its gross anatomy and function have been extensively studied, the metabolome of the cochlear nucleus remains poorly understood, particularly at different stages of auditory maturity. Here, we present a protocol for untargeted metabolomics analysis of the rat cochlear nucleus, then discuss differences in the metabolome of the rat cochlear nucleus between postnatal day (PD) 14 (hearing onset) and PD60 (hearing maturation). Cochlear nucleus samples collected from rats at PD14 or PD60 were analyzed by liquid chromatography-tandem mass spectrometry (LCMS). In total, 344 metabolites were identified. Principal component analysis and orthogonal partial least-square discriminant analysis showed that the metabolic profiles at these two stages had distinct distribution patterns. Moreover, 91 significantly differential metabolites (62 upregulated and 29 downregulated) were identified at PD60 vs. PD14. N-acetylaspartylglutamic acid (NAAG), γ-aminobutyric acid (GABA), taurine, adenosine monophosphate (AMP), and choline were significantly upregulated at PD60. Pathway enrichment analysis suggested that alanine, aspartate, and glutamate metabolism; glycine, serine, and threonine metabolism; the mammalian target of rapamycin (mTOR) signaling pathway; and the AMP-activated protein kinase (AMPK) signaling pathway may be involved in key developmental events during maturation of the cochlear nucleus. Taken together, the metabolic profiles identified in this study could lead to the identification and understanding of specific key biomarkers and metabolic pathways involved in the maturation of hearing. Moreover, LC-MS-based metabolomics provides an alternative approach for the characterization of auditory maturation and auditory diseases.

Spontaneous Calcium Oscillations through Differentiation: A Calcium Imaging Analysis of Rat Cochlear Nucleus Neural Stem Cells.

Causal therapies for the auditory-pathway and inner-ear diseases are still not yet available for clinical application. Regenerative medicine approaches are discussed and examined as possible therapy options. Neural stem cells could play a role in the regeneration of the auditory pathway. In recent years, neural stem and progenitor cells have been identified in the cochlear nucleus, the second nucleus of the auditory pathway. The current investigation aimed to analyze cell maturation concerning cellular calcium activity. Cochlear nuclei from PND9 CD rats were microscopically dissected and propagated as neurospheres in free-floating cultures in stem-cell medium (Neurobasal, B27, GlutaMAX, EGF, bFGF). After 30 days, the dissociation and plating of these cells took place under withdrawal of the growth factors and the addition of retinoic acid, which induces neural cell differentiation. Calcium imaging analysis with BAPTA-1/Oregon Green was carried out at different times during the differentiation phase. In addition, the influence of different voltage-dependent calcium channels was analyzed through the targeted application of inhibitors of the L-, N-, R- and T-type calcium channels. For this purpose, comparative examinations were performed on CN NSCs, and primary CN neurons. As the cells differentiated, a significant increase in spontaneous neuronal calcium activity was demonstrated. In the differentiation stage, specific frequencies of the spontaneous calcium oscillations were measured in different regions of the individual cells. Initially, the highest frequency of spontaneous calcium oscillations was ascertainable in the maturing somata. Over time, these were overtaken by calcium oscillations in the axons and dendrites. Additionally, in the area of the growth cones, an increasing activity was determined. By inhibiting voltage-dependent calcium channels, their expression and function in the differentiation process were confirmed. A comparable pattern of maturation of these channels was found in CN NSCs and primary CN neurons. The present results show that neural stem cells of the rat cochlear nucleus differentiated not only morphologically but also functionally. Spontaneous calcium activities are of great relevance in terms of neurogenesis and integration into existing neuronal structures. These functional aspects of neurogenesis within the auditory pathway could serve as future targets for the exogenous control of neuronal regeneration.

Different Neurogenic Potential in the Subnuclei of the Postnatal Rat Cochlear Nucleus.

