Open Access

Expression of glycine receptors and gephyrin in rat medial vestibular nuclei and flocculi following unilateral labyrinthectomy

  • Authors:
    • Wen Zhou
    • Liu-Qing Zhou
    • Hong Shi
    • Yang-Ming Leng
    • Bo Liu
    • Su-Lin Zhang
    • Wei-Jia Kong
  • View Affiliations

  • Published online on: September 27, 2016     https://doi.org/10.3892/ijmm.2016.2753
  • Pages: 1481-1489
  • Copyright: © Zhou et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The medial vestibular nucleus (MVN) and the cerebellar flocculus have been known to be the key areas involved in vestibular compensation (VC) following unilateral labyrinthectomy (UL). In this study, we examined the role of gephyrin and glycine receptor (GlyR) in VC using Sprague-Dawley rats, in an aim to gain deeper insight into the mechanisms responsible for VC. The expression of the α1 and β subunits of GlyR and gephyrin was immunohistochemically localized in rat MVN and flocculi. The mRNA and protein expression of GlyR (α1 and β subunits) and gephyrin was quantitatively determined by RT-qPCR and western blot analysis at 8 h, and at 1, 3 and 7 days following UL. It was found that in the ipsilateral MVN, the mRNA and protein expression of the β subunit of GlyR was significantly increased in comparison to the sham-operated (P<0.01) rats, and in comparison to the contralateral side (P<0.01) at 8 h following UL. In the ipsilateral flocculi, GlyR β protein expression was significantly elevated (P<0.01 for all), as compared to the sham-operated rats at 8 h, and at 1 and 3 days and to the contralateral side 8 h, 1 and 3 days following UL. No significant differences were observed in the mRNA and protein expression of GlyR α1 and gephyrin in the MVN or flocculi between the two sides (ipsilateral and contralateral) in the UL group, and between the sham-operated group and the UL group at any time point. The findings of our study thus suggest that GlyR plays a major role in the recovery of the resting discharge of the deafferented MVN neurons in the central vestibular system.

Introduction

Studies have demonstrated that unilateral labyrinthectomy (UL) causes severe oculomotor and postural symptoms in all animal models. The initial symptoms tend to subside within a few days. This rebalancing process is referred to as vestibular compensation (VC) (13). VC has been a well-established animal model used for the study of the deafferentation-induced plasticity of the central nervous system (CNS). It is generally believed that VC is a response to the substantial imbalance between the two sides of the medial vestibular nucleus (MVN), which are linked by the reciprocal commissural inhibitory system. This theory can well explain the drastic asymmetry in the resting discharge of MVN neurons following UL (4). Moreover, a number of studies have suggested that the cerebellum may also be involved in VC (59). The flocculus target neurons (FTNs), which are located at the rostral MVN, have been found to partake in vestibular adaptation (10). Additionally, the ipsilateral flocculus is essential for the enhanced intrinsic excitability of MVN neurons following UL (10,11). Therefore, both the cerebellum and MVN may play a role in VC. However, to date, as regards the time course of 'rebalancing' between the bilateral neuronal activities of the vestibular nuclei, the exact cellular and molecular mechanisms remain poorly understood.

Several hypotheses have been proposed regarding the mechanisms of VC. It has been found that glycinergic FTNs surrounded by Purkinje cell terminals project axons to the ipsilateral abducens nucleus (12,13). The response of MVN neurons to vestibular nerve stimulation is modulated by glycinergic, as well as GABAergic inhibitory inputs (14). Bagnall et al demonstrated the existance of commissurally projecting glycinergic neurons in the MVN of mice (15). Li et al (16) reported that the glycine concentration was decreased in the dorsal part of the bilateral lateral vestibular nucleus followng unilateral vestibular ganglionectomy in rats. Vibert et al (17) found that the response of the MVN neurons to glycine in slices was reduced 3 days following labyrinthectomy in guinea pigs. Recently, Lim et al (18) observed a significantly elevated glycinergic quantal current amplitude in mouse contralateral MVN neurons and a higher current frequency in both ipsilateral and contralateral neurons 4 h post-UL. Therefore, the augmented resting discharge in the deafferented MVN neurons may be ascribed to changes in the number, affinity or effect of the central vestibular glycine receptors (GlyRs). Nonetheless, Eleore et al failed to observe any post-UL difference in expression of gephyrin and various GlyR subunits in the bilateral MVNs (19). Thus far, it remains unknown which type of neurons expresses GlyRs and gephyrin in the central vestibular system. In this study, we examined the role of gephyrin and GlyRs in VC, in an aim to gain deeper insight into the mechanisms of VC.

