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Age-related changes in [3H]strychnine binding in the vestibular nuclei of rats
Hearing Research 127 (1999) 103^107
Age-related changes in [3 H]strychnine binding in the vestibular nuclei of rats Meiho Nakayama a
1;a;c
, Donald M. Caspary a;b , Horst R. Konrad a , Joseph C. Milbrandt b , Robert H. Helfert a;b; *
Division of Otolaryngology, Department of Surgery, Southern Illinois University School of Medicine, P.O. Box 19230, Spring¢eld, IL 62794-1312, USA b Department of Pharmacology, Southern Illinois University School of Medicine, Spring¢eld, IL 62794-1312, USA c Department of Otolaryngology, Aichi Medical University, Aichi-ken 48011, Japan Received 7 March 1998; received in revised form 22 August 1998; accepted 23 September 1998
Abstract Glycine plays an important role as a neurotransmitter in the four vestibular nuclei (VN). The objective of this study was to determine if the levels of glycine-receptor binding in the VN change as a function of age. Quantitative receptor autoradiography was performed on brainstem sections from three age groups (3, 18 and 26 months) of Fischer 344 rats to assess binding in the VN. Glycine receptors were localized using [3 H]strychnine binding. Strychnine binding declined monotonically with increasing age, such that the level of strychnine binding in each of the VN in the 28-month-old animals was approximately one-half that in the 3-montholds. The age-related decrease in levels of strychnine binding suggest altered glycinergic function in the VN, which may in turn contribute to disturbances in equilibrium observed in the elderly. z 1999 Elsevier Science B.V. All rights reserved. Key words: Aging; Brain; Glycine receptor; Receptor autoradiography
The vestibular nuclei (VN) consist of the superior vestibular nucleus (VS), the lateral vestibular nucleus (VL), the medial vestibular nucleus (VM) and the descending vestibular nucleus (VD). They play important roles as brainstem internuncial pools in the major vestibular pathways, and are therefore essential for a wide variety of equilibrium functions (reviewed in Sharpe and Johnston, 1993; Shepard and Telian, 1996; Markham, 1996) The VS and VM house the second order neurons of the vestibulo-ocular re£ex (VOR). The basic elements of the VOR are the receptor organ (ampullary crest), which transduce rotational head movements, and a three-neuron arc consisting of a bipolar primary afferent neuron, secondary `central processor' neuron, and an oculomotor e¡ector neuron. The VOR functions * Corresponding author. Tel.: +1 (217) 524-0860; Fax: +1 (217) 524-1793; E-mail: [email protected] 1 Send reprint requests to: Department of Otolaryngology, Aichi Medical University, Aichi-ken 48011, Japan.
to hold images stable on the retina during head movement. Vestibulospinal re£exes transduce angular and linear head movements and head position in respect to gravity. These are mediated primarily by the VL and VD to produce head-turning movements, postural re£exes and aid in locomotion. Glycine is a major inhibitory neurotransmitter in the mammalian brainstem and spinal cord, and receptorautoradiographic, pharmacologic and immunocytochemical studies suggest that it functions extensively as a neurotransmitter in the VN (Zarbin et al., 1981; Frostholm and Rotter, 1985; Probst et al., 1986 ; Walberg et al., 1990; Smith et al., 1991 ; Spencer and Baker, 1992 ; Lapeyre and De Waele, 1995). In cats, small and medium-sized, glycine immunoreactive neurons are distributed throughout the VN except in the VS (Walberg et al., 1990 ; Spencer and Baker, 1992). All of the VN, particularly VM, contain punctate glycine immunolabeling apposed to somata and neural pro¢les in the neuropil (Walberg et al., 1990), suggesting that VN neurons
0378-5955 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 9 8 ) 0 0 1 7 7 - 4
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M. Nakayama et al. / Hearing Research 127 (1999) 103^107
Fig. 1. Autoradiograph of total (left) and nonspeci¢c (right) [3 H]strychnine binding in two brainstem sections at the level of the vestibular nuclei. dcn, dorsal cochlear nucleus; lso, lateral superior olive; nVd, descending trigeminal nucleus; nVII, facial nucleus; vcn, ventral cochlear nucleus; VD descending vestibular nucleus; VL, lateral vestibular nucleus; VM, medial vestibular nucleus; VS, superior vestibular nucleus.
