Unilateral labyrinthectomy downregulates glutamate receptor δ-2 expression in the rat vestibulocerebellum

doi:10.1016/S0169-328X(98)00228-9

Molecular Brain Research

Volume 61, Issues 1–2, 30 October 1998, Pages 170-178

https://doi.org/10.1016/S0169-328X(98)00228-9 Get rights and content

Abstract

The differential display method was applied to identify genes expression of which is altered in the flocculus after unilateral labyrinthectomy (UL). Total RNA from sham operated and labyrinthectomized rat flocculi was isolated, amplified by PCR using an arbitrary primer set and separated by electrophoresis on a polyacrylamide gel. PCR products the amounts of which were significantly lower in samples from labyrinthectomized animals than those from controls, were cut out of the gel and sequenced. One of the cDNA fragments showed 100% nucleotide sequence identity to the rat glutamate receptor (GluR) δ-2 subunit mRNA. In situ hybridization autoradiography showed that GluR δ-2 mRNA expression was intensely located to the floccular Purkinje cell layers. Furthermore, northern blot analysis showed that the δ-2 mRNA expression was decreased for at least two days after UL in accordance with diminishing UL-induced behavioral deficits. Therefore, it is suggested that the downregulation of δ-2 mRNA is involved in vestibular compensation. UL-induced spontaneous nystagmus (SN) was then examined in GluR δ-2 mutant mice. The frequency of SN in mutant mice was significantly more than that in wild mice until 12 h after UL. GluR δ-2-associated synaptic efficacy may be changed for the induction of vestibular compensation at the initial stage.

Introduction

Vestibular compensation has been used as one of the best models of post-lesional plasticity in the central nervous system (CNS) [28]. Immediately after unilateral labyrinthectomy (UL), the resting activity in the ipsilesional medial vestibular second-order neurons is markedly reduced, resulting in an imbalance between intervestibular nuclear activities [36]. This imbalance causes severe oculomotor (nystagmus) and postural (barrel rotation, head tilt) disturbances. Despite permanent loss of the ipsilesional vestibular peripheral inputs, these symptoms gradually diminish together with the recovery of resting activity in the ipsilesional medial vestibular second-order neurons [7]. This process of progressive rebalancing is due to the functional reorganization of the central vestibular system and is one of the main features of vestibular compensation [37].

The vestibulocerebellum is an important component involved in the induction of vestibular compensation 11, 15. Furthermore, we have recently reported that changes in flocculus-mediated inhibitory control of the medial vestibular nucleus (MVe) after UL induced the initial processes of vestibular compensation 19, 20, 23. To understand the molecular basis underlying vestibular compensation, it is vital to identify molecules expression of which is altered in the cerebellar flocculus during vestibular compensation. Current methods to distinguish mRNA in comparative studies rely largely on subtractive hybridization [26]. This technique, although it has been used successfully in isolating a number of important genes 4, 38, is rather difficult to perform reproducibly and requires large amounts of RNA. Liang and Pardee [27]have recently developed an alternative approach, mRNA differential display, which circumvents these problems. The essence of the differential display method is to use an anchored oligo-dT primer for reverse transcription, followed by polymerase chain reaction (PCR), using a combination of arbitrary primers. The amplified cDNA subpopulations of mRNAs are then displayed on a DNA sequencing gel. By means of this method, several genes were successfully detected in vivo 8, 18, 21, 22, 24, 31, 39.

In the present study, we used the differential display method and demonstrated that glutamate receptor (GluR) δ-2 subunit mRNA expression is downregulated in the rat flocculus after UL.

Section snippets

Animals and tissue preparation

Forty-four male Wistar rats weighing about 150 g were used. Animals were divided into two groups. One group of 16 animals was anesthetized with ether and underwent unilateral labyrinthectomy (UL). The right tympanic membrane, malleus and incus were removed by the retroauricular approach under an operating microscope. The stapes crura were fractured and the stapes foot plate was removed to open the oval window. Then, a small opening was made in the bony horizontal semicircular canal with a small

Differential display

mRNA from the ipsi- and contra-flocculi 2 days after unilateral labyrinthectomy (UL) and the sham operated rat flocculus was compared by the differential display method. The patterns of the mRNA displayed on a DNA sequencing gel are shown in Fig. 1. The band differing in intensity between the ipsi-/contra- and control rat flocculi (arrowhead in Fig. 1) was cut out of the gel through the film.

