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Aging in the rat medial nucleus of the trapezoid body I. Light microscopy
Neuroblology ofA~,,ing, Vol. 3, pp. 187-195, 1982. Printed in the U.S.A.
Aging in the Rat Medial Nucleus of the Trapezoid Body I. Light Microscopy MICHAEL
A. C A S E Y A N D M A R T I N L. F E L D M A N
D e p a r t m e n t o f A n a t o m y , Boston University School o f Medicine, Boston, M A 02118 R e c e i v e d 21 J u l y 1982 CASEY, M. A. AND M. L. FELDMAN. Aging in the rat medial nucleus of the trapezoid body. 1. Light microscopy. NEUROBIOL. AGING 3(3) 187-195, 1982.--The medial nucleus of the trapezoid body (MTB), a cell group in the superior olivary complex, was examined in an age-graded series of rats for neuron loss, changes in the giant synaptic endings (chalices of Held) on MTB neurons, and accumulation of age pigment. Neuron counts were done on protargol-stained paraffin sections of MTBs from a series of 17 rats aged 2-3, 6, 18, and 24 months. Between 2-3 and 24 months a 34% decrease in the mean number of MTB neurons was observed. Significant loss Ip <0.05) was first evident in the early portion of the life span, between 2-3 and 6 months. In thionin-stained sections, there was no change with aging in the proportions of three MTB neuron types: principal cells (~82%), elongate cells (~15%), and stellate cells (~3%). In young adult rats, 25-:26% of all MTB neurons were associated with identifiable chalices of Held in protargol stained sections. This ratio did not vary significantly with aging. Age pigment accumulation in the MTB was examined in 2 p.m Araldite sections stained with toluidine blue. Age pigment deposits were larger and more numerous in the MTBs of old animals, but not as extensive as has been described previously in many other parts of the nervous system. This study is the first to report neuron loss in an animal brainstem nucleus. Aging Neuron loss Superior olive Brainstem aging Medial nucleus of the trapezoid body Aging auditory system
T H E medial nucleus of the trapezoid body (MTB) is the largest cell group, in terms of cell number, of the superior olivary complex in rats [21]. As shown in Fig. 1, it is situated in the vential medulla at the level of exit of the facial nerve. The principal source of afferents to the MTB is the projection from the contralateral ventral cochlear nucleus (VCN) via the trapezoid body [17, 18, 19, 20, 32]. The major auditory projection of the MTB is to the ipsilateral lateral superior olive [8, 17, 20, 32] and to the nuclei of the lateral lemniscus [12]. The MTB thus occupies a key position in the classical ascending auditory pathway. While no specific function has clearly been attributed to the MTB, it is believed to play a role in the localization of auditory stimuli by relaying input from the contralateral VCN to the ipsilateral lateral superior olive [2 I]. Since recent studies have shown that peripheral auditory structures degenerate with aging in rats [9, 10, 23, 24], this study was.. initiated to determine whether similar effects occur in central portions of the system. The MTBs of an age-graded series of rats were examined for neuron loss, changes in the giant synaptic endings (chalices of Held) on rat MTB neurons [17], and age pigment accumulation. This is the first aging study of the MTB, and indeed one of the first to address the question of neuron loss in an animal brainstem nucleus. METHOD Male
Sprague-Dawley-derived
albino
rats
C o p y r i g h t © 1982 A N K H O
from
the
Age pigment
Rats
Charles River Breeding Laboratories (Wilmington, MA) were studied. The rats were divided into four age groups: 2-3 months, 6 months, 18 months, and 24 months. The rats over 12 months of age were retired breeders, and were maintained by Charles River in a separate aging colony. All animals were reared with food and water available ad lib, and no antibiotics were administered. Under deep chloral hydrate anesthesia (36% aqueous, 0.1 cc/100 g body weight), each rat was tracheotomized and artificially respirated with a mixture of 95% 0., and 5% COz via an endotracheal cannula. Following thoracotomy the aorta was cannulated via the left ventricle and the animal was perfused with Bodian's flush and fixative [3] or with aldehydes [30]. Brainstems from the Bodian-fixed animals were removed, processed routinely, and embedded in paraffin. The tissue blocks were cut in the transverse plane at a thickness of 16 /xm. Every fourth section was mounted and Bodian-stained with protargol [2]. Additional sets of paraffin sections were mounted and Nissl-stained with thionin for study of cell shapes and Nissl patterns. The aldehyde-fixed brainstems were cut into small blocks, post-fixed in buffered osmium tetroxide, dehydrated in ethanols and propylene oxide, and embedded in Araldite. Tissue embedded in Araldite was cut at 2 /xm and stained with a mixture of toluidine blue and pyronin B. Neuron counts for each of 17 animals were done on all mounted protargol-stained sections containing the MTB. The nuclei of neurons in the MTB on one side of each