are the recipients of glycinergic synaptic input. Autoradiographic studies have revealed substantial levels of [3 H]strychnine binding in the vestibular nuclei of mice, rats, and humans (Zarbin et al., 1981; Frostholm and Rotter, 1985; Probst et al., 1986). Bath application of glycine to brain slices from guinea pigs results in a reversible decrease in the spontaneous ¢ring rate of VM neurons, which is abolished in the presence of the glycine-receptor antagonist, strychnine (Smith et al., 1991 ; Lapeyre and De Waele, 1995). In cats, the in vivo iontophoretic application of glycine to the VM reduces spontaneous activity in many VN neurons, including those in VM (Furuya et al., 1992). Given the extensive role glycine appears to play in VN function, it is assumed that decrements in glycinergic inhibition in the VN could cause functional impairment of balance. Such alterations in glycine neurotransmission may occur in the VN as a consequence of aging. Age-related declines in strychnine binding levels have been observed in brainstem homogenates from rats (Hunter et al., 1989). More speci¢cally, age-related decreases in glycine receptors have been observed in the cochlear nucleus (CN) of Fischer 344 (F344) rats and mice as evidenced by a decline in strychnine binding (Milbrandt and Caspary, 1995; Willott et al., 1997). The objective of this study was to determine if aging
similarly e¡ects glycine receptors in the VN of F344 rats in an e¡ort to further examine the causative roles glycine losses may play in presbystasis, the loss of equilibrium that occurs in the aged. All research was carried out using an animal use protocol approved by the Laboratory Animal Care and Use Committee at Southern Illinois University School of Medicine (USPHS Assurance #A-3209-01). Five male F344 rats in each of three age groups were used in this study. The age groups consisted of 3-month- (young adult), 18-month- (mature adult), and 26-month-old (aged) animals. They were swiftly decapitated, and the brains were immediately removed and frozen on dry ice. Serial transverse frozen sections through the portion of brainstem containing VN were cut at a thickness of 20 Wm with a cryostat, thawmounted on gelatin-subbed slides, and stored at 320³C for a time period not exceeding 48 h. The receptor binding methods used in this study are detailed in Milbrandt and Caspary (1995). Strychnine has been shown to be a selective inhibitory receptor ligand at low concentration, with little or no binding to central dopamine, cholinergic, and noradrenergic sites. Strychnine binding sites were localized by incubating the sections 20 minutes in 50 mM sodium-potassium phosphate bu¡er (pH 7.4, RT) containing 8 nM
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M. Nakayama et al. / Hearing Research 127 (1999) 103^107
[3 H]strychnine (22^26 Ci/mM ; New England Nuclear, Boston, MA). The single concentration of strychnine was chosen based on previous studies (Zarbin et al., 1981 ; Milbrandt and Caspary, 1995). Nonspeci¢c binding was determined in adjacent sections by the addition of 10 mM glycine to the ligand bu¡er. All of the sections were processed during a single run of the binding protocol. Autoradiograms were generated by apposing the sections, along with plastic-embedded 14 C standards (ARC, St. Louis, MO) containing known amounts of radioactivity to tritium-sensitive ¢lm in an X-ray cassette. These standards were previously calibrated against 3 H-brain paste standards containing known amounts of radioactivity and protein (Pan et al., 1983), thus permitting the conversion of areal optical densities measured from the ¢lms into femtomoles (fmol) per milligram protein values. After an exposure period of 4^5 weeks, the ¢lms were developed and the average optical density of each VN was obtained by taking multiple density readings from each structure using a morphometric workstation running BIOQUANT BQ System IV software (RpM Biometrics, Nashville, TN). The boundaries of the individual VN were de¢ned based on observations from adjacent Nissl-stained sections. An Analysis of Variance (ANOVA) was run to identify age-related di¡erences in [3 H]strychnine binding, and subsequent pairwise comparisons were made using Tukeys HSD. The area of the entire VN complex was measured from each analyzed brainstem image and the resulting data compared among age groups to determine if any alteration in synaptic density may be attributed to a corresponding change in VN size. Strychnine binding was observed in all of the VN (Fig. 1). In the 3-month-old animals, the VM exhibited the greatest level of [3 H]strychnine binding, followed by the VS, VL, and VD (Table 1). For all VN, statistically signi¢cant di¡erences in strychnine binding were noted among the three age groups (VM: F = 70.54, df = 2,12, P 6 0.0001 ; VS : F = 105.43, df = 2,12, P 6 0.0001; VL : F = 40.91, df = 2,12, P 6 0.0001; VD: F = 57.54, df = 2,12, P 6 0.0001). In all nuclei, [3 H]strychnine binding was reduced in the 18- and 26-month-old rats when compared to the 3-month-olds, and in 26-month-old