DNA sequencing

The nucleotide sequence of the cDNA is shown in Fig. 2. Searching the GenBank and EMBL DNA databases

Discussion

In the present study, we used the differential display method introduced by Liang and Pardee [27]to examine downregulation of gene expression in the labyrinthectomized rat flocculus. About 100 bands were obtained per lane using an arbitrary primer set, AP-4 and AP-13 (Fig. 1). Several bands which differed in intensity between the ipsi-/contra- and sham operated rat flocculi were cut out of gels. One of these cDNA fragments showed 100% identity in nucleotide sequence to the recently isolated and

Acknowledgements

This work was partly supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan, and Japan Society for the Promotion of Science (Tokyo Japan).

References (41)

K. Araki et al.
Selective expression of the glutamate receptor channel δ2 subunit in cerebellar Purkinje cells
Biochem. Biophys. Res. Commun.
(1993)
M.F. Bear et al.
Synaptic plasticity: LTP and LTD
Current Opinion Neurobiol.
(1994)
P. Chomczynski et al.
Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction
Anal. Biochem.
(1987)
G.M. Haddad et al.
Compensation of nystagmus after VIII the nerve lesions in vestibulo-cerebellectomized cats
Brain Res.
(1977)
T. Hirano et al.
Involvement of the glutamate receptor δ2 subunit in the long-term depression of glutamate responsiveness in cultured rat Purkinje cells
Neurosci. Lett.
(1994)
M. Ito et al.
Protein kinases and phosphatase inhibitors mediating long-term desensitization of glutamate receptors in cerebellar Purkinje cells
Neurosci. Res.
(1992)
N. Kashiwabuchi et al.
Impairment of motor coordination, Purkinje cell synapse formation, and cerebellar long-term depression in GluR δ2 mutant mice
Cell
(1995)
T. Kitahara et al.
Effects of MK801 on Fos expression in the rat brainstem after unilateral labyrinthectomy
Brain Res.
(1995)
T. Kitahara et al.
Up-regulation of ferritin heavy chain mRNA expression in the rat skeletal muscle after denervation: detected by means of differential display
Neurosci. Res.
(1995)
T. Kitahara et al.
Role of the flocculus in the development of vestibular compensation: immunohistochemical studies with retrograde tracing and flocculectomy using Fos expression as a marker in the rat brainstem
Neuroscience
(1997)

H. Lomeli et al.

The rat δ-1 and δ-2 subunits extend the excitatory amino acid receptor family

FEBS Lett.

(1993)

K.W. Roche et al.

Glutamate receptor phosphorylation and synaptic plasticity

Curr. Opin. Neurobiol.

(1994)

P.F. Smith et al.

Neuronal activity in the ipsilateral medial vestibular nucleus of the guinea pig following unilateral labyrinthectomy

Brain Res.

(1988)

P.F. Smith et al.

Mechanism of recovery following unilateral labyrinthectomy: a review

Brain Res. Rev.

(1989)

E. Vreugdenhil et al.

Kainic acid-induced gene expression in the rat hippocampus is severly affected by adrenalectomy

Neurosci. Lett.

(1996)

M. Yamazaki et al.

Molecular cloning of a cDNA encoding a novel member of the mouse glutamate receptor channel family

Biochem. Biophys. Res. Commun.

(1992)

A. Ajima et al.

A unique role of protein phosphatases in cerebellar long- term depression

Neuroreport

(1995)

N.H. Barmack et al.

Influence of unilateral labyrinthectomy on expression of protein kinase C in a cerebellar vestibular pathway in rat

Soc. Neurosci. Abstr.

