Eur Arch Otorhinolaryngol DOI 10.1007/s00405-014-3479-3

OTOLOGY

Expression of immediate-early genes in the dorsal cochlear nucleus in salicylate-induced tinnitus Shou-Sen Hu • Ling Mei • Jian-Yong Chen Zhi-Wu Huang • Hao Wu

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Received: 7 February 2014 / Accepted: 25 December 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Spontaneous neuronal activity in dorsal cochlear nucleus (DCN) may be involved in the physiological processes underlying salicylate-induced tinnitus. As a neuronal activity marker, immediate-early gene (IEG) expression, especially activity-dependent cytoskeletal protein (Arc/Arg3.1) and the early growth response gene-1 (Egr-1), appears to be highly correlated with sensoryevoked neuronal activity. However, their relationships with tinnitus induced by salicylate have rarely been reported in the DCN. In this study, we assessed the effect of acute and chronic salicylate treatment on the expression of N-methyl D-aspartate receptor subunit 2B (NR2B), Arg3.1, and Egr1. We also observed ultrastructural alterations in the DCN synapses in an animal model of tinnitus. Levels of mRNA and protein expression of NR2B and Arg3.1 were increased in rats that were chronically administered salicylate (200 mg/kg, twice daily for 3, 7, or 14 days). These levels returned to baseline 14 days after cessation of treatment. However, no significant changes were observed in Egr-1 gene expression in any groups. Furthermore, rats subjected to long-term salicylate administration showed more presynaptic vesicles, thicker and longer postsynaptic densities, and increased synaptic interface curvature. Alterations of Arg3.1 and NR2B may be responsible for the changes in the synaptic ultrastructure. These changes confirm that

S.-S. Hu  L. Mei  J.-Y. Chen  Z.-W. Huang (&)  H. Wu Department of Otolaryngology-Head and Neck Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, 1665 Kongjiang Road, Shanghai 200092, China e-mail: [email protected] S.-S. Hu  L. Mei  J.-Y. Chen  Z.-W. Huang  H. Wu Ear Institute, Shanghai Jiao Tong University School of Medicine, 1665 Kongjiang Road, Shanghai 200092, China

salicylate can cause neural plasticity changes at the DCN level. Keywords Tinnitus  Arg3.1  Egr-1  NR2B  Dorsal cochlear nucleus

Introduction Tinnitus is a disturbing sensation of sound in the absence of external stimulation. Salicylate, which can cause temporary tinnitus, has been widely used in animal models to investigate the neural correlates of tinnitus at different sites along the auditory pathway [1, 2]. As a primary acoustic nucleus, the dorsal cochlear nucleus (DCN) occupies a pivotal position in the hierarchy of functional processes leading to the emergence of tinnitus percepts. Evidence suggests that the DCN is an important brain center involved in the triggering and modulation of tinnitus [3, 4]. Damage to the auditory input pathway leads to hyperactivity in the dorsal cochlear nucleus (DCN) [5, 6]. This suggests that the dorsal cochlear nucleus may undergo neural plasticity changes. Plastic changes in the nervous system include alterations in the expression of the receptors and protein synthesis in the nerve cells [7]. As neuronal activity marker, immediate-early gene (IEG) activation is considered a result of normal synaptic activity. Some IEGs, especially activity-dependent cytoskeletal protein (Arc/Arg3.1) or the early growth response gene-1 (Egr-1), appear to be highly correlated with sensory-evoked neuronal activities [8] and are induced rapidly in neurons by patterned synaptic activity that activates N-methyl-D-aspartate (NMDA) receptors [9]. The Arg3.1 is postulated to provide continuous and precise control over synaptic