In patients suffering from hearing loss, the reduced or absent neural input induces morphological changes in the cochlear nucleus (CN). Neural stem cells have recently been identified in this first auditory relay. Afferent nerve signals and their impact on the immanent neural stem and progenitor cells already impinge upon the survival of early postnatal cells within the CN. This auditory brainstem nucleus consists of three different subnuclei: the anteroventral cochlear nucleus (AVCN), the posteroventral cochlear nucleus (PVCN), and the dorsal cochlear nucleus (DCN). Since these subdivisions differ ontogenetically and physiologically, the question arose whether regional differences exist in the neurogenic niche. CN from postnatal day nine Sprague-Dawley rats were microscopically dissected into their subnuclei and cultivated in vitro as free-floating cell cultures and as whole-mount organ cultures. In addition to cell quantifications, immunocytological and immunohistological studies of the propagated cells and organ preparations were performed. The PVCN part showed the highest mitotic potential, while the AVCN and DCN had comparable activity. Specific stem cell markers and the ability to differentiate into cells of the neural lineage were detected in all three compartments. The present study shows that in all subnuclei of rat CN, there is a postnatal neural stem cell niche, which, however, differs significantly in its potential. The results can be explained by the origin from different regions in the rhombic lip, the species, and the various analysis techniques applied. In conclusion, the presented results provide further insight into the neurogenic potential of the CN, which may prove beneficial for the development of new regenerative strategies for hearing loss.

Effects of long-term Doppler ultrasound exposure on cochlea and cochlear nucleus in prenatal period in an experimental model

Background New generation Doppler ultrasonography (DUSG) application effects on cochlea and cochlear nucleus (CN) are unclear. We aimed to investigate the effects of new generation DUSG application at different frequencies in prenatal period on cochlea and CN in rats. Objective Twenty-four pregnant female rats were divided into three groups (n = 8). Group 1 was the control group and was not subjected to any treatment. Group 2 was determined as the USG every day (USGED) treatment group. Group 2 has received DUSG application every day from the 4th to 18th day (20 min/15 per day). Group 3 has received DUSG application as “2 days/one dose as every other day application” (USG2D1) from the 4th to 18th day (20 min/8 every other day). Twenty-four female rats were sacrificed in 21 days. Also, 24 pups were sacrificed after two days. First day after born, the cochlear activities of the right ears of all pups were examined using DPOAEs. Second day, neural tissues from CN were evaluated histopathologically and immunohistochemically. Results There was no any statistical difference between the groups in respect of histopathologically. USGED group showed mild caspase-3 positive neurons and glial cells. However, there was no significant difference between the USGED and other groups (p>.05). Similarly, the rats applied with USG2D1 had mild caspase-3 expression, but no significant difference between the USG2D1 and other groups (p>.05). Differences in DPOAE amplitudes, and therefore in cochlear activity, between the groups were revealed. The decrease in cochlear activity between the groups involved frequencies at 2, 8, 16, and 32 kHz (p<.05). Conclusions Multiple administration of new generation DUSG to pregnant rats has not shown harmful effects on the cochlear neural tissue. High frequencies are more sensitive in cochlea to apply DUSG.

Effects of brainstem lesions on amino acid levels in the rat cochlear nucleus

There is evidence for glutamate, γ-amino butyric acid (GABA), and glycine as neurotransmitters of centrifugal pathways to the cochlear nucleus, but the quantitative extent of their contributions to amino acid neurotransmission in cochlear nucleus regions has not been known. We used microdissection of freeze-dried tissue sections of rat cochlear nucleus, with mapping of sample locations, combined with a high performance liquid chromatography (HPLC) assay, to measure amino acid levels in cochlear nucleus subregions of rats with unilateral lesions of centrifugal pathways to the cochlear nucleus. In rats with lesions transecting all or almost all pathways to the cochlear nucleus from brain stem regions, GABA, aspartate, and glutamate levels were reduced, compared to contralateral values, in almost all ipsilateral cochlear nucleus regions. The largest reductions, in dorsal (DCN), anteroventral (AVCN), and posteroventral (PVCN) cochlear nucleus regions, approached 50% for GABA, 40% for aspartate, and 30% for glutamate. In contrast, glutamine and taurine levels were typically higher in lesioned-side cochlear nucleus regions than contralaterally. Effects on glycine levels were mixed but usually included increased lesioned-side values compared to contralateral, probably reflecting a balance between increases during protein breakdown and decreases of free glycine in transected pathways. More limited lesions transecting just dorsal pathways showed much less effect on amino acid levels. Lesion of the ipsilateral trapezoid body connection plus ipsilateral superior olivary nuclei resulted in decreases of GABA, aspartate, and glutamate levels especially in ventral cochlear nucleus regions. No clear contralateral effects of this lesion could be shown. The results most strongly support centrifugal GABAergic pathways to the cochlear nucleus, providing almost half of GABAergic neurotransmission in most regions. Our results support and extend previously published measurements of lesion effects on GABA uptake and release in cochlear nucleus subdivisions.