(1995)

P. Basset et al.

A novel metalloproteinase gene specifically expressed in stromal cells of breast carcinomas

Nature

(1990)

C.L. Darlington et al.

Molecular mechanisms of brainstem plasticity

Mol. Neurobiol.

(1991)

Cited by (25)

Vestibular motor control
2023, Handbook of Clinical Neurology
The vestibular system is an essential sensory system that generates motor reflexes that are crucial for our daily activities, including stabilizing the visual axis of gaze and maintaining head and body posture. In addition, the vestibular system provides us with our sense of movement and orientation relative to space and serves a vital role in ensuring accurate voluntary behaviors. Neurophysiological studies have provided fundamental insights into the functional circuitry of vestibular motor pathways. A unique feature of the vestibular system compared to other sensory systems is that the same central neurons that receive direct input from the afferents of the vestibular component of the 8th nerve can also directly project to motor centers that control vital vestibular motor reflexes. In turn, these reflexes ensure stabilize gaze and the maintenance of posture during everyday activities. For instance, a direct three-neuron pathway mediates the vestibulo-ocular reflex (VOR) pathway to provide stable gaze. Furthermore, recent studies have advanced our understanding of the computations performed by the cerebellum and cortex required for motor learning, compensation, and voluntary movement and navigation. Together, these findings have provided new insights into how the brain ensures accurate self-movement during our everyday activities and have also advanced our knowledge of the neurobiological mechanisms underlying disorders of vestibular processing.
6.39 - Vestibular Compensation as a Distributed Process
2020, The Senses: A Comprehensive Reference: Volume 1-7, Second Edition
Vestibular compensation is the process by which the central nervous system (CNS) attempts to adapt to the loss of unilateral or bilateral peripheral vestibular sensory inputs. Although it is well accepted that the bilateral vestibular nuclei are critical for the compensation, it has been suspected for decades that the full spectrum of compensatory processes involves many different areas of the CNS, and that therefore vestibular compensation is a “distributed process”. This concept, originally advanced by Llinas and Walton (1979), has been supported by numerous studies over the last decades, including recent positron-emission tomography and functional magnetic resonance imaging studies.
Sensorimotor investigations for the Mars Gravity Biosatellite: A rotating spacecraft for partial gravity research
2006, Brain Research
Citation Excerpt :
As demonstrated by Takumi et al. (2002), hypergravity stimulus causes differential mRNA expression of at least six genes, including up-regulation of MAP1A, which may stabilize the actin–microtubule complex. Unilateral labryrinthectomy and its subsequent compensation have also been shown to modulate gene expression in the rat vestibulocerebellum (Kitahara et al., 1998; Zheng et al., 2001). Further exploration of gravity-dependent changes with genomic and proteomic tools offers substantial opportunities for new developments.
The Mars Gravity Biosatellite will offer investigators a unique environment for sensorimotor research. Fifteen mice will fly for 5 weeks in low Earth orbit before being returned safely to the ground. Chronic 35-rpm rotation will produce artificial gravity equal to that on the surface of Mars (0.38 g). This groundbreaking flight will be the longest rodent spaceflight investigation and the first to explore the effects of accelerations between weightlessness and Earth's 1 g.
The δ2 glutamate receptor: 10 Years later
2003, Neuroscience Research
The orphan glutamate receptor δ2 (GluRδ2) is predominantly expressed in Purkinje cells and plays a crucial role in cerebellar functions: mice that lack the GluRδ2 gene display ataxia and impaired synaptic plasticity. However, when expressed alone or with other glutamate receptors, GluRδ2 does not form functional glutamate-gated ion channels nor does it bind to glutamate analogs. Therefore, the mechanisms by which GluRδ2 participates in cerebellar functions have been elusive. Studies of mutant mice such as lurcher, hotfoot, and GluRδ2 knockout mice have provided clues to the structure and function of GluRδ2. GluRδ2 has a channel pore similar to that of other glutamate receptors; the channel is functional at least when the lurcher mutation is present. GluRδ2 must be transported to the Purkinje cell surface to function; the absence of surface GluRδ2 causes the ataxic phenotype of hotfoot mice. In GluRδ2-null mice, the presence of naked spines not innervated by parallel fibers may influence the sustained innervation of mutant Purkinje cells by multiple climbing fibers. From these results, several hypotheses about mechanisms by which GluRδ2 functions are proposed in this article. Further characterization of GluRδ2's functions will provide key insights into normal and abnormal cerebellar functions.