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FIG. 1. Ventral medulla, in cross-section at the level of the superior olivary complex, of a young adult rat. The boundary of the mcdtai nucleus of the trapezoid body (MTB) is indicated by the interrupted line. BA, basilar artery; LSO, tatcrat superior ~livary nucleus; P't pyramidal tract; TB, fibers of the trapezoid body; VII. exiting fibers of the facial nerve. Protargol stain, + 55
brainstem were counted at a magnification of 400 × with the aid of a grid reticle. Since every fourth section was mounted, the total number of MTB neurons was estimated by multiplying the actual number of nuclei counted by a factor of 4. The correction method of Abercrombie [I] was used. As each neuron was counted, it and the surrounding neuropil were examined for the presence of an identifiable chalice of Held (see Fig. 2). Thus, the percentage of MTB neurons with identifiable chalices and the estimated total number of identifiable chalices in the MTB were determined. It is important to emphasize that, due to plane of section considerations, the obtained chalice counts underrepresent the number of chalices actually present, and are consequently relative rather than absolute values. For this reason. the designation "identifiable chalice" has been used in this study. It is assumed, based on the observations made (see R E S U L T S ) , that the identifiability of chalices in the present material does not change with age. One-way analysis of variance (ANOVA) was used to test for age-dependent effects in the data. When A N O V A s were significant (p<0.05), Scheff6 multiple comparison tests were done a posteriori to detect significant (p<0.05) differences between individual age groups. RESULTS
Qualitative comparison of the thionin-stained MTB sec-
tions from 3 and 24 month old rats (Fig. 3) revealed an apparent loss of neurons over this age range. Quantitatively, this impression was confirmed: the number of neurons in the MTB of Sprague-Dawley rats is reduced with aging (Fig. 4). The observed neuron loss was statistically significant (p <0.05) by 6 months of age and an additional 20% reduction in the mean number of neurons was observed in 18 month old animals. At 24 months, the mean ( - S E M ) number of MTB neurons (1776+_110) was lower than the mean at 18 months, (1945+_127), but the difference as determined with the Scheffe method was not statistically significant. A 34% decrease in the mean number of MTB neurons was observed between 2-3 and 24 months of age (2700+_48 vs. 1776___110; Fig. 4). As was the case with MTB neurons, the number of identifiable chalices of Held steadily declined with age (Fig. 4). Although the decrease at 6 months was not significant (1159+_30 vs. 1033+_57) according to the Scheff6 method, that seen at 18 months was (1159_+30 vs. 799_.+48, p<0,05)+ The mean number of identifiable chalices in 24 month old rats (739__.40) was lower than but not significantly different from that in 18 month old rats (799+_48). At each of the four ages studied, 25-26% of the cells counted in protargolstained sections exhibited chalices. Thus there was no age effect on the percentage of MTB neurons associated with identifiable chalices. No chalices were observed in isolation
NEURON
LOSS IN THE
RAT SUPERIOR
OLIVE
189
FIG. 2. (a) Chalices of Held (arrows) in the M T B , x960; (b) two principal n e u r o n s of the MTB, neither showing evidence o f a chalice of Held in the plane of focus illustrated, × 1040; (c) the s a m e two n e u r o n s illustrated in (b) but at a different plane o f focus showing chalices of Held, × 1040. Protargol stain.
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in the neuropil, i.e., not associated with neuronal cell bodies. In addition, no age-related change was seen in either the form of the chalices (size, number of branches, degree of envelopment of the postsynaptic soma) or their staining properties. Similar to the findings reported in cats [29], three MTB neuron types based on shape were identified in the present study. These cell types were most readily differentiated in the thionin-stained material. The most abundant type of neuron, the principal cell, was rounded or slightly ovoid with an eccentric nucleus (Fig. 5). Principal cells accounted for about 82% of all neurons in the rat MTB (Fig. 6 and Table 1). About 16% of all MTB neurons were elongate or spindleshaped (Figs. 5, 6 and Table 1). Large stellate cells (Fig. 5) made up about 2.5% of the MTB neuronal population (Fig. 6 and Table 1). These proportions of principal, elongate, and stellate cells were relatively consistent along the rostrocaudal extent of the MTB (Fig. 6) and did not vary systematically with age (Table 1). The Nissl patterns of the three cell types were not strikingly different. Both clumped and diffuse forms of Nissl substance were present in MTB neurons. No age-related variations in Nissl patterns were observed• While aging did not significantly alter the ratio of the three neuron types in the MTB, the estimated total number of principal and elongate cells did diminish with age (Fig. 7). The numbers of principal and elongate cells decreased significantly by 18 months of age (Table 2 and Fig. 7). Between 2-3 and 24 months, principal cells decreased by 32'% and elongate cells by 47%. The number of stellate cells, however did not change significantly with age (Fig. 7 and Table 2).
