Cell Tissue Res (1999) 295:477-484
© Springer-Verlag 1999
REGULAR ARTICLE
Jennifer K. Brueckner · Laura P. Ashby Jennifer R. Prichard · John D. Porter
Vestibulo-ocular pathways modulate extraocular muscle myosin expression patterns
Received: 1 April 1998 / Accepted: 22 September 1998
Abstract The genetic and epigenetic influences that establish and maintain the unique phenotype of the extraocular muscles (EOMs) are poorly understood. The vestibulo-ocular reflex (VOR) represents an important input into the EOMs, as it stabilizes eye position relative to the environment and provides a platform for function of all other eye movement systems. A role for vestibular cues in shaping EOM maturation was assessed in these studies using the ototoxic nitrile compound 3’,3’-iminodipropionitrile (IDPN) to eliminate the receptor hair cells that drive the vestibulo-ocular reflex. Intraperitoneal injections of IDPN were followed by a 2-week survival period, after which myosin heavy chain (MyHC) analysis of the EOMs was performed. When IDPN was administered to juvenile rats, the proportion of eye muscle fibers expressing developmental and fast myosins was increased, while EOM-specific MyHC mRNA levels were downregulated. By contrast, IDPN treatment in adult rats affected only the proportion of fibers expressing developmental MyHC isoforms, leaving the EOM-specific MyHC mRNA unaltered. These data provide evidence that the VOR modulates EOM-specific MyHC expression in development. The lack of significant
This work was supported by the National Eye Institute (J.D.P.), the Knights Templar Eye Foundation (J.K.B.), an undergraduate fellowship from the Howard Hughes Medical Institute (L.P.A.), National Science Foundation’s Research Experience for Undergraduates (J.R.P.), an unrestricted departmental grant from Research to Prevent Blindness, Evenor Armington Fund, CWRU Visual Sciences Research Fund, and a CORE grant from the National Eye Institute (P30 EY11373). J. K. Brueckner · L. P. Ashby · J. R. Prichard Department of Anatomy and Neurobiology, University of Kentucky, Chandler Medical Center, 800 Rose Street, Lexington, KY 40536-0084, USA J. D. Porter (✉) Department of Ophthalmology, University Hospitals of Cleveland, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, OH 44106-5068, USA Tel.: +1 216 844 7053; Fax: +1 216 844 4792; e-mail:
[email protected]
changes in EOM-specific MyHC expression in adult EOM following IDPN administration suggests that there may be a critical period during development when alterations in vestibular activity have significant and permanent consequences for the eye muscles. Key words Extraocular muscle · Muscle development · Myosin heavy chain · Vestibular development · Rat (Sprague Dawley)
Introduction As the effectors for five distinct eye movement control systems, the extraocular muscles (EOMs) represent a functionally diverse skeletal muscle group. This functional heterogeneity is manifested in the myosin heavy chain (MyHC) phenotype of the EOMs. The retention of developmental myosin isoforms in mature EOMs, expression of a tissuespecific isoform, and presence of longitudinal variations in the expression of some myosin isoforms are distinguishing features of EOMs (Wieczorek et al. 1985; Jacoby et al. 1990; Brueckner et al. 1996). The regulation of this unique myosin phenotype is not well understood, although roles for both genetic and epigenetic mechanisms have been considered. Given the unusual functional demands associated with this muscle group, epigenetic influences may be the key to understanding how the EOM phenotype is established (Porter et al. 1997). Both the visual and vestibular systems play a significant role in shaping oculomotor behavior (Berthoz et al. 1975; Van Hof-van Duin 1976; Coliewijn 1977; Rothblat et al. 1978). We have previously shown that the visual system exerts an important regulatory influence upon EOM development (Brueckner and Porter 1998). The vestibular system, however, is the phylogenetically oldest of the five eye movement control systems responsible for modulating oculomotor activity and might be expected to play an important role in shaping EOM properties. The vestibulo-ocular reflex (VOR) is the primary mechanism whereby the visual
478 150
% of control
Fig. 1 Neuronal number in the abducens nucleus of IDPN-treated and control rats expressed as a percentage of control for adult and juvenile (P20) rat abducens nuclei. n=3 rats for each group
Materials and methods Animal care and behavioral testing
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0