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strength, cellular excitability, and consolidation of enduring synaptic plasticity [10, 11]. Egr-1 shows some characteristics similar to those of Arc and also plays crucial roles in neural plasticity [12]. NMDA receptors, which receive the excitatory signal released from the presynaptic terminal into the postsynaptic neuron, are closely related in many aspects of plasticity changes. As modulatory subunits, the roles played by NR2B in the DCN of salicylate-induced tinnitus rats are still unknown. Therefore, we investigated the expression of NR2B and immediate-early genes (Egr-1, Arg3.1) in the DCN in a salicylate-induced tinnitus model. Furthermore, as the neural plasticity may involve changes in the synaptic structure, we also observed ultrastructural alterations of synaptic endings in the DCN in an animal model of tinnitus induced by salicylate. These results will help us understand the role of neural plasticity in DCN involvement in tinnitus. The gap prepulse inhibition of acoustic startle (GPIAS) paradigm was used to detect salicylate-induced tinnituslike behavior in rats [13–15].

Materials and methods Animals and reagents Experimental procedures involving animal handling were approved by the Animal Care and Use Committee of the Shanghai Jiao Tong University School of Medicine. A total of 57 male Sprague–Dawley rats, aged 2–3 months (250–350 g), were randomly divided into four groups: (a) controls (n = 12); (b) acute treatment group with salicylate injected once (n = 6); (c) chronic treatment groups with daily injections of salicylate for 3 days (n = 6) (S3); 7 days (n = 9) (S7); or 14 days (n = 12) (S14); (4) recovery groups with 14 days (n = 6) (S14 ? R14) and 28 days (n = 6) (S14 ? R28) recovery after chronic salicylate administration. Sodium salicylate (Sigma-Aldrich, St. Louis, USA) was dissolved in saline to 200 mg/ml. The acute group received a single intraperitoneal (i.p.) injection of salicylate (400 mg/kg). Rats were heavily anesthetized with sodium pentobarbital (40 mg/kg, intraperitoneally) and killed 2 h after the injection. Rats in the chronic treatment groups were given an injection i.p. of salicylate (200 mg/kg) daily at 08:00 h and at 16:00 h for 3 (S3), 7 (S7), or 14 consecutive days (S14) and were killed at 08:00 h on days 4 (S3), 8 (S7), or 15 (S14), respectively. The S14 ? R14 and S14 ? R28 groups were given i.p. injections for 14 and 28 consecutive days with 14 and 28 days’ recovery, respectively, after salicylate treatment ceased. The control group was given an injection i.p. of saline (200 mg/kg) twice daily at 8:00 and 16:00 for 14 consecutive days. The

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method of grouping rats was based on our previously study [16, 17]. Gap detection testing Tinnitus was assessed using the GPIAS paradigm as described in detail in previous reports [13, 14]. This procedure utilized the acoustic startle reflex test in animals treated with salicylate. GPIAS testing began 1 h before being killed. Each rat was placed in an acoustically transparent cage which rested on a sensitive piezoelectric transducer that generated a voltage proportional to the magnitude of the startle response evoked by sound stimuli generated digitally by digital signal processor (RZ6, Tucker Davis Technologies). The amplitude of the startle response was collected by a computer and analyzed offline. GPIAS sessions were composed of 20 gap trials and 20 no-gap trials. Rats underwent gap detection testing with different band-pass-filtered (1,000 Hz bandwidth) sounds centered at 6, 12, and 16 kHz at 65 dB SPL. Startle responses were elicited by a 20-ms burst of white noise at 100 dB SPL. The gap in the narrowband noise began 100 ms before the onset of the broadband startling noise. The interval between each startle sound was 30–35 s and each test lasted *30 min [14]. Percentage GPIAS was calculated by computing the average ratio of trials with a gap versus trials with no gap for each frequency using the formula: ½ðAvgTnogap  AvgTgapÞ= AvgTnogap  100 % where AvgTgap was the average amplitude during gap trials and AvgTnogap was the average amplitude of trials with no gap [15, 18]. Real-time polymerase chain reaction (RT-PCR) Rats were killed and the DCN were rapidly dissected [4, 19]. Total RNA of each DCN was extracted using TRIzol reagent (TaKaRa) according to the manufacturer’s protocol, followed by reverse transcription to cDNA using a Reverse Transcription Kit (TaKaRa, DRR036A). Primers for NR2B, Egr-1, Arg3.1, and GAPDH were obtained from Shanghai Sangon Biological Engineering Technology& Services Co, Ltd (China). The PCR primer sequences were as follows: NR2B, 50 -TGGCTATCCTGCAGCTGTTTG-30 and 50 -TGGCTGCTCATCACCTCATTC-30 ; Egr-1, 50 GAACAACCCTACGAGCACCTG-30 and 50 -GCCACAA AGTGTTGCCACTG-30 ; Arg3.1, 50 -CTGCCACAGAAGC AGGGTGA-30 and 50 -AGGGTGCCCACCACATACTGA30 ; GAPDH, 50 -GGCACAGTCAAGGCTGAGAATG-30 and 50 -ATGGTGGTGAAGACGCCAGTA-30 . PCR amplification was performed using SYBRÒ Premix Ex TaqTM (TaKaRa, DRR420A).

