Specific immediate early gene expression induced by high doses of salicylate in the cochlear nucleus and inferior colliculus of the rat / Expressão específica de genes precoces imediatos induzida por doses elevadas de salicilato no núcleo coclear e colículo inferior de rato

Abstract Introduction: Salicylate at high doses induces tinnitus in humans and experimental animals. However, the mechanisms and loci of action of salicylate in inducing tinnitus are still not well known. The expression of Immediate Early Genes (IEG) is traditionally associated with long-term neuronal modifications but it is still not clear how and where IEGs are activated in animal models of tinnitus. Objectives: Here we investigated the expression of c-fos and Egr-1, two IEGs, in the Dorsal Cochlear Nucleus (DCN), the Inferior Colliculus (IC), and the Posterior Ventral Cochlear Nucleus (pVCN) of rats. Methods: Rats were treated with doses known to induce tinnitus in rats (300 mg/kg i.p. daily, for 3 days), and c-fos and Egr-1 protein expressions were analyzed using western blot and immunocytochemistry. Results: After administration of salicylate, c-fos protein expression increased significantly in the DCN, pVCN and IC when assayed by western blot. Immunohistochemistry staining showed a more intense labeling of c-fos in the DCN, pVCN and IC and a significant increase in c-fos positive nuclei in the pVCN and IC. We did not detect increased Egr-1 expression in any of these areas. Conclusion: Our data show that a high dose of salicylate activates neurons in the DCN, pVCN and IC. The expression of these genes by high doses of salicylate strongly suggests that plastic changes in these areas are involved in the genesis of tinnitus.

Resumo Introdução: Salicilato em doses elevadas induz zumbido nos seres humanos e em animais experimentais. No entanto, os mecanismos e loci de ação do salicilato na indução de zumbido ainda não são bem conhecidos. A expressão dos genes precoces imediatos (GPIs) está tradicionalmente associada a alterações neuronais em longo prazo, mas ainda não está claro como e onde os GPIs são ativados em modelos animais de zumbido. Objetivos: No presente estudo investigamos a expressão de c-fos e Egr-1, dois GPIs, no núcleo coclear dorsal (NCD), colículo inferior (CI) e núcleo coclear ventral posterior (NCVp) de ratos. Métodos: Os ratos foram tratados com doses que, conhecidamente, induzem zumbido em ratos (300 mg/kg IP/dia, por três dias) e as expressões das proteínas c-fos e Egr-1 foram analisadas por meio de Western blot e imunoistoquímica. Resultados: Após a administração de salicilato, a expressão da proteína c-fos aumentou significativamente no NCD, NCVp e CI, quando analisados por Western blot. A coloração imunoistoquímica mostrou uma marcação mais intensa de c-fos no NCD, NCVp e CI e um aumento significativo de núcleos positivos de c-fos no NCVp e CI. Não detectamos aumento da expressão de Egr-1 em qualquer dessas áreas. Conclusão: Nossos dados mostram que uma dose alta de salicilato ativa neurônios no NCD, NCVp e CI. A expressão desses genes por doses altas de salicilato sugere que as alterações plásticas nessas áreas estão envolvidas na gênese do zumbido.

Specific immediate early gene expression induced by high doses of salicylate in the cochlear nucleus and inferior colliculus of the rat.

INTRODUCTION: Salicylate at high doses induces tinnitus in humans and experimental animals. However, the mechanisms and loci of action of salicylate in inducing tinnitus are still not well known. The expression of Immediate Early Genes (IEG) is traditionally associated with long-term neuronal modifications but it is still not clear how and where IEGs are activated in animal models of tinnitus. OBJECTIVES: Here we investigated the expression of c-fos and Egr-1, two IEGs, in the Dorsal Cochlear Nucleus (DCN), the Inferior Colliculus (IC), and the Posterior Ventral Cochlear Nucleus (pVCN) of rats. METHODS: Rats were treated with doses known to induce tinnitus in rats (300mg/kg i.p. daily, for 3 days), and c-fos and Egr-1 protein expressions were analyzed using western blot and immunocytochemistry. RESULTS: After administration of salicylate, c-fos protein expression increased significantly in the DCN, pVCN and IC when assayed by western blot. Immunohistochemistry staining showed a more intense labeling of c-fos in the DCN, pVCN and IC and a significant increase in c-fos positive nuclei in the pVCN and IC. We did not detect increased Egr-1 expression in any of these areas. CONCLUSION: Our data show that a high dose of salicylate activates neurons in the DCN, pVCN and IC. The expression of these genes by high doses of salicylate strongly suggests that plastic changes in these areas are involved in the genesis of tinnitus.