GlyR consists of four α subunits (α1–α4) and one β subunit. The α subunit is an indispensable subunit capable of forming functional homomeric channels. The β subunit orchestrates ligand binding. Consequently, the subunit stoichiometry is 3α:2β (20,21). The α1 subunit is ubiquitous in the adult brain, while the α2 subunit is highly expressed in the embryonic brain and its expression diminishes with development (22). The α3 and α4 subunits are rare (23). The β subunit is expressed throughout the embryonic and adult brain (24,25) and one of its roles is synaptic anchoring of the GlyR through binding to gephyrin (26,27). To better understand the role of gephyrin and GlyR in VC, in this study, we investigated changes in the expression of gephyrin and the α1 and β subunits of GlyR in MVN neurons and flocculi at different time points following UL in rats.

Materials and methods

Animal experiments

A total of 99 male Sprague-Dawley (SD) rats (weighing 200–250 g) was used. Among these, 3 rats were used for immunohistochemistry, 48 for western blot analysis and the other 48 for reverse transcription-quantitative PCR (RT-qPCR). The animals were obtained from the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). All experimental procedures were conducted in strict accordance with 'Guide for the Care and Use of Laboratory Animals' (no. 80-23, NIH publications, revised 1996) formulated by the National Institutes of Health and approved by the Institutional Review Committee on animal research of our college. For western blot analysis and RT-qPCR, the animals were randomly divided into 2 groups as follows: a UL group (n=24) and a sham-operated group (n=24). A total of 6 animals from each group was sacrificed under anesthesia (ketamine-chlorpromazine mixture 10:1, 2 ml/kg, intraperitoneal injection) at 8 h, and at 1, 3 and 7 days following UL or sham operation. The 4 post-operative time points were selected as representative ones of the restoration of VC based on previous studies and animal studies (13,28,29).

Immunofluorescence double-labeling

As aforementioned, in 3 rats, the α1 or β subunits of GlyR and gephyrin on Purkinje cells (including axon and terminals) in the MVN and flocculi were stained using immunofluorescence double-labeling. The 3 rats were anesthetized deeply with ketamine-chloprromazine mixture (10:1, 2 ml/kg, intraperitoneal injection) and perfused transcardially with 0.9% physiological saline and then with 4% paraformaldehyde (in 0.1 M phosphate buffer). After decapitating the bodies, the temporal bones were removed and the brains were dissected. The cerebellum and brain stem portions were fixed in the same fixative for 12 h at 4°C and then rinsed with distilled water for 30 min, dehydrated with a graded alcohol series, cleared in xylene, immersed in paraffin and then embedded in paraffin. Finally, following the rat brain atlas of Paxinos and Watson (30), the cerebellum sections with flocculus and the brainstem sections with MVN were sectioned at 5 µm and collected on poly-L-lysine-coated glass slides. The boundaries of the MVN and flocculus were identified based on the brain atlas (30). Purkinje cells (including axons and terminals) in the flocculus and MVN were immunolabeled with, calbindin, an intracellular calcium-binding protein, and a reliable marker for labeleing these cells (13,31). For immunofluorescence labeling, the cerebellum and brain stem samples which were sectioned (into 5-µm-thick slices), were formalin-fixed, paraffin-embedded, de-waxed, re-hydrated, with antigens retrieved, as previously described (32). The brain sections with flocculi were then incubated with the following primary antibodies: mouse anti-calbindin monoclonal antibody (1:30; ab82812; Abcam, Cambridge, UK) and rabbit polyclonal antibody against the α1 subunit of GlyR (1:100; AB5052; Millipore, Billerica, MA, USA) or the β subunit of GlyR (1:50; sc-20134; Santa Cruz Biotechnology, Inc., Paso Robles, CA, USA) or gephyrin (1:100; ab25784; Abcam). Secondary antibodies included Alexa 488 (green) conjugated to donkey anti-mouse IgG (1:300; 715-545-150) and Alexa 594 (red) conjugated to donkey anti-rabbit IgG (1:300; 711-585-152) (both from Jackson Immunoresearch Laboratories Inc., West Grove, PA, USA). In the end, the sections were counterstained with a DAPI mix (Beyotime Institute of Biotechnology, Haimen, China). Subsequently, the sections were observed under a laser scanning confocal microscope (Nikon, Tokyo, Japan) using laser beams of 490 and 590 nm with appropriate emission filters, 520–530 nm and 600–630 nm, respectively. Control tests were incubated with PBS instead of the primary antibody and no immunostaining was observed. The size and brightness of the images were adjusted using Adobe Photoshop 5.0 software (Adobe Systems, Inc., San Jose, CA, USA) during the preparation of the figures.