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field is stabilized during head rotations, thereby preventing visual blur. Vestibularly induced eye movements that cancel the visual field motion that would accompany such head rotations act to achieve visual stabilization. Target-acquiring eye movements, including saccades and pursuit, then can be executed upon the stable foundation provided by the VOR. Vestibular system structure and function have been studied extensively during the postnatal period of rodent development (Karhunen 1973; Taber Pierce 1973; Curthoys 1979; Lannou et al. 1979; Altman and Bayer 1980; Cazin et al. 1980). Physiological studies show that vestibular neuronal maturation is progressive throughout the first few postnatal weeks of life (Curthoys 1982), concurrent with the development of physiological properties of oculomotor motor neurons (Tzsuki et al. 1995) and myosin expression patterns in the EOMs (Brueckner et al. 1996). Behaviorally, the VOR is evident even before birth in rodents and can be elicited in newborn rats at thresholds only modestly higher than in adults (Curthoys 1979). The presence of an intact, albeit immature, VOR at a stage when the EOMs are still maturing suggests a potentially important role for vestibular activity in determining the EOM phenotype. Vestibular function has also been studied through the administration of ototoxic nitrile compounds, including 3’,3’iminodipropionitrile (IDPN; Llorens and Dememes 1994; Llorens et al. 1994; Llorens and Rodriguez-Farre 1997). IDPN kills the sensory receptors, or hair cells, in the semicircular canals, utricle, and saccule, effectively deafferenting the vestibular system (Llorens et al. 1993). When administered to rodents, this agent produces behavioral effects consistent with a vestibular lesion, including repetitive and abnormal head movements, retropulsion, circling, and hyperactivity (Llorens et al. 1993). Although the development of the vestibular system and its regulation by activity-dependent mechanisms has been well characterized, the contribution of vestibular cues to the maturation of the terminal component of the oculomotor system, the EOMs, is poorly understood. In these studies, we examined the effects of eliminating vestibular cues (via IDPN administration) on the contractile properties of EOM; specifically, MyHC isoform expression was used as an index of phenotypic change in the EOMs following this vestibular insult.
Sprague-Dawley rats (Harlan) were maintained in a 22°C room with a 12-h light/dark cycle and unlimited access to standard chow and water. Animal protocols were approved by the Institutional Animal Care and Use Committee. Two experimental groups [postnatal day 7 (P7) rats and adult rats] were used in these studies. Rats were injected intraperitoneally with 3’,3’-iminodipropionitrile (IDPN) at a dosage of 1.5 mg/g body weight. Controls were injected with an equivalent volume of 0.9% saline as vehicle. A 2-week survival period followed injection, after which IDPN-treated rats and age-matched, vehicle-treated controls were examined with a battery of behavioral tests (see below) and then sacrificed using CO2 asphyxiation. Behavioral analysis The toxic effect of IDPN on the rodent vestibular system has been well documented both histologically and behaviorally (Llorens et al. 1993). To assess the efficacy of IDPN in disrupting vestibular function in our experiments, three behavioral tests were administered in triplicate for each subject: tail hanging, air-righting reflex, and contact inhibition of the righting reflex. The tail-hanging test involved lifting rats by the tail and observing their forelimb response. Normal rats extend their forelimbs towards the ground, thus exhibiting a landing response, while vestibularly compromised rats bend ventrally. In the air-righting reflex, animals were held supine and dropped from a height of 30 cm onto a foam cushion. Normal rats right themselves in air, while compromised rats land on their back or side. In order to test contact inhibition of the righting reflex, rats were placed supine on a horizontal surface and another horizontal surface (a plastic grid) was lightly placed in contact with the soles of the supine feet. Normal rats righted themselves while vestibular-deficient rats remained on their back with feet facing up and walked along the grid surface. Protein analysis Monoclonal myosin heavy chain antibodies were used in immunocytochemical analysis of the effects of IDPN treatment on EOM phenotype. Primary antibodies included: developmental MyHC (Novocastra Labs), fast MyHC (Novocastra Labs), IIA/IIX MyHC (A4.74, Developmental Studies