Expression and Localization of Kv1.1 and Kv3.1b Potassium Channels in the Cochlear Nucleus and Inferior Colliculus after Long-Term Auditory Deafferentation.

Deafness affects the expression and distribution of voltage-dependent potassium channels (Kvs) of central auditory neurons in the short-term, i.e., hours to days, but the consequences in the expression of Kvs after long-term deafness remain unknown. We tested expression and distribution of Kv1.1 and Kv3.1b, key for auditory processing, in the rat cochlear nucleus (CN), and in the inferior colliculus (IC), at 1, 15 and 90 days after mechanical lesion of the cochlea, using a combination of qRT-PCR and Western blot in the whole CN, along with semi-quantitative immunocytochemistry in the AVCN, where the role of both Kvs in the control of excitability for accurate auditory timing signal processing is well established. Neither Kv1.1/Kv3.1b mRNA or protein expression changed significantly in the CN between 1 and 15 days after deafness. At 90 days post-lesion, however, mRNA and protein expression for both Kvs increased, suggesting that regulation of Kv1.1 and Kv3.1b expression is part of cellular mechanisms for long-term adaptation to auditory deprivation in the CN. Consistent with these findings, immunocytochemistry showed increased labeling intensity for both Kvs in the AVCN at day 90 after cochlear lesion. This increase argues that up-regulation of Kv1.1 and Kv3.1b in AVCN neurons may be required to adapt intrinsic excitability to altered input over the long term after auditory deprivation. Contrary to these findings in the CN, expression levels of Kv1.1 and Kv3.1b in the IC did not undergo major changes after cochlear lesion. In particular, there was no evidence of long-term up-regulation of either Kv1.1 or Kv3.1b, supporting that such post-lesion adaptive mechanism may not be needed in the IC. These results reveal that post-lesion adaptations do not necessarily involve stereotyped plastic mechanisms along the entire auditory pathway.

Inhibitory effect of 980-nm laser on neural activity of the rat's cochlear nucleus.

.Near-infrared radiation (NIR) has been described as one of the highest-resolution tools for neuromodulation. However, the poor tissue penetration depth of NIR has limited its further application on some of the deeper layer neurons in vivo. A 980-nm short-wavelength NIR (SW-NIR) with high penetration depth was employed, and its inhibitory effect on neurons was investigated in vivo. In experiments, SW-NIR was implemented on the rat’s cochlear nucleus (CN), the auditory pathway was activated by pure-tones through the rat’s external auditory canal, and the neural responses were recorded in the inferior colliculus by a multichannel electrode array. Neural firing rate (FR) and the first spike latency (FSL) were analyzed to evaluate the optically induced neural inhibition. Meanwhile, a two-layered finite element, consisting of a fluid layer and a gray matter layer, was established to model the optically induced temperature changes in CN; different stimulation paradigms were used to compare the inhibitory efficiency of SW-NIR. Results showed that SW-NIR could reversibly inhibit acoustically induced CN neural activities: with the increase of laser radiant exposures energy, neural FR decreased significantly and FSL lengthened steadily. Significant inhibition occurred when the optical pulse stimulated prior to the acoustic stimulus. Results indicated that the inhibition relies on the establishment time of the temperature field. Moreover, our preliminary results suggest that short-wavelength infrared could regulate the activities of neurons beyond the neural tissues laser irradiated through neural networks and conduction in vivo. These findings may provide a method for accurate neuromodulation in vivo.

Targets of olivocochlear collaterals in cochlear nucleus of rat and guinea pig.

Descending auditory pathways can modify afferent auditory input en route to cortex. One component of these pathways is the olivocochlear system which originates in brainstem and terminates in cochlea. Medial olivocochlear (MOC) neurons also project collaterals to cochlear nucleus and make synaptic contacts with dendrites of multipolar neurons. Two broadly distinct populations of multipolar cells exist: T-stellate and D-stellate neurons, thought to project to inferior colliculus and contralateral cochlear nucleus, respectively. It is unclear which of these neurons receive direct MOC collateral input due to conflicting results between in vivo and in vitro studies. This study used anatomical techniques to identify which multipolar cell population receives synaptic innervation from MOC collaterals. The retrograde tracer Fluorogold was injected into inferior colliculus or cochlear nucleus to label T-stellate and D-stellate neurons, respectively. Axonal branches of MOC neurons were labeled by biocytin injections at the floor of the fourth ventricle. Fluorogold injections resulted in labeled cochlear nucleus multipolar neurons. Biocytin abundantly labeled MOC collaterals which entered cochlear nucleus. Microscopic analysis revealed that MOC collaterals made some putative synaptic contacts with the retrogradely labeled neurons but many more putative contacts were observed on unidentified neural targets. This suggest that both T- and D-stellate neurons receive synaptic innervation from the MOC collaterals on their somata and proximal dendrites. The prevalence of these contacts cannot be stated with certainty because of technical limitations, but the possibility exists that the collaterals may also make contacts with neurons not projecting to inferior colliculus or the contralateral cochlear nucleus.