UL

With the UL group, the labyrinthectomy was performed on the right side by following the technique previously reported by Zhou et al (32) and Kitahara et al (33). Briefly, the right tympanic bulla was carefully opened via a retroauricular surgical approach. The stapedial artery was cauterized at two points. The contents of the canal ampullae, the utricle and the saccule were aspirated and the wound was sutured. For the sham-operation group, the tympanic bulla was cut open, with the tympanic membrane and ossicles left intact.

RT-qPCR

At 8 h, and 1, 3 and 7 days following UL, 48 rats (n=6 per group) were sacrificed under anesthesia (ketamine-chlorpromazine mixture 10:1, 2 ml/kg, intraperitoneal injection). Following decapitation, the bilateral flocculi were carefully removed from the skull. The brains were immediately placed into 0.9% ice-cold saline for 1 min. According to the rat brain atlas of Paxinos and Watson (30), horizontal brainstem slices including the MVN were taken in order to dissect each nucleus. To take a slice including the MVN, a brainstem slice (1.5 mm thickness) just caudal from the abducens nucleus was dissected, and the bilateral MVN were dissected separately under microscopic guidance using a sharp blade, as previously described by Horii et al (34). All these procedures were performed on a chilled plate in order to prevent possible RNA degradation. Total RNA was extracted from the brain tissues using an RNeasy mini kit (Axygen, Hangzhou, China). cDNA was reverse transcribed using a PrimeScript™ RT reagent kit with gDNA Eraser (Takara Biotechnology Co., Ltd., Dalian, China). Quantitative (real-time) PCR (qPCR) was performed on a real-time PCR system (model 7300; Applied Biosystems, Foster City, CA, USA). The primer pairs for GlyR α1, GlyR β, gephyrin and an internal control (β-actin) were as follows: GlyR α1 forward, 5′-GTGCTCACCATGACCACACAGA-3′ and reverse, 5′-GACACAAAGTTGACAGCGGCATA-3′; GlyR β forward, 5′-TGTATGCACCAACCCTGCTGA-3′ and reverse, 5′-AGCCACACATCCAGTGCCTTC-3′; gephyrin forward, 5′-CTGGACCCTCGCCCAGAATA-3′ and reverse, 5′-CTGTCTTTGGAGGTAGCATCAGCA-3′; β-actin forward, 5′-CCTGGAGAAGAGCTATGAGC-3′ and reverse, 5′-ACAGGATTCCATACCCAGG-3′. The thermal cycling conditions were as follows: 1 min at 95°C, and then 40 cycles of 15 sec at 95°C, 20 sec at 60°C, and 35 sec at 72°C. Dissociation curves were plotted after each run to ensure primer specificity. The differences (ΔCt) in the Ct values between the target mRNA and the internal control (β-actin) were calculated. The changes in the relative mRNA levels were analyzed using the 2−ΔΔCt method, as described in previous studies (32,35,36).