Serotonin modulates response properties of neurons in the dorsal cochlear nucleus of the mouse.

The neurochemical serotonin (5-hydroxytryptamine, 5-HT) is involved in a variety of behavioral functions including arousal, reward, and attention, and has a role in several complex disorders of the brain. In the auditory system, 5-HT fibers innervate a number of subcortical nuclei, yet the modulatory role of 5-HT in nearly all of these areas remains poorly understood. In this study, we examined spiking activity of neurons in the dorsal cochlear nucleus (DCN) following iontophoretic application of 5-HT. The DCN is an early site in the auditory pathway that receives dense 5-HT fiber input from the raphe nuclei and has been implicated in the generation of auditory disorders marked by neuronal hyperexcitability. Recordings from the DCN in awake mice demonstrated that iontophoretic application of 5-HT had heterogeneous effects on spiking rate, spike timing, and evoked spiking threshold. We found that 56% of neurons exhibited increases in spiking rate during 5-HT delivery, while 22% had decreases in rate and the remaining neurons had no change. These changes were similar for spontaneous and evoked spiking and were typically accompanied by changes in spike timing. Spiking increases were associated with lower first spike latencies and jitter, while decreases in spiking generally had opposing effects on spike timing. Cases in which 5-HT application resulted in increased spiking also exhibited lower thresholds compared to the control condition, while cases of decreased spiking had no threshold change. We also found that the 5-HT2 receptor subtype likely has a role in mediating increased excitability. Our results demonstrate that 5-HT can modulate activity in the DCN of awake animals and that it primarily acts to increase neuronal excitability, in contrast to other auditory regions where it largely has a suppressive role. Modulation of DCN function by 5-HT has implications for auditory processing in both normal hearing and disordered states.

Salicylate-Induced Hearing Loss Trigger Structural Synaptic Modifications in the Ventral Cochlear Nucleus of Rats via Medial Olivocochlear (MOC) Feedback Circuit.

Lesion-induced cochlear damage can result in synaptic outgrowth in the ventral cochlear nucleus (VCN). Tinnitus may be associated with the synaptic outgrowth and hyperactivity in the VCN. However, it remains unclear how hearing loss triggers structural synaptic modifications in the VCN of rats subjected to salicylate-induced tinnitus. To address this issue, we evaluated tinnitus-like behavior in rats after salicylate treatment and compared the amplitude of the distortion product evoked otoacoustic emission (DPOAE) and auditory brainstem response (ABR) between control and treated rats. Moreover, we observed the changes in the synaptic ultrastructure and in the expression levels of growth-associated protein (GAP-43), brain-derived neurotrophic factor (BDNF), the microglial marker Iba-1 and glial fibrillary acidic protein (GFAP) in the VCN. After salicylate treatment (300 mg/kg/day for 4 and 8 days), analysis of the gap prepulse inhibition of the acoustic startle showed that the rats were experiencing tinnitus. The changes in the DPOAE and ABR amplitude indicated an improvement in cochlear sensitivity and a reduction in auditory input following salicylate treatment. The treated rats displayed more synaptic vesicles and longer postsynaptic density in the VCN than the control rats. We observed that the GAP-43 expression, predominantly from medial olivocochlear (MOC) neurons, was significantly up-regulated, and that BDNF- and Iba-1-immunoreactive cells were persistently decreased after salicylate administration. Furthermore, GFAP-immunoreactive astrocytes, which is associated with synaptic regrowth, was significantly increased in the treated groups. Our study revealed that reduced auditory nerve activity triggers synaptic outgrowth and hyperactivity in the VCN via a MOC neural feedback circuit. Structural synaptic modifications may be a reflexive process that compensates for the reduced auditory input after salicylate administration. However, massive increases in excitatory synapses in the VCN may represent a detrimental process that causes central hyperactivity, leading to tinnitus.