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The PCR protocol was 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 34 s, and a final dissociation stage, using the ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). We assumed that the amplification efficiency of the target and reference was approximately equal. Relative quantification and calculations were done with the comparative threshold (Ct) cycle method (2-DDCt) [20].

antibody (Cell Signaling Technology, 4153S), 1:50 rabbit anti-NMDAR2B antibody (Cell Signaling Technology, 4212S). Photomicrographs for coronal sections of the DCN were taken and analyzed using image J software to evaluate the number of immunoreactive neurons (IRN). IRN were counted on six sections, separately for the two hemispheres. Density of IRN was expressed as mean ± standard deviation of the mean of the number of positive neurons/sections.

Western blot assay Transmission electron microscopy (TEM) Total proteins were extracted from samples and their concentrations were determined using ultraviolet spectrophotometer (DR/4000UV-VIS, Hach, USA). Equal quantities of protein were loaded, and 12 % (w/v) SDS-PAGE was used to electrophorese Arc or Egr-1 protein and 8 % (w/v) SDSPAGE was used to electrophorese NR2B protein. Proteins were then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in Tris-buffered saline, 0.1 % Tween20, and 5 % skimmed milk powder, and then incubated with primary antibodies overnight and washed in Tris-buffered saline/0.1 % Tween20. Secondary antibodies were diluted in blocking buffer and incubated with the membranes for 2 h at room temperature. Finally, the immunoreactive bands were visualized by the SuperSignal Chemilumnescent Substrate system (Pierce). The images of Western blot analysis were quantified by Image Lab software, and band intensities of Arc, Egr-1, and NR2B were expressed relative to GAPDH. The following antibodies were used: 1:1000 rabbit polyclonal anti-Arc antibody (Abcam, ab23382), 1:1000 rabbit anti-Egr-1 antibody (Cell Signaling Technology, 4153S), 1:1000 rabbit antiNMDAR2B antibody (Cell Signaling Technology, 4212S), 1:5000 goat anti-rabbit IgG-HRP (Jackson).

Changed expression of IEGs may be caused by alteration of synapses in the DCN. To test this hypothesis, nine rats (three from Controls, S7 and S14 groups) were used for the TEM study. The rats were anesthetized heavily with 2 % sodium pentobarbital, perfused through the ascending aorta with 2 % glutaraldehyde. After perfusion, brains were removed from the skull and tissue blocks containing the DCN were dissected and washed in 0.1 M PB. The tissue samples were then immersed in 2 % glutaraldehyde and 1 % osmium tetroxide for 2 h at 4 °C. Subsequently, the tissue blocks were routinely dehydrated using a graded ethanol series. After displacing ethanol by propylene oxide, the tissues were embedded with Epon. A series of consecutive ultrathin sections were prepared to a thickness of 70 lm using a diamond knife in the coronal plane. Subsequently, they were stained with lead citrate and observed under CM-120 TEM (Philips, Eindhoven, North Brabant, the Netherlands). The Image J program was used for quantitative analysis on six sections in the superficial layers of the DCN, separately for the two hemispheres [21]. The number of synaptic vesicles, the thickness of postsynaptic density (PSD), the width of synaptic cleft, and the curvature of synaptic interface were measured [22].