Western blot analysis

Western blot analysis was performed as previously described (32,36). Briefly, protein from the MVN and flocculi was extracted according to the stereotaxic rat brain atlas of Paxinos and Watson (30). Total protein concentrations were determined using a BCA protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Ten micrograms of each sample were resolved on an 8% SDS-PAGE gel. The separated proteins in the trimmed SDS gel were transferred onto PVDF membranes using a transblotting apparatus (Bio-Rad Laboratories, Inc., Hercules, CA, USA), as previously described (32,36,37,38). GlyR α1 (48 kDa) and GlyR β (58 kDa) were transferred for 1 h; gephyrin (93 kDa) was transferred for 1.5 h. Prestained protein markers (10–170 kDa; Bio-Rad Laboratories, Inc.) were always run on the same gel. The blots were blocked in 5% fat-free milk in TBS containing Tween-20 for 1 h. The membranes were incubated at 4°C overnight with affinity-purified polyclonal antibodies against the GlyR α1 (AB5052) and gephyrin (AB5725) (Millipore) diluted at 1:1,000; with monoclonal antibody against the GlyR β (sc-365819 Santa Cruz Biotechnology, Inc.) diluted at 1:400. The samples were then incubated with secondary anti-rabbit (111-035-003) or anti-mouse (115-035-003) antibodies (diluted at 1:3,000; both from Jackson Immunoresearch Laboratories Inc.) for 1 h. The monoclonal antibody against GAPDH (GTX627408, GeneTex, Irvine, CA, USA) served as an internal control. The optical density of the protein bands was measured by utilizing the Bio-Rad Quantity One software package (Bio-Rad Laboratories, Inc.), as previously described (32,36,39).

Statistical analysis

Data are expressed as the means ± SEM and were statistically analyzed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). Statistical significance was evaluated by employing the single analysis of variance (ANOVA), or the Student-Newman-Keuls multiple comparison test or the paired Student's t-test. Statistical significance was set at P<0.05.

Results

Immunohistochemical findings

Strong immunoreactivity for the α1 and β subunits of GlyR and gephyrin was found in all the MVN neurons. The neurons labeled with the α1 and β subunits of GlyR and gephyrin were densely surrounded by axonal terminals of Purkinje cells in the MVN (Fig. 1A–I). The same cell can be identified by the arrows. The images shown in panels A'–C', D'–F' and G'–I' are the enlarged images of the cells shown in panels A–C, D–F and G–I, respectively.

In the flocculi, the α1 and β subunits of GlyR, and gephyrin (Fig. 2A–I) were moderately to intensely stained in the layers of granular cells and Purkinje cells. The same cell can be identified by the arrows. The images shown in panels A'–C', D'–F' and G'–I' are the enlarged images of the cells shown in panels A–C, D–F and G–I, respectively.

UL-induced changes in the mRNA expression of GlyRs and gephyrin in MVN and floccule

At any time point following UL, no significant differences were observed in the mRNA expression of GlyR α1 (Fig. 3A and D) and gephyrin (Fig. 3C and F) in the MVN or flocculi between the two sides (ipsilateral and contralateral) in the UL group, and between the sham-operated and UL group.

However, the mRNA expression of GlyR β in the ipsilateral MVN in the UL group was significantly increased as compared to the sham-operated group (P<0.01) and to the contralateral MVN (P<0.01) 8 h following UL (Fig. 3B). On the other hand, there were no significant differences in GlyR β mRNA expression between the contralateral MVN in the UL group and the sham-operated group (Fig. 3B). Furthermore, in the ipsilateral flocculi, the mRNA expression of GlyR β was elevated as compared to the contralateral flocculi and the sham-operated group at 8 h (P<0.05 vs. contralateral side; P<0.05 vs. sham-operated group), 1 day (P<0.01 vs. contra-lateral side; P<0.01 vs. sham-operated group) and 3 days (P<0.05 vs. contralateral side; P<0.05 vs. sham-operated group) following UL (Fig. 3E). By contrast, no significant changes in GlyR β mRNA expression were observed between the contralateral flocculi and the sham-operated group (Fig. 3E).