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animals relative to the 18-month-old group (Table 1). Tukey HSD post hoc analysis revealed that all of the di¡erences were signi¢cant to the level of P = 0.012. The percentage changes in bound strychnine described below were extracted from the data in Table 1. In three of the four VN (VM, VL, and VD) the reductions in strychnine binding were remarkably similar (18 months vs. 3 months : VM, 19.98% þ 2.65% (mean þ S.E.M.), VL, 18.21% þ 4.45%, VD, 19.76% þ 3.14% ; 26 months vs. 3 months: VM, 46.01% þ 2.44%, VL, 47.14% þ 3.00%, VD, 46.86% þ 3.25%). The VS, however, appeared to undergo a greater reduction in the 26-month-old group relative to the 3-month-old group (56.10% þ 2.64%). No di¡erences in VN area were detectable among the three age groups, suggesting that the observed di¡erences in receptor binding were not related to changes in VN size. The results of this study indicate that glycine receptors are distributed in all of the VN as evidenced by the presence of strychnine binding. The levels of strychnine binding in the VM and VS appear to be particularly high, suggesting that glycine may have a major role as an inhibitory neurotransmitter in these nuclei. Here, the integrity of glycine receptors would be essential for the regulation of the VOR. In many regards the levels of strychnine binding described in this study are in agreement with observations by other investigators. In humans and mice, as well as rats, binding for strychnine is clearly present in the VN, and in all three species the level of strychnine binding is highest in the VM relative to the other VN (Zarbin et al., 1981; Frostholm and Rotter, 1985; Probst et al., 1986). Further support for the existence of glycine receptors in the VN is provided by Sato et al. (1991), who showed that mRNA for the K1 subunit of the glycine receptor is strongly expressed by cells in all four VN. Each of the four vestibular nuclei underwent an agerelated reduction in strychnine binding. These observations suggest that a decline in central vestibular function may occur with age, and that glycine-receptor loss, or changes in its binding properties, may be an underlying cause. A similar study performed in the CN of F344 rats employed multiple doses of strychnine to obtain measures of glycine receptor number and a¤nity,
Table 1 Speci¢c [3 H]strychnine binding in the VN by age group Region VM VS VL VD
[3 H]Strychnine bound (fmol/mg protein)a 3 months
18 months
26 months
201.00 þ 6.24 167.44 þ 5.03 141.32 þ 5.02 129.68 þ 3.78
160.84 þ 5.33* 136.85 þ 4.53* 115.58 þ 6.29*** 104.05 þ 4.07*
108.52 þ 4.91*,** 73.50 þ 4.42*,** 74.70 þ 4.24*,** 68.91 þ 4.21*,**
a All values are expressed as mean þ S.E.M. *P 6 0.01 relative to 3-month-olds. **P 6 0.01 relative to 18-month-olds. ***P = 0.012 relative to 3-month-olds.
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and found signi¢cant age-related reductions in glycine receptor number along with trends toward a decrease in glycine receptor a¤nity (Milbrandt and Caspary, 1995). These ¢ndings were extended by an in situ hybridization study (Krenning et al., 1998) that provided evidence not only that glycine receptors are reduced in number in the CN of aged rats, but that they may also possess a lowered a¤nity for strychnine due to changes in their subunit composition. The subunit changes may alter the functional state of the glycine receptor (Krenning et al., 1998). The causal basis for a loss of glycine receptors in the VN of aged F344 rats remains to be established, as it is not known to what extent the causes are peripheral or central in origin. Losses of glycine receptors in the VN with age may be attributed at least in part to neuronal loss. A recent postmortem study in humans (aged 40^93 years) described a loss of neurons in the VN, occurring at a rate of approximately three percent per decade (Lopez et al., 1997). Coincident with this loss was a modest decrease in the volume of the VN (ibid.). Interestingly, Lopez et al. (1997) observed the greatest cell loss in VS, the nucleus in which this study demonstrated the greatest reduction in strychnine binding. We were unable to detect di¡erences in VN size among the three age groups of rats used in the present study, which would tend to argue against substantial decreases in their neuronal populations. However, the con¢rmatory cell counts were not performed. Further research will be required to determine if the observed decrease in strychnine binding in the VN is related to cell loss. Banay-Schwartz et al. (1989) observed a signi¢cant age-related reduction in the levels of glycine in the cochlear nucleus and several other brain areas, but not in the VN. This lack of change in the VN indicates that glycinergic inputs to the VN may remain intact during aging. Alternatively, a loss of input to the VN may be o¡set by a compensatory increase in levels of releasable glycine in the surviving presynaptic terminals. Such a compensatory process may be found in dopaminergic projections to the striatum of