Hybridoma Bank), and a slow MyHC (A4.840, Developmental Studies Hybridoma Bank). For immunocytochemical demonstration of MyHC isoforms, rats were sacrificed by CO2 asphyxiation and the orbital contents were immediately dissected and frozen in isopentane chilled in liquid nitrogen. Twelve-micrometer cryostat sections were collected from three levels within each muscle: proximal (at the muscle’s origin near the tendinous ring in the posterior aspect of the orbit), mid-belly (innervation zone), and distal (proximal to the myotendinous junction, before the muscle attaches to the eye). The Vectastain ABC kit (Vector Labs, CA) was used for immunoperoxidase staining following incubation with the primary antibody. Sections were dehydrated and coverslipped for quantitative fiber analysis. As a control for systemic consequences of IDPN, the MyHC protein content of rat diaphragms was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, based on the method of Talmadge and Roy (1993). Myosin was extracted from diaphragms as previously described (Brueckner et al. 1996) and 5 µg myosin protein was added per lane and MyHC isoforms were electrophoretically separated on an 8% polyacrylamide-separating gel and a 4% stacking gel in the presence of SDS (Laemmli 1970; Carraro and Cantini 1983; Talmadge and Roy 1993). Electrophoresis proceeded at 70 V for 29 h at 40°C. Following electrophoresis, gels were fixed for 2 h, stained with Coomassie blue R250 (BioRad) overnight, and destained the following day. Bands from each gel were captured digitally and images were imported into NIH Image software for densitometric analysis.
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A
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Proximal Middle
Slow MyHCs 40
∗
% of total fibers
% of total fibers
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Developmental MyHCs 40
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Distal
EOM Region
C
D
Fast MyHC
% of total fibers
% of total fibers
Proximal Middle
Distal
IIA / IIX MyHCs 45
30 20 10 0
Quantitative analysis of the abducens nucleus EOM Region
40
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Distal
EOM Region IDPN EOM
tal RNA was isolated by phenol/chloroform extraction using Trizol (Gibco). Following denaturation and separation by electrophoresis, RNA was transferred to nytran, which was hybridized and washed as previously described (Brueckner et al. 1996). Following hybridization, membranes were exposed to a storage phosphor screen and densitometric analysis was performed by ImageQuant software (Molecular Dynamics). Membranes were stripped and hybridized subsequently with the 18S probe as a loading control.
30
15
0
Proximal Middle
Distal
EOM Region
To ensure that any effects elicited by IDPN administration were the result of selective degeneration of sensory vestibular receptor cells, and not of EOM motoneurons, neurons in the abducens nucleus were quantified in IDPN-treated and control rat brains from each age group (n=3 rats/group). For this study, developing and adult IDPN-treated and control rats were perfused with 4% paraformaldehyde. The brain and brainstem were dissected and paraffin embedded for ultramicrotome sectioning. These sections were stained with hematoxylin and eosin for neuron quantification. Within the cytoarchitectural boundaries of the abducens nucleus, all neurons exhibiting nuclei with prominent nucleoli were counted in 5-µm serial sections throughout the abducens nucleus. Since we were interested in whether relative changes occurred in control vs. experimental groups, we did not attempt corrections for split nucleoli, assuming an equal effect upon counts in both groups. To ensure the consistency of this analysis, a single trained observer blindly performed all counts.
Control EOM
Statistical analysis Fig. 2A–D Proportion of total fibers expressing different MyHC isoforms in IDPN-treated and control-developing EOMs. Immunocytochemical analysis was performed on eye muscle sections from IDPN-treated and age-matched control rat EOMs (P20). Four MyHC antibodies were used in this study, including developmental (A), slow (B), fast (C), and IIA/IIX (D) MyHC antibodies. n=5 rats per group
Statistical methods were used to determine the impact of the vestibular hair cell receptor lesion on myosin expression in developing and adult rat EOMs. In each case, mean±standard error of the mean is reported for experimental and control groups. Statistical differences in myosin gene and protein expression between groups were determined using t-tests. Differences were considered statistically significant at P