UL-induced changes in the protein levels of GlyRs and gephyrin in MVN and floccule

In the MVN, the protein expression of both GlyR α1 (Fig. 4A, panels a and b) and gephyrin (Fig. 4C, panels a and b) exhibited no obvious difference at any time point following UL.

In the flocculi, the protein expression of both GlyR α1 (Fig. 5A, panels a and b) and gephyrin (Fig. 5C, panels a and b) exhibited no obvious difference at any time point following UL.

However, the protein expression of the GlyR β in the ipsilateral MVN was significantly increased in comparison to the sham-operated group (P<0.01) and to the contralateral side (P<0.01) at 8 h following UL (Fig. 4B, panels a and b). On the other hand, no significant difference was observed in the protein expression of GlyR β between the contralateral MVN and the sham-operated group (Fig. 4B, panel a). Furthermore, in the ipsilateral flocculi, GlyR β protein expression was significantly increased (P<0.01 for all), as compared to the sham-operated group at 8 h (Fig. 5B, panel a), 1 day (Fig. 5, panel a) and 3 days (Fig. 5, panel a) following UL, and in comparison to the contralateral side at 8 h (Fig. 5B, panel b), 1 day (Fig. 5B, panel b) and 3 days (Fig. 5, panel b) following UL. No significant difference in GlyR β protein expression was observed between the contralateral flocculi and the sham-operated group (Fig. 5B, panel a).

Fig. 6A shows an immunoblot of GlyR β expression in the ipsilateral side in the MVN at 8 h following UL as compared to the contralateral side in the UL group and to the sham-operated group. Following UL, GlyR β expression markedly increased in the MVN in the ipsilaterial side compared to the contra-lateral side and to the sham-operated group. Fig. 6B–D shows an immunoblot of GlyR β in the ipsilateral side in the flocculus at 8 h, and at 1 and 3 days following UL compared to the contralateral side, and to the sham-operated group. Following UL, GlyR β expression was also markedly increased in the ipsilateral side.

Discussion

This study demonstrated that the GlyR β subunit underwent a significant change in expression at the early stage of VC. Following UL, the expression levels of the GlyR α1 subunit and gephyrin in the MVN and flocculi did not exhibit any significant differences at either the mRNA or protein level. These findings suggested that the GlyR α1 subunit or gephyrin may not be involved in VC. Alternatively, the GlyR α1 subunit or gephyrin may experience some functional, not quantitative, changes in the MVN and flocculi during VC as a result of modification of receptor-binding affinity and its efficacy. However, a previous animal study (19) reported that no asymmetry in the mRNA expression between the two sides of the MVN was detected on autoradiographs at 5 h, and at 1, 3, 8, 30 and 60 days following UL, and in terms of the intensity of immuofluorescence staining at 5 h, and at 1, 3 and 8 days following UL. The discrepancies between our study and the research in question may be that: i) the findings may be differ when different post-UL time points were used; ii) the different experimental procedures used may yield different results; and iii) the role of GlyR subunits may vary as a consequence of deafferentation, and the antibody was unable to distinguish between the α1 and β subunits of GlyR.