mice, where a three-fold increase in dopamine synthesis per nigrostriatal neuron was observed along with a substantial loss of nigrostriatal neurons (Tatton et al., 1991). In any case, the lack of change in glycine levels argues against inhibitory dea¡erentation as a major cause of an age-related loss of glycine receptors in the VN. A loss of glycine receptors in the VN could also occur subsequent to reduced primary a¡erent input. In aged F344 rats, hair cell loss and other morphologic abnormalities are present in the cristae (Nakayama et al., 1994). Given that the cristae project primarily to the VM and VS (Stein and Carpenter, 1967), it is conceivable that glycine receptor expression in these nuclei could downregulate to balance a decrement in primary a¡erent input. On the other hand, much of the primary
a¡erent input to the VL and VD originates from the maculae, which appear to be una¡ected by aging (Nakayama et al., 1994). Therefore, the reductions in strychnine binding observed in these nuclei are less likely to be explained by peripheral changes. Central mechanisms may play a role here. A reduction in the number and e¤cacy of glycine receptors would likely result in a net loss of glycinergic inhibition. The disequilibrium su¡ered by many in the elderly population could, in part, be the result of inappropriate neural signals due to the loss of inhibition in this system. The speci¢c cause of the observed reduction in strychnine binding in the VN with age is not known. Studies exploring both responses near threshold as well as at high stimulus magnitudes and studies across the entire frequency range transduced by the vestibular system are needed to better elucidate the relationships among anatomic alterations in the sense organ, transmitter-related de¢cits in the vestibular nuclei, and the response changes seen in vestibular function. Future studies should also explore the possibility that primary degenerative changes in the secondary vestibular neurons may be an underlying cause of the receptor changes. Acknowledgments The authors gratefully acknowledge the technical assistance provided by Teresa J. Sommer. Supported by USPHS Grants DC-02247 and DC-00151.
References Banay-Schwartz, M., Lajtha, A., Palkovits, M., 1989. Changes with aging in the levels of amino acids in rat CNS structural elements ^ II. Taurine and small neutral amino acids. Neurochem. Res. 14, 563^570. Frostholm, A., Rotter, A., 1985. Glycine receptor distribution in mouse CNS: autoradiographic localization of [3 H]strychnine binding sites. Brain Res. Bull. 15, 473^486. Furuya, N., Yabe, T., Koizumi, T., 1992. Neurotransmitters in the vestibular commissural system of the cat. Ann. NY Acad. Sci. 22, 594^601. Hunter, C., Chung, E., Van Woert, M.H., 1989. Age-dependent changes in brain glycine concentration and strychnine-induced seizures in the cat. Brain Res. 482, 247^251. Krenning, J., Hughes, L.F., Caspary, D.M., Helfert, R.H., 1998. Agerelated glycine receptor subunit changes in the cochlear nucleus of Fischer-344 rats. Laryngoscope 108, 26^31. Lapeyre, P.N., De Waele, C., 1995. Glycinergic inhibition of spontaneously active guinea-pig medial vestibular nucleus neurons in vitro. Neurosci. Lett. 188, 155^158. Lopez, I., Honrubia, V., Baloh, R.W., 1997. Aging and the human vestibular nucleus. J. Vestib. Res. 7, 77^85. Markham, C.H., 1996. How does the brain generate horizontal vestibular nystagmus? In: Baloh, R.W., Halmagyi, G.M. (Eds.), Disorders of the Vestibular System. Oxford University Press, New York, pp. 48^61.
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M. Nakayama et al. / Hearing Research 127 (1999) 103^107 Milbrandt, J.C., Caspary, D.M., 1995. Age-related reduction of [3 H]strychnine binding sites in the cochlear nucleus of the Fischer 344 rat. Neuroscience 67, 713^719. Nakayama, M., Helfert, R.H., Konrad, H.R., Caspary, D.M., 1994. Scanning electron microscopic evaluation of age-related changes in the rat vestibular epithelium. Otolaryngol. Head Neck Surg. 111, 799^806. Pan, H.S., Frey, K.A., Young, A.B., Penny, J.B., 1983. Changes in [3 H]muscimol binding in substantia nigra, entopeduncular nucleus, globus pallidus, and thalamus after striatal lesions as demonstrated by quantitative receptor autoradiography. J. Neurosci. 3, 1189^1198. Probst, A., Cortes, R., Palacios, J.M., 1986. The distribution of glycine receptors in the human brain. A light microscopic autoradiographic study using [3 H]strychnine. Neuroscience 17, 11^35. Sato, K., Zhang, J.H., Saika, T., Sato, M., Tada, K., Tohyama, M., 1991. Localization of glycine receptor alpha 1 subunit mRNAcontaining neurons in the rat brain: an analysis using in situ hybridization histochemistry. Neuroscience 43, 381^395. Sharpe, J.A., Johnston, J.L., 1993. The vestibulo-ocular re£ex: clinical, anatomic, and physiologic correlates. In: Sharpe, J.A., Barber, H.O. (Eds.), The Vestibulo-Ocular Re£ex and Vertigo. Raven Press, New York, pp. 15^39. Shepard, N.T., Telian, S.A., 1996. Practical Management of the Balance Disorder Patient. Singular, San Diego, pp. 1^16.