Effects of early and late treatment with L-baclofen on the development and maintenance of tinnitus caused by acoustic trauma in rats.

Subjective tinnitus is a chronic neurological disorder in which phantom sounds are perceived. Recent evidence supports the hypothesis that tinnitus is related to neuronal hyperactivity in auditory brain regions, and consequently drugs that increase GABAergic neurotransmission in the CNS, such as the GABA(B) receptor agonist L-baclofen, may be effective as a treatment. The aim of this study was to investigate the effects of early (5 mg/kg s.c., 30 min and then every 24 h for 5 days following noise exposure) and late treatment (3 mg/kg/day s.c. for 4.5 weeks starting at 17.5 weeks following noise exposure) with l-baclofen on the psychophysical attributes of tinnitus in a conditioned lick suppression model following acoustic trauma in rats. Acoustic trauma (a 16-kHz, 115-dB pure tone presented unilaterally for 1h) resulted in a significant decrease in the suppression ratio (SR) compared to sham controls in response to 20-kHz tones at 2, 10 and 17.5 weeks post-exposure (P ≤ 0.009, P ≤ 0.02 and P ≤ 0.03, respectively). However, l-baclofen failed to prevent the development of tinnitus when administered during the first 5 days following the acoustic trauma and also failed to reverse it when treatment was carried out every day for 4.5 weeks. We also found that treatment with L-baclofen did not alter the expression of the GABA(B)-R2 subunit in the cochlear nucleus of noise-exposed animals.

Reversible neurotoxicity of kanamycin on dorsal cochlear nucleus.

The time course of aminoglycoside neurotoxic effect on cochlear nucleus is still obscure. We examined dynamic pathological changes of dorsal cochlear nucleus (DCN) and investigated whether apoptosis or autophagy was upregulated in the neurotoxic course of kanamycin on DCN after kanamycin treatment. Rats were treated with kanamycin sulfate/kg/day at a dose of 500mg by subcutaneous injection for 10 days. Dynamic pathological changes, neuron density and neuron apoptosis of the DCN were examined at 1, 7, 14, 28, 56, 70 and 140 days after kanamycin treatment. The expressions of JNK1, DAPK2, Bcl-2, p-Bcl-2, Caspase-3, LC3B and Beclin-1 were also detected. Under transmission electron microscopy, the mitochondrial swelling and focal vacuoles as well as endoplasmic reticulum dilation were progressively aggravated from 1 day to 14 days, and gradually recovered from 28 days to 140 days. Meanwhile, both autophagosomes and autolysosomes were increased from 1 day to 56 days. Only few neurons were positive to the TUNEL staining. Moreover, neither the expressions of caspase-3 and DAPK2 nor neurons density of DCN changed significantly. LC3-II was drastically increased at 7 days. Beclin-1 was upgraded at 1 and 7 days. P-Bcl-2 increased at 1, 7, 14 and 28 days. JNK1 increased at 7 days, and Bcl-2 was downgraded at 140 days. LC3-B positive neurons were increased at 1, 7 and 14 days. These data demonstrated that the neurons damage of the DCN caused by kanamycin was reversible and autophagy was upregulated in the neurotoxic course of kanamycin on DCN through JNK1-mediated phosphorylation of Bcl-2 pathway.

The generation of direction selectivity in the auditory system.