Asymmetric Fos labeling in lobule X of the cerebellum following transtympanic tetrodotoxin (TTX) in the rat
2003, Neuroscience Letters
Unilateral transtympanic administration of the sodium channel blocker tetrodotoxin (75 μl of 3 mM TTX in 0.1 M citrate buffer, pH 5.0) produced behavioral symptoms indicative of unilateral vestibular disruption. Following survival times of 2 and 24 h immunocytochemistry for Fos revealed asymmetric labeling in the granular and molecular layers of lobule X of the cerebellum and in the medial cerebellar nucleus. The granular and molecular layers as well as the medial cerebellar nucleus ipsilateral to TTX treatment contained elevated levels of Fos relative to the same regions contralaterally and when compared to controls receiving equal volume injections of vehicle. The asymmetric changes in neuronal activity as measured by Fos (genetic activity) in lobule X implicate this region of the vestibulocerebellum in recovery from transient vestibular disruption in the intact system.
Molecular mechanisms of recovery from vestibular damage in mammals: Recent advances
2000, Progress in Neurobiology
The aim of this review is to summarise and critically evaluate studies of vestibular compensation published over the last 2 years, with emphasis on those concerned with the molecular mechanisms of this process of lesion-induced plasticity. Recent studies of vestibular compensation have confirmed and extended the previous findings that: (i) compensation of the static ocular motor and postural symptoms occurs relatively rapidly and completely compared to the dynamic symptoms, many of which either do not compensate substantially or else compensate variably due to sensory substitution and the development of sensori-motor strategies which suppress or minimize symptoms; (ii) static compensation is associated with, and may be at least partially caused by a substantial recovery of resting activity in the ipsilateral vestibular nucleus complex (VNC), which starts to develop very quickly following the unilateral vestibular deafferentation (UVD) but does not correlate perfectly with the development of some aspects of static compensation (e.g., postural compensation); and (iii) many complex biochemical changes are occurring in the VNC, cerebellum and even areas of the central nervous system like the hippocampus, following UVD. However, despite many recent studies which suggest the importance of excitatory amino acid receptors such as the N-methyl-D-aspartate receptor, expression of immediate early gene proteins, glucocorticoids, neurotrophins and nitric oxide in the vestibular compensation process, how these various factors are linked and which of them may have a causal relationship with the physiological changes underlying compensation, remains to be determined.

View all citing articles on Scopus

View full text

Research reportUnilateral labyrinthectomy downregulates glutamate receptor δ-2 expression in the rat vestibulocerebellum

Abstract

Introduction

Section snippets

Animals and tissue preparation

Differential display

DNA sequencing

Discussion

Acknowledgements

Biochem. Biophys. Res. Commun.

Current Opinion Neurobiol.

Anal. Biochem.

Brain Res.

Neurosci. Lett.

Neurosci. Res.

Cell

Brain Res.

Neurosci. Res.

Neuroscience

FEBS Lett.

Curr. Opin. Neurobiol.

Brain Res.

Brain Res. Rev.

Neurosci. Lett.

Biochem. Biophys. Res. Commun.

A unique role of protein phosphatases in cerebellar long- term depression

Neuroreport

Influence of unilateral labyrinthectomy on expression of protein kinase C in a cerebellar vestibular pathway in rat

Soc. Neurosci. Abstr.

A novel metalloproteinase gene specifically expressed in stromal cells of breast carcinomas

Nature

Molecular mechanisms of brainstem plasticity

Mol. Neurobiol.

Research report
Unilateral labyrinthectomy downregulates glutamate receptor δ-2 expression in the rat vestibulocerebellum