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Smith, P.F., Darlington, C.L., Hubbard, J.I., 1991. Evidence for inhibitory amino acid receptors on guinea pig medial vestibular nucleus neurons in vitro. Neurosci. Lett. 121, 244^246. Spencer, R.F., Baker, R., 1992. GABA and glycine as inhibitory neurotransmitters in the vestibuloocular re£ex. Ann. NY Acad. Sci. 22, 602^611. Stein, M.B., Carpenter, M.B., 1967. Central projections of portions of the vestibular ganglia innervating speci¢c parts of the labyrinth in the rhesus monkey. Am. J. Anat. 120, 281^313. Tatton, W.G., Greenwood, C.E., Salo, P.T., Seniuk, N.A., 1991. Transmitter synthesis increases in substantia nigra neurons of the aged mouse. Neurosci. Lett. 131, 179^182. Walberg, F., Ottersen, O.P., Rinvik, E., 1990. GABA, glycine, aspartate, glutamate and taurine in the vestibular nuclei: an immunocytochemical investigation in the cat. Exp. Brain Res. 79, 547^563. Willott, J.F., Milbrandt, J.C., Bross, L.S., Caspary, D.M., 1997. Glycine immunoreactivity and receptor binding in the cochlear nucleus of C57BL/6J and CBA/CaJ mice: e¡ects of cochlear impairment and aging. J. Comp. Neurol. 385, 405^414. Zarbin, M.A., Wamsley, J.K., Kuhar, M.J., 1981. Glycine receptor: light microscopic autoradiographic localization with [3 H]strychnine. J. Neurosci. 1, 532^547.
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Age-related changes in [3 H]strychnine binding in the vestibular nuclei of rats Meiho Nakayama a
1;a;c
, Donald M. Caspary a;b , Horst R. Konrad a , Joseph C. Milbrandt b , Robert H. Helfert a;b; *
Division of Otolaryngology, Department of Surgery, Southern Illinois University School of Medicine, P.O. Box 19230, Spring¢eld, IL 62794-1312, USA b Department of Pharmacology, Southern Illinois University School of Medicine, Spring¢eld, IL 62794-1312, USA c Department of Otolaryngology, Aichi Medical University, Aichi-ken 48011, Japan Received 7 March 1998; received in revised form 22 August 1998; accepted 23 September 1998
Abstract Glycine plays an important role as a neurotransmitter in the four vestibular nuclei (VN). The objective of this study was to determine if the levels of glycine-receptor binding in the VN change as a function of age. Quantitative receptor autoradiography was performed on brainstem sections from three age groups (3, 18 and 26 months) of Fischer 344 rats to assess binding in the VN. Glycine receptors were localized using [3 H]strychnine binding. Strychnine binding declined monotonically with increasing age, such that the level of strychnine binding in each of the VN in the 28-month-old animals was approximately one-half that in the 3-montholds. The age-related decrease in levels of strychnine binding suggest altered glycinergic function in the VN, which may in turn contribute to disturbances in equilibrium observed in the elderly. z 1999 Elsevier Science B.V. All rights reserved. Key words: Aging; Brain; Glycine receptor; Receptor autoradiography
The vestibular nuclei (VN) consist of the superior vestibular nucleus (VS), the lateral vestibular nucleus (VL), the medial vestibular nucleus (VM) and the descending vestibular nucleus (VD). They play important roles as brainstem internuncial pools in the major vestibular pathways, and are therefore essential for a wide variety of equilibrium functions (reviewed in Sharpe and Johnston, 1993; Shepard and Telian, 1996; Markham, 1996) The VS and VM house the second order neurons of the vestibulo-ocular re£ex (VOR). The basic elements of the VOR are the receptor organ (ampullary crest), which transduce rotational head movements, and a three-neuron arc consisting of a bipolar primary afferent neuron, secondary `central processor' neuron, and an oculomotor e¡ector neuron. The VOR functions * Corresponding author. Tel.: +1 (217) 524-0860; Fax: +1 (217) 524-1793; E-mail: [email protected] 1 Send reprint requests to: Department of Otolaryngology, Aichi Medical University, Aichi-ken 48011, Japan.