Immunohistochemistry Statistical analysis The immunostaining procedure was performed on six rats (three from Controls and S14 groups) DCN paraffinembedded sections. Sections were deparaffinized in xylene and rapidly rehydrated through graded alcohols. Excess liquid was removed and sections were washed in PBS (pH 7.4) with 0.05 % Tween 20 (PBS-T). To reduce nonspecific binding, normal goat serum (1 % in PBS) was applied to slides for 30 min at 37 °C. The sections were then incubated with primary antibody on consecutive sections. After the sections were rinsed with PBS-T, they were then incubated for 1 h at room temperature with secondary antibodies. The immunoreactions were visualized using 0.015 % H2O2 in 3,30 -diaminobenzidinetetrahydrochloride (DAB)/Tris-buffered saline for 10 min at room temperature. The following antibodies were used: 1:50 rabbit antiArc antibody (Abcam, ab23382), 1:50 rabbit anti-Egr-1

Data were described as mean ± standard deviation (SD). According to the data distribution and the homogeneity of variance, unpaired, two-sided Student’s t tests, one-way analysis of variance (ANOVA) followed by Student– Newman–Keuls (SNK) post hoc tests were used for data comparison between groups. A level of P \ 0.05 was considered statistically significant.

Results Salicylate-induced tinnitus-like behavior in rats Chronic treatment groups (S3, S7, and S14) showed a statistically significant decrease in GPIAS values relative

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Fig. 1 Effects of salicylate in gap prepulse inhibition of acoustic startle (GPIAS) values. Chronic treatment groups (S3, S7 and S14) showed a significant decrease in GPIAS values compared to the control group at 12 and 16 kHz but not at 6 kHz. There was no difference of GPIAS values among the acute treatment, recovery (S14?R14, S14?R28), and control groups

to the control group at 12 and 16 kHz but not at 6 kHz, indicating that these animals were experiencing tinnitus. However, there was no difference in GPIAS values in any other group, indicating that tinnitus-like behavior disappeared 14 days after treatment with salicylate ceased (Fig. 1). Expression of NR2B in the DCN The expression of NR2B in the DCN was evaluated using RT-PCR, Western blot assay, and immunohistochemistry. Figure 2 shows the relative mRNA and protein expression levels of NR2B under different salicylate treatment paradigms. Compared with the control group, levels of mRNA and protein expression of NR2B were upregulated in rats that were administered salicylate chronically (S3, S7, and S14) (P \ 0.05). This increase was not found in the acute group, and was eliminated in the S14 ? R14 and S14 ? R28 groups after cessation of salicylate treatment. The expression of NR2B IRN in the DCN of the sodium salicylate group (S14) was significantly higher than in the control group (Fig. 3).

Fig. 2 Expression of NR2B, Arg3.1, and Egr-1 in the DCN. The expression levels of NR2B and Arg3.1 were significantly higher in rats that were chronically administered with salicylate (S3, S7, and S14) (P \ 0.05) compared to any other group, indicated by real-time PCR and Western blot assays (*P \ 0.05, **P \ 0.01). However, Egr-1 gene expression showed no significant changes in any groups

Compared with the control group, Egr-1 gene expression showed no significant changes in any groups (Fig. 2). Furthermore, no difference was found between the S14 group and control group in Egr-1 IRN in the DCN (Fig. 3).

Expression of Arg3.1 and Egr-1 in the DCN Ultrastructural alterations of synaptic endings Compared with the control group, Arg3.1 gene expression was upregulated in the DCN of rats that were administered salicylate chronically (S3, S7, and S14) (P \ 0.05). There were no significant changes in the acute, S14 ? R14, and S14 ? R28 groups after cessation of salicylate treatment (Fig. 2). The expression of Arg3.1 IRN in the DCN of the sodium salicylate group (S14) was significantly higher than in the control group (Fig. 3).