Recently, it has been demonstrated that the suppression of GlyR function decreased the excitability of MVN neurons in mice (40). Inhibitory commissural neurons, which connect the two sides of the MVN, have been shown to be GABAergic and glycinergic (15,41). Several types of neurons in the MVN receive Purkinje cell synapses from the cerebellar flocculus (19). These FTNs involve two types of glycinergic neurons (13). Our immunohistochemical findings were consistent with two previous studies concerning the distribution of GlyR and gephyrin in MVN (19,42). Our study went one step further, in that we found that these GlyR- and gephyrin-expressing neurons were densely surrounded by Purkinje cell terminals. Presumably, GlyR and gephyrin may be present in FTNs. Lu et al (43) observed that post-synaptic GlyRs, when facing GABAergic nerve endings, could be activated by synaptic GABA release in auditory synapses. This raises the possibility that post-synaptic GlyRs, particularly the β subunit of GlyR, may be activated by GABA in the FTNs. Li and Xu (44) reported that GlyR mediated the downregulation of the GABAA receptors. When the GABAergic inhibition is dominant, GlyRs should modulate excitability by suppressing the GABAergic effect (24). In addition, Grudzinska et al (25) suggested that in heteromeric GlyRs, the role of the β subunit may be more essential than that of the α1 subunit, therefore the α1:β stoichiometry may be 2:3 instead of 3:2 (20,21). Therefore, such an increase in the expression of the β subunit of GlyR following UL may decrease floccular inhibition on ipsilateral FTNs and contralateral commissural inhibition on ipsilateral MVN neurons (Fig. 7). Thus, a rebalance of the resting activity may be achieved between the ipsilateral and contralateral MVN.

The mRNA and protein expression of the GlyR β subunit was significantly increased in the ipsilateral flocculi at 8 h, and at 1 and 3 days following UL. This increase may be ascribed to the different contribution of the α1 and β subunits to channel opening (25). Moreover, we also found the β subunit of GlyR present in cell bodies and axon terminals of Purkinje cells. Purkinje cells are a major type of inhibitory neurons that release GABA, which acts as a neurotransmitter in the cerebello-vestibular pathways (45). Zhang et al (46) reported that GlyRs regulated synaptic plasticity by altering GABAergic neuro-transmission. Johnston et al (10) hypothesized that following UL, cerebellar cortical plasticity could induce vestibular neuronal plasticity. On the basis of these findings, we came to the conclusion that, following UL, the upregulated expression of the β subunit of GlyR may contribute to cerebellar cortical plasticity. As a result, a rebalance of the neuronal activity was attained between the ipsilateral and contralateral MVN.

In conclusion, GlyR may play a major role in the recovery of the resting discharge of the deafferented MVN neurons in the central vestibular system. However, the mechanisms through which the β subunit of GlyR functions in the central vestibular system during VC warrants further investigation.

Abbreviations:

CNS

central nervous system

FTNs

flocculus target neurons

GlyRs

glycine receptors

MVN

medial vestibular nucleus

UL

unilateral labyrinthectomy

VC

vestibular compensation

Acknowledgments

This study was supported by grants from the National 12th-Five Year Research Program of China (no. 2012BAI12B02), the Key Project of the National 11th-Five Year Research Program of China (no. 2007BAI18B13) and the National Natural Science Foundation of China (no. 81500792).

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November-2016
Volume 38 Issue 5

Print ISSN: 1107-3756
Online ISSN:1791-244X

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Spandidos Publications style
Zhou W, Zhou L, Shi H, Leng Y, Liu B, Zhang S and Kong W: Expression of glycine receptors and gephyrin in rat medial vestibular nuclei and flocculi following unilateral labyrinthectomy. Int J Mol Med 38: 1481-1489, 2016
APA
Zhou, W., Zhou, L., Shi, H., Leng, Y., Liu, B., Zhang, S., & Kong, W. (2016). Expression of glycine receptors and gephyrin in rat medial vestibular nuclei and flocculi following unilateral labyrinthectomy. International Journal of Molecular Medicine, 38, 1481-1489. https://doi.org/10.3892/ijmm.2016.2753
MLA
Zhou, W., Zhou, L., Shi, H., Leng, Y., Liu, B., Zhang, S., Kong, W."Expression of glycine receptors and gephyrin in rat medial vestibular nuclei and flocculi following unilateral labyrinthectomy". International Journal of Molecular Medicine 38.5 (2016): 1481-1489.
Chicago
Zhou, W., Zhou, L., Shi, H., Leng, Y., Liu, B., Zhang, S., Kong, W."Expression of glycine receptors and gephyrin in rat medial vestibular nuclei and flocculi following unilateral labyrinthectomy". International Journal of Molecular Medicine 38, no. 5 (2016): 1481-1489. https://doi.org/10.3892/ijmm.2016.2753