to hold images stable on the retina during head movement. Vestibulospinal re£exes transduce angular and linear head movements and head position in respect to gravity. These are mediated primarily by the VL and VD to produce head-turning movements, postural re£exes and aid in locomotion. Glycine is a major inhibitory neurotransmitter in the mammalian brainstem and spinal cord, and receptorautoradiographic, pharmacologic and immunocytochemical studies suggest that it functions extensively as a neurotransmitter in the VN (Zarbin et al., 1981; Frostholm and Rotter, 1985; Probst et al., 1986 ; Walberg et al., 1990; Smith et al., 1991 ; Spencer and Baker, 1992 ; Lapeyre and De Waele, 1995). In cats, small and medium-sized, glycine immunoreactive neurons are distributed throughout the VN except in the VS (Walberg et al., 1990 ; Spencer and Baker, 1992). All of the VN, particularly VM, contain punctate glycine immunolabeling apposed to somata and neural pro¢les in the neuropil (Walberg et al., 1990), suggesting that VN neurons
0378-5955 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 9 8 ) 0 0 1 7 7 - 4
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Fig. 1. Autoradiograph of total (left) and nonspeci¢c (right) [3 H]strychnine binding in two brainstem sections at the level of the vestibular nuclei. dcn, dorsal cochlear nucleus; lso, lateral superior olive; nVd, descending trigeminal nucleus; nVII, facial nucleus; vcn, ventral cochlear nucleus; VD descending vestibular nucleus; VL, lateral vestibular nucleus; VM, medial vestibular nucleus; VS, superior vestibular nucleus.
are the recipients of glycinergic synaptic input. Autoradiographic studies have revealed substantial levels of [3 H]strychnine binding in the vestibular nuclei of mice, rats, and humans (Zarbin et al., 1981; Frostholm and Rotter, 1985; Probst et al., 1986). Bath application of glycine to brain slices from guinea pigs results in a reversible decrease in the spontaneous ¢ring rate of VM neurons, which is abolished in the presence of the glycine-receptor antagonist, strychnine (Smith et al., 1991 ; Lapeyre and De Waele, 1995). In cats, the in vivo iontophoretic application of glycine to the VM reduces spontaneous activity in many VN neurons, including those in VM (Furuya et al., 1992). Given the extensive role glycine appears to play in VN function, it is assumed that decrements in glycinergic inhibition in the VN could cause functional impairment of balance. Such alterations in glycine neurotransmission may occur in the VN as a consequence of aging. Age-related declines in strychnine binding levels have been observed in brainstem homogenates from rats (Hunter et al., 1989). More speci¢cally, age-related decreases in glycine receptors have been observed in the cochlear nucleus (CN) of Fischer 344 (F344) rats and mice as evidenced by a decline in strychnine binding (Milbrandt and Caspary, 1995; Willott et al., 1997). The objective of this study was to determine if aging
similarly e¡ects glycine receptors in the VN of F344 rats in an e¡ort to further examine the causative roles glycine losses may play in presbystasis, the loss of equilibrium that occurs in the aged. All research was carried out using an animal use protocol approved by the Laboratory Animal Care and Use Committee at Southern Illinois University School of Medicine (USPHS Assurance #A-3209-01). Five male F344 rats in each of three age groups were used in this study. The age groups consisted of 3-month- (young adult), 18-month- (mature adult), and 26-month-old (aged) animals. They were swiftly decapitated, and the brains were immediately removed and frozen on dry ice. Serial transverse frozen sections through the portion of brainstem containing VN were cut at a thickness of 20 Wm with a cryostat, thawmounted on gelatin-subbed slides, and stored at 320³C for a time period not exceeding 48 h. The receptor binding methods used in this study are detailed in Milbrandt and Caspary (1995). Strychnine has been shown to be a selective inhibitory receptor ligand at low concentration, with little or no binding to central dopamine, cholinergic, and noradrenergic sites. Strychnine binding sites were localized by incubating the sections 20 minutes in 50 mM sodium-potassium phosphate bu¡er (pH 7.4, RT) containing 8 nM
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M. Nakayama et al. / Hearing Research 127 (1999) 103^107