Quantitative distribution of choline acetyltransferase activity in rat trapezoid body

There is evidence for a function of acetylcholine in the cochlear nucleus, primarily in a feedback, modulatory effect on auditory processing. Using a microdissection and quantitative microassay approach, choline acetyltransferase activity was mapped in the trapezoid bodies of rats, in which the activity is relatively higher than in cats or hamsters. Maps of series of sections through the trapezoid body demonstrated generally higher choline acetyltransferase activity rostrally than caudally, particularly in its portion ventral to the medial part of the spinal trigeminal tract. In the lateral part of the trapezoid body, near the cochlear nucleus, activities tended to be higher in more superficial portions than in deeper portions. Calculation of choline acetyltransferase activity in the total trapezoid body cross-section of a rat with a comprehensive trapezoid body map gave a value 3–4 times that estimated for the centrifugal labyrinthine bundle, which is mostly composed of the olivocochlear bundle, in the same rat. Comparisons with other rats suggest that the ratio may not usually be this high, but it is still consistent with our previous results suggesting that the centrifugal cholinergic innervation of the rat cochlear nucleus reaching it via a trapezoid body route is much higher than that reaching it via branches from the olivocochlear bundle. The higher choline acetyltransferase activity rostrally than caudally in the trapezoid body is consistent with evidence that the centrifugal cholinergic innervation of the cochlear nucleus derives predominantly from locations at or rostral to its anterior part, in the superior olivary complex and pontomesencephalic tegmentum.

Auditory responses to short-wavelength infrared neural stimulation of the rat cochlear nucleus.

Pulsed Infrared laser is well-known for its high spatial resolution and contact-free stimulation mode. In order to improve the current auditory brainstem implants (ABI), 980 nm-short-wavelength infrared light was applied to stimulate the cochlear nucleus of rats. Single neural activity from contralateral inferior colliculus was recorded during laser stimulation. Animal experimental results showed that 980 nm laser could not directly activate neurons in cochlear nucleus and a photo-acoustic response was observed during stimulation, however, 980 nm laser produces an inhibitory effect on neural responses to sounds; After we added carbon nanoparticles onto the surface of animal cochlear nucleus, 980 nm laser could directly stimulate neurons and no interference between adjacent recording channels was observed. These findings indicated that with the enhancement of carbon nanoparticles, short-wavelength infrared neural stimulation (SW-INS) might be an effective method to overcome the defects of current ABIs. With this method, a new type of optical ABI with transcutaneous stimulating method is quite promising.

Noise-Induced "Toughening" Effect in Wistar Rats: Enhanced Auditory Brainstem Responses Are Related to Calretinin and Nitric Oxide Synthase Upregulation.

An appropriate conditioning noise exposure may reduce a subsequent noise-induced threshold shift. Although this “toughening” effect helps to protect the auditory system from a subsequent traumatic noise exposure, the mechanisms that regulate this protective process are not fully understood yet. Accordingly, the goal of the present study was to characterize physiological processes associated with “toughening” and to determine their relationship to metabolic changes in the cochlea and cochlear nucleus (CN). Auditory brainstem responses (ABR) were evaluated in Wistar rats before and after exposures to a sound conditioning protocol consisting of a broad-band white noise of 118 dB SPL for 1 h every 72 h, four times. After the last ABR evaluation, animals were perfused and their cochleae and brains removed and processed for the activity markers calretinin (CR) and neuronal nitric oxide synthase (nNOS). Toughening was demonstrated by a progressively faster recovery of the threshold shift, as well as wave amplitudes and latencies over time. Immunostaining revealed an increase in CR and nNOS levels in the spiral ganglion, spiral ligament, and CN in noise-conditioned rats. Overall, these results suggest that the protective mechanisms of the auditory toughening effect initiate in the cochlea and extend to the central auditory system. Such phenomenon might be in part related to an interplay between CR and nitric oxide signaling pathways, and involve an increased cytosolic calcium buffering capacity induced by the noise conditioning protocol.