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Quantitatively, the ultrastructure of DCN neurons showed an increased number of synaptic vesicles (P \ 0.05), thicker postsynaptic densities (PSD) (P \ 0.05), and increased synaptic interface curvature (P \ 0.05) in chronic treatment group with long-term administration of salicylate (S7 and S14), compared with the control group (Fig. 4; Table 1).

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Fig. 4 Ultrastructural alterations of synapses. Compared with the control group, synapses of rats that received long-term administration of salicylate (S7 and S14) showed more presynaptic vesicles (white arrows), thicker and longer PSD (black arrows), and increased synaptic interface curvature and fewer microtubules and neurofilament (arrowheads). Scale bar 0.2 lm

Fig. 3 The expression of NR2B, Arg3.1, and Egr-1 was evaluated using IHC staining in the DCN (9400). Comparison of the IRN of NR2B-, Arg3.1-, and Egr-1-positive cells between two groups (control group and S14). NR2B and Arg3.1 IRN were significantly higher frequent after long-term administration of salicylate (S14) compared to control groups in DCN (*P \ 0.05)

Discussion Plasticity changes include both short-term changes in the strength or efficacy of neurotransmission and long-term changes in the structure of the synapses [23]. They also include changes in the expression of the receptors and protein synthesis in nerve cells. In this study, we have demonstrated that long-term administration of salicylates significantly alters the normal levels of NR2B, Arg3.1, Egr-1, and synaptic structure in the DCN of the rats over time. Our findings suggest that these changes in protein expression may serve as part of the molecular mechanism underlying significant physiological and morphological changes which lead to neural plasticity in the DCN following salicylate administration.

The NMDA receptors are central players in many aspects of plasticity changes [24]. NR2B, as the subunit of the NMDA receptor, is localized in the forebrain, hippocampus, cerebral cortex, striatum, thalamus [25], small cell cap, and fusiform cell layer in the DCN [26]. This is consistent with our immunohistochemical observations. We found that the mRNA and protein expression of the NR2B in the DCN increased significantly after long-term administration of salicylates (Fig. 2). The results are similar, with significant increases in spontaneous activity typically appearing in the DCN after acoustic overstimulation or cisplatin treatment [27]. The upregulation of the NR2B-containing NMDARs increases levels of charge transfer and calcium influx [28], which may serve to increase levels of the overall excitability neurotransmission in DCN. We further found that the NR2B upregulation was reversible. After cessation of salicylate injections, the NR2B expression returned to its original level (Fig. 2). This demonstrates that the NR2B expression can be regulated to restore the balance after cessation of stimulate. Long-lasting forms of synaptic plasticity require new mRNA and protein synthesis. Immediate-early genes have long been thought to account for de novo macromolecular synthesis. The expression of Arg3.1 and Egr-1 was monitored in our tinnitus models. Arg3.1 plays a critical role in the consolidation of enduring synaptic plasticity and in several forms of long-term memory [11]. Similarly, Egr-1 is essential for the persistence of late-phase long-term potentiation (LTP) in the hippocampus and for the consolidation of several forms of long-term memory [12]. Our study has shown that Arg3.1 genes were upregulated in the DCN after chronic administration of salicylate, despite an absence of changes of Egr-1. These results may indicate, in part, promotion of long-term plastic changes through alterations in the transcription and translation of Arg3.1 in the DCN. Because elevated expression of Arg3.1 mRNA in the dendrites of neurons is readily observed following neural activation, Arg3.1 expression is highly dynamic and

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Eur Arch Otorhinolaryngol Table 1 Comparisons of synaptic parameters in different groups N = 15 Synaptic vesicles (number/lm2) Cleft width (lm) Thickness of the PSD (lm) Synaptic curvature