[3 H]strychnine (22^26 Ci/mM ; New England Nuclear, Boston, MA). The single concentration of strychnine was chosen based on previous studies (Zarbin et al., 1981 ; Milbrandt and Caspary, 1995). Nonspeci¢c binding was determined in adjacent sections by the addition of 10 mM glycine to the ligand bu¡er. All of the sections were processed during a single run of the binding protocol. Autoradiograms were generated by apposing the sections, along with plastic-embedded 14 C standards (ARC, St. Louis, MO) containing known amounts of radioactivity to tritium-sensitive ¢lm in an X-ray cassette. These standards were previously calibrated against 3 H-brain paste standards containing known amounts of radioactivity and protein (Pan et al., 1983), thus permitting the conversion of areal optical densities measured from the ¢lms into femtomoles (fmol) per milligram protein values. After an exposure period of 4^5 weeks, the ¢lms were developed and the average optical density of each VN was obtained by taking multiple density readings from each structure using a morphometric workstation running BIOQUANT BQ System IV software (RpM Biometrics, Nashville, TN). The boundaries of the individual VN were de¢ned based on observations from adjacent Nissl-stained sections. An Analysis of Variance (ANOVA) was run to identify age-related di¡erences in [3 H]strychnine binding, and subsequent pairwise comparisons were made using Tukeys HSD. The area of the entire VN complex was measured from each analyzed brainstem image and the resulting data compared among age groups to determine if any alteration in synaptic density may be attributed to a corresponding change in VN size. Strychnine binding was observed in all of the VN (Fig. 1). In the 3-month-old animals, the VM exhibited the greatest level of [3 H]strychnine binding, followed by the VS, VL, and VD (Table 1). For all VN, statistically signi¢cant di¡erences in strychnine binding were noted among the three age groups (VM: F = 70.54, df = 2,12, P 6 0.0001 ; VS : F = 105.43, df = 2,12, P 6 0.0001; VL : F = 40.91, df = 2,12, P 6 0.0001; VD: F = 57.54, df = 2,12, P 6 0.0001). In all nuclei, [3 H]strychnine binding was reduced in the 18- and 26-month-old rats when compared to the 3-month-olds, and in 26-month-old
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animals relative to the 18-month-old group (Table 1). Tukey HSD post hoc analysis revealed that all of the di¡erences were signi¢cant to the level of P = 0.012. The percentage changes in bound strychnine described below were extracted from the data in Table 1. In three of the four VN (VM, VL, and VD) the reductions in strychnine binding were remarkably similar (18 months vs. 3 months : VM, 19.98% þ 2.65% (mean þ S.E.M.), VL, 18.21% þ 4.45%, VD, 19.76% þ 3.14% ; 26 months vs. 3 months: VM, 46.01% þ 2.44%, VL, 47.14% þ 3.00%, VD, 46.86% þ 3.25%). The VS, however, appeared to undergo a greater reduction in the 26-month-old group relative to the 3-month-old group (56.10% þ 2.64%). No di¡erences in VN area were detectable among the three age groups, suggesting that the observed di¡erences in receptor binding were not related to changes in VN size. The results of this study indicate that glycine receptors are distributed in all of the VN as evidenced by the presence of strychnine binding. The levels of strychnine binding in the VM and VS appear to be particularly high, suggesting that glycine may have a major role as an inhibitory neurotransmitter in these nuclei. Here, the integrity of glycine receptors would be essential for the regulation of the VOR. In many regards the levels of strychnine binding described in this study are in agreement with observations by other investigators. In humans and mice, as well as rats, binding for strychnine is clearly present in the VN, and in all three species the level of strychnine binding is highest in the VM relative to the other VN (Zarbin et al., 1981; Frostholm and Rotter, 1985; Probst et al., 1986). Further support for the existence of glycine receptors in the VN is provided by Sato et al. (1991), who showed that mRNA for the K1 subunit of the glycine receptor is strongly expressed by cells in all four VN. Each of the four vestibular nuclei underwent an agerelated reduction in strychnine binding. These observations suggest that a decline in central vestibular function may occur with age, and that glycine-receptor loss, or changes in its binding properties, may be an underlying cause. A similar study performed in the CN of F344 rats employed multiple doses of strychnine to obtain measures of glycine receptor number and a¤nity,
Table 1 Speci¢c [3 H]strychnine binding in the VN by age group Region VM VS VL VD
[3 H]Strychnine bound (fmol/mg protein)a 3 months
18 months
26 months
201.00 þ 6.24 167.44 þ 5.03 141.32 þ 5.02 129.68 þ 3.78
160.84 þ 5.33* 136.85 þ 4.53* 115.58 þ 6.29*** 104.05 þ 4.07*
108.52 þ 4.91*,** 73.50 þ 4.42*,** 74.70 þ 4.24*,** 68.91 þ 4.21*,**
a All values are expressed as mean þ S.E.M. *P 6 0.01 relative to 3-month-olds. **P 6 0.01 relative to 18-month-olds. ***P = 0.012 relative to 3-month-olds.