Glutamic acid decarboxylase levels in the cochlear nucleus of rats with acoustic trauma-induced chronic tinnitus

Tinnitus is the perception of phantom sounds, a phenomenon believed to be due to abnormal neuronal activity in auditory regions of the CNS such as the brainstem cochlear nucleus (CN). One possible mechanism for the abnormal neuronal activity in the CN, supported by recent animal studies, is a decrease in GABAergic inhibition. One possible explanation for this is a decrease in the enzyme responsible for the synthesis of GABA, glutamic acid decarboxylase (GAD). In this study, we used immunohistochemistry to analyse the levels of GAD in the dorsal and ventral CN of rats that had been exposed to noise trauma and had been confirmed to exhibit psychophysical evidence of tinnitus (at 17.5 weeks post-exposure) using a conditioned behavioural task. At 22 weeks following noise trauma or sham treatment, the levels of GAD in the dorsal and ventral CN were not significantly different. This result suggests that acoustic trauma that can cause chronic tinnitus is not associated with changes in GAD in the CN at 22 weeks post-exposure.

Target- and input-dependent organization of AMPA and NMDA receptors in synaptic connections of the cochlear nucleus.

We examined the synaptic structure, quantity, and distribution of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)- and N-methyl-D-aspartate (NMDA)-type glutamate receptors (AMPARs and NMDARs, respectively) in rat cochlear nuclei by a highly sensitive freeze-fracture replica labeling technique. Four excitatory synapses formed by two distinct inputs, auditory nerve (AN) and parallel fibers (PF), on different cell types were analyzed. These excitatory synapse types included AN synapses on bushy cells (AN-BC synapses) and fusiform cells (AN-FC synapses) and PF synapses on FC (PF-FC synapses) and cartwheel cell spines (PF-CwC synapses). Immunogold labeling revealed differences in synaptic structure as well as AMPAR and NMDAR number and/or density in both AN and PF synapses, indicating a target-dependent organization. The immunogold receptor labeling also identified differences in the synaptic organization of FCs based on AN or PF connections, indicating an input-dependent organization in FCs. Among the four excitatory synapse types, the AN-BC synapses were the smallest and had the most densely packed intramembrane particles (IMPs), whereas the PF-CwC synapses were the largest and had sparsely packed IMPs. All four synapse types showed positive correlations between the IMP-cluster area and the AMPAR number, indicating a common intrasynapse-type relationship for glutamatergic synapses. Immunogold particles for AMPARs were distributed over the entire area of individual AN synapses; PF synapses often showed synaptic areas devoid of labeling. The gold-labeling for NMDARs occurred in a mosaic fashion, with less positive correlations between the IMP-cluster area and the NMDAR number. Our observations reveal target- and input-dependent features in the structure, number, and organization of AMPARs and NMDARs in AN and PF synapses.

Auditory responses to electric and infrared neural stimulation of the rat cochlear nucleus

In an effort to improve the auditory brainstem implant, a prosthesis in which user outcomes are modest, we applied electric and infrared neural stimulation (INS) to the cochlear nucleus in a rat animal model. Electric stimulation evoked regions of neural activation in the inferior colliculus and short-latency, multipeaked auditory brainstem responses (ABRs). Pulsed INS, delivered to the surface of the cochlear nucleus via an optical fiber, evoked broad neural activation in the inferior colliculus. Strongest responses were recorded when the fiber was placed at lateral positions on the cochlear nucleus, close to the temporal bone. INS-evoked ABRs were multipeaked but longer in latency than those for electric stimulation; they resembled the responses to acoustic stimulation. After deafening, responses to electric stimulation persisted, whereas those to INS disappeared, consistent with a reported “optophonic” effect, a laser-induced acoustic artifact. Thus, for deaf individuals who use the auditory brainstem implant, INS alone did not appear promising as a new approach.