FIG. 4. Effect of age on the total number of neurons and identifiable chalices of Held in the rat MTB. Data points represent the means of N rats. Error bars represent_+l SEM. Analysis of variance (neurons): F(3,13)=21.823, p<0.001. Significant (,o<0.05) Scheff~ tests: 2-3 vs. 6 months, 2-3 vs. 18 months, 2-3 vs. 24 months, 6 vs. 18 months, and 6 vs. 24 months. Analysis of variance (chalices of Held): F(3,13)=24.527, p<0.001. Significant (p<0•05) Scheff~ tests: 2-3 vs. 18 months, 2-3 vs. 24 months, 6 vs. 18 months, and 6 vs. 24 months.
Systematic differences in cell body and nuclear size as a function of age were not apparent in qualitative observation of the present material• This notion was confirmed quantitatively by measuring somal and nuclear areas, using a Zeiss MOP-3 analyzer, in the MTBs of two young (2-3 month) and two aged (24 month) animals. For each animal, approximately 60 principal cells and their nuclei were measured• The means (_+standard deviation) for somal area were 163.7_+33.1 /zmz and 169.9_+28.5/~m 2 in the young animals, and 160.8_+30.1/zm2 and 171.8-+38.9/xm2 in the old animals. The mean nuclear areas obtained were 63.2-_ + 11.4/zm 2 and 66.2-+ 12.1/~m 2 in the young animals and 68.6-. + 14.2/xm 2 and 64.2-+ 10.5/~m 2 in the old animals. Age pigment was observed in some MTB neurons of both young and old rats (Fig. 8). When present, it appeared in the light microscope as dense granules of various sizes which in young animals were usually dispersed within the cytoplasm. Heavy clumped accumulations of age pigment were observed more often in old rats (Fig. 8). Heavy deposits in glial cells in the MTB were present only in old rats. In general, an age-related increase in neuronal pigment was observed, but it was not as dramatic as that seen in, for example, the spiral ganglion [10]. DISCUSSION Relatively few studies have dealt with age-related neuron loss in the nuclei of the brainstem. In humans the facial nucleus [34], trochlear nucleus [35], abducens nucleus [36], ventral cochlear nucleus [25], and the inferior olivary complex [27,28] reportedly do not lose neurons with age. The
N E U R O N LOSS IN T H E RAT S U P E R I O R O L I V E
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FIG. 5. Examples of the appearance, in Nissl stained sections, of the three neuron types in rat MTB, principal cells (a,b,c), elongate cells (d,e,f), and stellate cells (g,h,i arrows). Thionin stain, x720.
locus coeruleus undergoes neuron loss in humans [4, 26, 33, 37, 38], but not in rats [13]. Similarly, the red nucleus in rats does not lose neurons with age [5]. The present study has shown that the MTB in Charles River Sprague-Dawley albino rats undergoes substantial neuron loss with age. Comparable data in humans is lacking, owing chiefly to uncertainties about the identification of MTB neurons in human brainstem. The present study provides the first evidence of significant neuron loss in a non-human brainstem nucleus. Since only two previous studies have addressed the question of neuron loss in an animal brainstem nucleus [5,13], it re-. mains an open question whether loss of neurons with age occurs in other parts of the animal brainstem. Aging in the MTB may be affected by age-related degeneration of other parts of the auditory system. Hair cells and spiral ganglion cells in the rat cochlea are progressively lost throughout the lifespan of rats [23,24], and the auditory nerve, which contains the central processes of spiral ganglion cells, displays nerve fiber degeneration with aging [11]. Quantitative ultrastructural studies of neurons in the rat VCN have revealed loss of synaptic endings from axons originating in the spiral ganglion, indicating a loss of periph-
eral input to the VCN with age [9]. In addition to this reduction in input from the periphery, synapses originating from other sources are also reduced with age [9]. Since the major input to the MTB is from the contralateral VCN [17, 18, 20, 32] a decreased input to the MTB from the partially deafferented VCN is possible in aged animals. Meaningful evaluation of this possibility will of course require considerable additional information about the transneuronal effects of age-related decreases in auditory nerve input on the postsynaptic VCN cells and their axons, in addition to information about other aging changes. The question of cell loss in the aging rat VCN, for example, still remains unresolved. Even though observation of VCN neurons in old animals has revealed cytological alterations [1 I], these have not been studied systematically enough to permit predictions about their consequences in the MTB. While it is possible to speculate about such consequences on the basis of lesion studies in non-aged animals (e.g., [22,31]), it is important to recognize that the findings from such studies may not be applicable to the naturally occurring changes encountered in old animals. Aging changes differ in several respects from the effects of lesions in young animals. They are usually less
192
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TABt.E 1 THE EFFECTOF AGE ON THE PERCENTAGESOF NEURON TYPES 1N THE MTB Mean (SEM) Percentage Age (months)
No. Rats
Principal Cells
5 3 4 5
2-3 6 18 24
8t.68 83.13 83.88 84.16
F(3,12)* = p=
(0.97) (1.56) (0.64) (I.16)
1.086 >0.25 INS)
Elongate Cells 15.78 14.33 13.28 13.08
(1.19) (1.53) (0.52) (I.01)
1.331 >0.25 (NS)
St el]aIc Cells 2.55(0.26) 2.53(0.39) 2.85(0.27) 2.76 (0.29) 0.79 >0.75 (NS)
*Prior to performing the analysis of variance, the percentages of each cell type m each animal were converted to arc sines, since percentages are not normally distributed.