Control

S7

4±3

S14 65 ± 21***

38 ± 12***

0.0082 ± 0.0024

0.0094 ± 0.0013

0.0092 ± 0.0015

0.032 ± 0.006

0.042 ± 0.009*

0.055 ± 0.008*

0.63 ± 0.12

0.95 ± 0.21*

0.85 ± 0.26*

N the number of photographs, * P \ 0.05, compared with control; *** P \ 0.001, compared with control

correlated with neuronal activity. Arg3.1 mRNA detection has now been validated as a marker of neuronal activity in the hippocampus (e.g., new environment exposures), the amygdala (e.g., fear-conditioning memory), and sensory cortices (e.g., sensory deprivation) [29–31]. The elevated expression of Arg3.1 has been shown to achieve heightened levels of neuronal activity in DCN. In addition, Arg3.1 is a direct effector protein at the synapse. Arg3.1 mRNA traffics to dendrites and accumulates at sites of synaptic activity, where it is locally translated and plays important roles in the homeostatic scaling of AMPA receptors and structural modifications at the synapse. The elevated expression of Arg3.1 means increased variability of the neuron structure. Some controversial results have been obtained concerning the regulation of Egr-1 expression after long-term synaptic plasticity. For example, Egr-1 does not seem to change in the CA1 region of the hippocampus when LTP is induced in vivo by high-frequency stimulation, but significantly increases after in vivo tetanic stimulation. No change in Egr-1 expression was reported after LTP was induced in slices of CA1 either in the literature [32]. It has been suggested that different mechanisms for the regulation of the Egr-1 gene may exist in different regions of the rat brain [33]. To date, there have been limited investigations into the time course of changes that occur in the DCN after salicylate injection. Our study has shown that NR2B and Arg3.1 were upregulated in the DCN after chronically administered salicylate. Over the time course, NR2B and Arg3.1 were very closely related, both experiencing fluctuations at similar time points. Arg3.1 protein is found to copurify with the NMDA receptor complex in the postsynaptic density (PSD) [34], and like many immediate-early genes, its induction in vivo requires NMDA receptor activation [35]. This suggests that NR2B and Arg3.1 promote long-term plastic changes together through alterations of transcription and translation in the DCN. Interestingly, our data have demonstrated that NR2B and Arg3.1 mRNA and protein levels are significantly increased after 14 days of salicylate administration but returned to normal 14 days after cessation of treatment. These findings are similar to those of reversibly

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increased cochleoneural activity [36], DPOAEs [16], and prestin in our previous studies [17]. These fluctuations over time indicate that the nervous system continually attempts to rebalance the changes that occur in response to salicylate and for at least 14 days following chronically administered salicylate in the DCN. Salicylate-treated rats showed synapses with more presynaptic vesicles, thicker and longer PSD, and increased synaptic interface curvature. Increased numbers of synaptic vesicles may result in an increase of neurotransmitter release and synaptic transmission. Our results are similar with previous results showing huge lucent pleomorphic vesicles in the dorsal cochlear nucleus of the chinchilla following acoustic trauma [37]. Most of the PSD are thicker, while some is longer and the synaptic interface is more bent. This provides further evidence that the PSD is influenced by the level of presynaptic input activity. These fine structural changes at the synapse may reflect a change in synaptic efficacy as it seems probable that some of the component molecules of the PSD will prove to be involved in mediating the postsynaptic events of synaptic transmission. Interestingly, fewer microtubules and neurofilaments were observed in some synapse. A compensatory reduction induced by hyperactivity is suggested to reduce the transport of vesicles and proteins.

Conclusions Chronically administered salicylate elicited temporary tinnitus, and increased reversibly the mRNA and protein expression level of the Arg3.1 and NR2B gene in the dorsal cochlear nucleus. Alterations of Arg3.1 and NR2B may be responsible for the changes in the synaptic ultrastructure. These changes confirm that salicylate can cause neural plasticity changes at the DCN level. Acknowledgments This study was supported by the National Natural Science Foundation of China (Grant Nos 81170917 and 30973298) and by the Creative Project of the Shanghai Municipal Education Committee (Grant No. 12ZZ103) to Zhi-Wu Huang. Conflict of interest of interest.

The authors do not have any possible conflicts

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