Effects of cochlear ablation on amino acid levels in the rat cochlear nucleus and superior olive

Amino acids have important roles in the chemistry of the auditory system, including communication among neurons. There is much evidence for glutamate as a neurotransmitter from auditory nerve fibers to cochlear nucleus neurons. Previous studies in rodents have examined effects of removal of auditory nerve input by cochlear ablation on levels, uptake and release of glutamate in cochlear nucleus subdivisions, as well as on glutamate receptors. Effects have also been reported on uptake and release of γ-aminobutyrate (GABA) and glycine, two other amino acids strongly implicated in cochlear nucleus synaptic transmission. We mapped the effects of cochlear ablation on the levels of amino acids, including glutamate, GABA, glycine, aspartate, glutamine, taurine, serine, threonine, and arginine, in microscopic subregions of the rat cochlear nucleus. Submicrogram-size samples microdissected from freeze-dried brainstem sections were assayed for amino acid levels by high performance liquid chromatography. After cochlear ablation, glutamate and aspartate levels decreased by 2 days in regions receiving relatively dense innervation from the auditory nerve, whereas the levels of most other amino acids increased. The results are consistent with a close association of glutamate and aspartate with auditory nerve fibers and of other amino acids with other neurons and glia in the cochlear nucleus. A consistent decrease of GABA level in the lateral superior olive could be consistent with a role in some lateral olivocochlear neurons. The results are compared with those obtained with the same methods for the rat vestibular nerve root and nuclei after vestibular ganglionectomy.

Effects of Salicylate on the Inflammatory Genes Expression and Synaptic Ultrastructure in the Cochlear Nucleus of Rats

Aspirin (salicylate), as a common drug that is frequently used for long-term treatment in a clinical setting, has the potential to cause reversible tinnitus. However, few reports have examined the inflammatory cytokines expression and alteration of synaptic ultrastructure in the cochlear nucleus (CN) in a rat model of tinnitus. The tinnitus-like behavior of rats were detected by the gap prepulse inhibition of acoustic startle (GPIAS) paradigm. We investigated the expression levels of the tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), N-methyl D-aspartate receptor subunit 2A (NR2A) mRNA and protein in the CN and compared synapses ultrastructure in the CN of tinnitus rats with normal ones. GPIAS showed that rats with long-term administration of salicylate were experiencing tinnitus, and the mRNA and protein expression levels of TNF-α and NR2A were up-regulated in chronic treatment groups, and they returned to baseline 14 days after cessation of treatment. Furthermore, compared to normal rats, repetitive salicylate-treated rats showed a greater number of presynaptic vesicles, thicker and longer postsynaptic densities, increased synaptic interface curvature. These data revealed that chronic salicylate administration markedly, but reversibly, induces tinnitus possibly via augmentation of the expression of TNF-α and NR2A and cause changes in synaptic ultrastructure in the CN. Long-term administration of salicylate causes neural plasticity changes at the CN level.

Differential molecular profiles of astrocytes in degeneration and re-innervation after sensory deafferentation of the adult rat cochlear nucleus

Ablating the cochlea causes total sensory deafferentation of the cochlear nucleus. Over the first postoperative week, degeneration of the auditory nerve and its synaptic terminals in the cochlear nucleus temporally overlaps with its re-innervation by axon collaterals of medial olivocochlear neurons. At the same time, astrocytes increase in size and density. We investigated the time courses of the expression of ezrin, polysialic acid, matrix metalloprotease-9 and matrix metalloprotease-2 within these astrocytes during the first week following cochlear ablation. All four proteins are known to participate in degeneration, regeneration, or both, following injury of the central nervous system. In a next step, stereotaxic injections of kainic acid were made into the ventral nucleus of the trapezoid body prior to cochlear ablation to destroy the neurons that re-innervate the deafferented cochlear nucleus by axon collaterals developing growth-associated protein 43 immunoreactivity. This experimental design allowed us to distinguish between molecular processes associated with degeneration and those associated with re-innervation. Under these conditions, astrocytic growth and proliferation showed an unchanged deafferentation-induced pattern. Similarly, the distribution and amount of ezrin and matrix metalloprotease-9 in astrocytes after cochlear ablation developed in the same way as under cochlear ablation alone. In sharp contrast, the astrocytic expression of polysialic acid and matrix metalloprotease-2 normally invoked by cochlear ablation collapsed when re-innervation of the cochlear nucleus was inhibited by lesioning medial olivocochlear neurons with kainic acid. In conclusion, re-innervation, including axonal growth and synaptogenesis, seems to prompt astrocytes to recompose their molecular profile, paving the way for tissue reorganisation after nerve degeneration and loss of synaptic contacts.