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M. Nakayama et al. / Hearing Research 127 (1999) 103^107
and found signi¢cant age-related reductions in glycine receptor number along with trends toward a decrease in glycine receptor a¤nity (Milbrandt and Caspary, 1995). These ¢ndings were extended by an in situ hybridization study (Krenning et al., 1998) that provided evidence not only that glycine receptors are reduced in number in the CN of aged rats, but that they may also possess a lowered a¤nity for strychnine due to changes in their subunit composition. The subunit changes may alter the functional state of the glycine receptor (Krenning et al., 1998). The causal basis for a loss of glycine receptors in the VN of aged F344 rats remains to be established, as it is not known to what extent the causes are peripheral or central in origin. Losses of glycine receptors in the VN with age may be attributed at least in part to neuronal loss. A recent postmortem study in humans (aged 40^93 years) described a loss of neurons in the VN, occurring at a rate of approximately three percent per decade (Lopez et al., 1997). Coincident with this loss was a modest decrease in the volume of the VN (ibid.). Interestingly, Lopez et al. (1997) observed the greatest cell loss in VS, the nucleus in which this study demonstrated the greatest reduction in strychnine binding. We were unable to detect di¡erences in VN size among the three age groups of rats used in the present study, which would tend to argue against substantial decreases in their neuronal populations. However, the con¢rmatory cell counts were not performed. Further research will be required to determine if the observed decrease in strychnine binding in the VN is related to cell loss. Banay-Schwartz et al. (1989) observed a signi¢cant age-related reduction in the levels of glycine in the cochlear nucleus and several other brain areas, but not in the VN. This lack of change in the VN indicates that glycinergic inputs to the VN may remain intact during aging. Alternatively, a loss of input to the VN may be o¡set by a compensatory increase in levels of releasable glycine in the surviving presynaptic terminals. Such a compensatory process may be found in dopaminergic projections to the striatum of mice, where a three-fold increase in dopamine synthesis per nigrostriatal neuron was observed along with a substantial loss of nigrostriatal neurons (Tatton et al., 1991). In any case, the lack of change in glycine levels argues against inhibitory dea¡erentation as a major cause of an age-related loss of glycine receptors in the VN. A loss of glycine receptors in the VN could also occur subsequent to reduced primary a¡erent input. In aged F344 rats, hair cell loss and other morphologic abnormalities are present in the cristae (Nakayama et al., 1994). Given that the cristae project primarily to the VM and VS (Stein and Carpenter, 1967), it is conceivable that glycine receptor expression in these nuclei could downregulate to balance a decrement in primary a¡erent input. On the other hand, much of the primary
a¡erent input to the VL and VD originates from the maculae, which appear to be una¡ected by aging (Nakayama et al., 1994). Therefore, the reductions in strychnine binding observed in these nuclei are less likely to be explained by peripheral changes. Central mechanisms may play a role here. A reduction in the number and e¤cacy of glycine receptors would likely result in a net loss of glycinergic inhibition. The disequilibrium su¡ered by many in the elderly population could, in part, be the result of inappropriate neural signals due to the loss of inhibition in this system. The speci¢c cause of the observed reduction in strychnine binding in the VN with age is not known. Studies exploring both responses near threshold as well as at high stimulus magnitudes and studies across the entire frequency range transduced by the vestibular system are needed to better elucidate the relationships among anatomic alterations in the sense organ, transmitter-related de¢cits in the vestibular nuclei, and the response changes seen in vestibular function. Future studies should also explore the possibility that primary degenerative changes in the secondary vestibular neurons may be an underlying cause of the receptor changes. Acknowledgments The authors gratefully acknowledge the technical assistance provided by Teresa J. Sommer. Supported by USPHS Grants DC-02247 and DC-00151.
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Smith, P.F., Darlington, C.L., Hubbard, J.I., 1991. Evidence for inhibitory amino acid receptors on guinea pig medial vestibular nucleus neurons in vitro. Neurosci. Lett. 121, 244^246. Spencer, R.F., Baker, R., 1992. GABA and glycine as inhibitory neurotransmitters in the vestibuloocular re£ex. Ann. NY Acad. Sci. 22, 602^611. Stein, M.B., Carpenter, M.B., 1967. Central projections of portions of the vestibular ganglia innervating speci¢c parts of the labyrinth in the rhesus monkey. Am. J. Anat. 120, 281^313. Tatton, W.G., Greenwood, C.E., Salo, P.T., Seniuk, N.A., 1991. Transmitter synthesis increases in substantia nigra neurons of the aged mouse. Neurosci. Lett. 131, 179^182. Walberg, F., Ottersen, O.P., Rinvik, E., 1990. GABA, glycine, aspartate, glutamate and taurine in the vestibular nuclei: an immunocytochemical investigation in the cat. Exp. Brain Res. 79, 547^563. Willott, J.F., Milbrandt, J.C., Bross, L.S., Caspary, D.M., 1997. Glycine immunoreactivity and receptor binding in the cochlear nucleus of C57BL/6J and CBA/CaJ mice: e¡ects of cochlear impairment and aging. J. Comp. Neurol. 385, 405^414. Zarbin, M.A., Wamsley, J.K., Kuhar, M.J., 1981. Glycine receptor: light microscopic autoradiographic localization with [3 H]strychnine. J. Neurosci. 1, 532^547.
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