Both human speech and animal vocal signals contain frequency-modulated (FM) sounds. Although central auditory neurons that selectively respond to the direction of frequency modulation are known, the synaptic mechanisms underlying the generation of direction selectivity (DS) remain elusive. Here we show the emergence of DS neurons in the inferior colliculus by mapping the three major subcortical auditory nuclei. Cell-attached recordings reveal a highly reliable and precise firing of DS neurons to FM sweeps in a preferred direction. By using in vivo whole-cell current-clamp and voltage-clamp recordings, we found that the synaptic inputs to DS neurons are not direction selective, but temporally reversed excitatory and inhibitory synaptic inputs are evoked in response to opposing directions of FM sweeps. The construction of such temporal asymmetry, resulting DS, and its topography can be attributed to the spectral disparity of the excitatory and the inhibitory synaptic tonal receptive fields.

Hyperbaric oxygenation with corticoid in experimental acoustic trauma.

Among possible therapies after acute acoustic trauma, hyperbaric oxygenation (HBO) combined with corticoid was found effective in several animal studies. Such evidence was obtained for moderate 20-25 dB losses. The aim of this study was to further assess this therapy for noise-induced hearing losses greater than previously examined. Sixty-five ears from thirty-six adult guinea pigs were used. Acoustically evoked responses from intracranial electrodes chronically implanted bilaterally into the ventral cochlear nucleus were used to assess acoustic sensitivity alterations. Trauma sound was a third-octave noise-band around 8 kHz presented bilaterally at 115 dB SPL for 45 min. One control group received no treatment, one group was treated with HBO only and another with corticoid only both starting within one day post-trauma, two groups were treated with both HBO and corticoid starting for one group within one day post-trauma, and for the second group at 6 days post-trauma. Acoustic thresholds were measured between the 6th and the 16th days after acoustic trauma. Animals treated with HBO alone or corticoid alone did not differ from controls. Combined HBO and corticoid therapy provided significant protection from noise-induced loss of auditory thresholds, especially when started one day post-exposure. Hearing loss reduction induced by HBO combined with corticoid was of similar magnitude (about 10-15 dB) as in previous studies although the induced hearing loss was considerably greater (about 40 dB instead of 20-25 dB).

Projections of the second cervical dorsal root ganglion to the cochlear nucleus in rats.

Physiological, anatomical, and clinical data have demonstrated interactions between somatosensory and auditory brainstem structures. Spinal nerve projections influence auditory responses, although the nature of the pathway(s) is not known. To address this issue, we injected biotinylated dextran amine into the cochlear nucleus or dorsal root ganglion (DRG) at the second cervical segment (C2). Cochlear nucleus injections retrogradely labeled small ganglion cells in C2 DRG. C2 DRG injections produced anterograde labeling in the external cuneate nucleus, cuneate nucleus, nucleus X, central cervical nucleus, dorsal horn of upper cervical spinal segments, and cochlear nucleus. The terminal field in the cochlear nucleus was concentrated in the subpeduncular corner and lamina of the granule cell domain, where endings of various size and shapes appeared. Examination under an electron microscope revealed that the C2 DRG terminals contained numerous round synaptic vesicles and formed asymmetric synapses, implying depolarizing influences on the target cell. Labeled endings synapsed with the stalk of the primary dendrite of unipolar brush cells, distal dendrites of presumptive granule cells, and endings containing pleomorphic synaptic vesicles. These primary somatosensory projections contribute to circuits that are hypothesized to mediate integrative functions of hearing.

Evidence for tinnitus-related plasticity in the auditory and limbic system, demonstrated by arg3.1 and c-fos immunocytochemistry.

Distributions of arg3.1 and c-fos immunoreactive neurons (IRN) in gerbil auditory cortex (AC) and amygdala showed characteristic differences when comparing systemic application of the tinnitus-eliciting drug salicylate with acoustic stimulation or saline injections. In AC, arg3.1 IRN induced by stimulation focused in regions corresponding to the frequency content of the stimulus. Injections of salicylate (350 mg/kg body weight) led to accumulation of arg3.1 IRN in the high frequency domain, while saline injections produced a diffuse distribution. After all treatments, c-fos IRN outnumbered arg3.1 IRN in AC and showed a broad distribution. In subcortical auditory structures arg3.1 IRN were absent in all but one brain. In ventral cochlear nucleus, c-fos IRN were always found after stimulation and often also after saline injections, whereas none were present when injecting salicylate. Similarly, in inferior colliculus, numbers of c-fos IRN were lowest after salicylate injections. In the amygdala, c-fos and arg3.1 IRN were increased substantially after salicylate injections compared to auditory stimulation or saline injections. In particular in its central nucleus, c-fos and arg3.1 IRN were found exclusively after the tinnitus-inducing treatment, suggesting that coactivation of the AC and the amygdala may by an essential feature of tinnitus-related activation.

Does exogenous GM1 ganglioside enhance the effects of electrical stimulation in ameliorating degeneration after neonatal deafness?

This study examined the combined effects of administration of exogenous GM1 ganglioside and electrical stimulation on the cochlear nucleus (CN) of cats deafened neonatally by ototoxic drugs. Five normal hearing adult cats served as controls. Another 12 cats were deafened bilaterally by daily injections of neomycin sulfate (60 mg/kg) for 17-21 days after birth until auditory brainstem testing demonstrated profound hearing loss. Six of the deaf animals comprised the GM1 group, which received daily injections of GM1 ganglioside (30 mg/kg) for 28-38 days during the period after profound deafness was confirmed, and prior to receiving a cochlear implant. The non-GM1 group (n=6) received no treatment during this interim period. All the deafened animals underwent unilateral cochlear implantation at 6-9 weeks postnatal and received several months (mean duration, 32 weeks) of chronic electrical stimulation (4 h/day, 5 days/week). Stimulation was delivered by intracochlear bipolar electrodes, using electrical signals that were designed to be temporally challenging to the central auditory system. Results showed that in the neonatally deafened animals, both the GM1 and non-GM1 groups, the volume of the CN was markedly reduced (to 76% of normal), but there was no difference between the animals that received GM1 and those that did not. The cross sectional areas of spherical cell somata in both GM1 and non-GM1 groups also showed a highly significant reduction in size, to < or =75% of normal after neonatal deafening. Moreover, in both the GM1 and non-GM1 groups, the spherical cells in the CN ipsilateral to the implanted cochlea were significantly larger (6%) than cells in the control, unstimulated CN. Again, however, there was no significant difference between the GM1 group and the non-GM1 group in spherical cell size. These results contrast sharply with previous reports that exogenous GM1 prevents CN degeneration after neonatal conductive hearing loss and partially prevents spiral ganglion cell degeneration when administered immediately after ototoxic drug deafening in adult animals. Taken together, findings to date suggest that GM1 may be effective in preventing degeneration only if the GM1 is administered immediately at the time hearing loss occurs.

Generators of the brainstem auditory evoked potential in cat. II. Correlating lesion sites with waveform changes.

Brainstem regions involved in generating the brainstem auditory evoked potential (BAEP) were identified by examining the effects of lesions on the click-evoked BAEP in cats. An excitotoxin, kainic acid, was injected into various parts of the cochlear nucleus (CN) or into the superior olivary complex (SOC). The locations of the resulting lesions were correlated with the changes produced in the various extrema of the BAEP waveforms. The results indicate that: (1) the earliest BAEP extrema (P1, N1 (recorded between vertex and the earbar ipsilateral to the stimulus) and P1a, P1b, (vertex to contralateral earbar)) are generated by cells with somata peripheral to the CN; (2) P2 is primarily generated by posterior anteroventral CN (AVCNp) and anterior posteroventral CN (PVCNa) cells; (3) SOC, anterior anteroventral CN (AVCNa), AVCNp, and PVCNa cells are involved in generating P3; (4) AVCNa cells are the main CN cells involved in P4, N4, and P5 generation; (5) both ipsilateral and contralateral SOC cells have a role in generating monaurally evoked P4 and P5; and (6) P5 is generated by cells with characteristic frequencies below 10 kHz. From (2) and (4), it is clear that P2 and P4-P5 are generated by cells in distinct, parallel pathways.