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SECTION NUMBER FIG. 6. Percentages of principal (O), elongate (O), and stellate (A) cells throughout the rostro-caudal extent of the MTB of a 3 month old rat (top graph) and a 24 month old rat (bottom graph), Each data point represents the percentage of a given cell type observed in a given section.
severe in magnitude, they occur more gradually, and they affect cellular elements which may already be compromised by other age-dependent changes in a variety of ways. It is therefore important to avoid the uncritical assumption that MTB changes in old animals are attributable to the same causes and processes present in a young animal after, say, experimental cochlear ablation. The results of the present study indicate that aging affects the chalices of Held on MTB principal neurons. Whether chalice degeneration is a primary or independent event, is
due to possible cell changes in the VCN, or is secondary to the degeneration and loss of postsynaptic neurons is unknown. It is, however, important to note that identifiable chalices not associated with MTB neurons were not observed in the neuropil of the MTB in aged rats. Further, the tempo and extent of identifiable chalice loss parallels that of the neuronal loss (Fig. 4). These facts make it probable that neuron loss and a decrease in the number of observed chalices are related, and it is hypothesized that individual principal neurons and their chalices may disappear at approximately the same time. It is possible that, with aging, chalices undergo morphological changes, such as shrinkage or neurofilament loss, and can no longer be readily identified at the light microscope level in protargol-stained sections. If so, progressively smaller percentages of MTB neurons associated with visible chalices would probably have been observed in the old rats. Instead, these percentages were identical to those in young rats. This, coupled with a failure to qualitatively detect age-related changes in chalice form or stainability, suggests that chalice morphology does not change sufficiently to affect the comparability of the counts in young and old animals. Because principal cells account for about 82% of all neurons in the rat MTB, and a 32% decrease in the number of principal cells was observed in rats at 24 months of age, it is clear that their loss contributed the most to total neuron loss in the aged MTB. The population of elongate cells, however, was more severely affected by aging on a percentage basis, displaying a 47% loss at 24 months of age. The relatively rare stellate MTB cells did not decrease significantly with age. It is thus reasonable to conclude that, as a population, steltate ceils in the MTB are the most resistant to the effects of aging. As is true of neurons in most other parts of the nervous system, some neurons in the rat MTB accumulate cytoplasmic age pigment during the lifespan. Pigment granules were present m some MTB ceils in young adult rats. but relatively large deposits were observed only in some cells of the oldest animals. In old rats. glial cells with heavy pigment deposits were also occasionally seen. The situation thus appears to be similar to that reported for aged rhesus monkeys [6], in which relatively little pigment was observed in neurons of the medial nucleus of the superior olive (MSO) when compared with other parts of the nervous system. However, the glia in the MSO were reported to be heavy
N E U R O N LOSS IN T H E RAT SUPERIOR OLIVE
193 TABLE 2
STATISTICAL ANALYSIS OF DIFFERENTIAL NEURON LOSS IN THE MTB
Analysis of Variance Cell Type Principal Elongate Stellate Significant (p<0.05) Scheff6 Tests Principal Cells: Elongate Ceils:
% Loss* 32% 47% 29%
F(3,12)t 14.557 9.131 2.958
p <0.001 <0.005 <0.100 (NS)
2-3 vs. 18 MO 2-3 vs. 24 MO
*Losses are expressed as percent reductions observed between the youngest (2-3 months) and the oldest (24 months) ages studied. tThe analysis of variance was performed on the total cell counts, not the percentage of cell loss in the preceeding column.
accumulators of pigment [6]. Although quantitative comparative analyses were not performed in the present study, the qualitative observations during the course of this study suggest that pigment accumulation is not a strikingly prominent component of the aging picture in the rat MTB. By relaying contralateral impulses from the VCN to the LSO, the MTB presumably functions in sound localization [21]. The substantial amount of neuron loss with aging observed in the present study suggests that, in addition to other possible changes in auditory behavior, the ability to localize sounds in the azimuth, may be affected in old rats. In a behavioral comparison of 12 month and 30 month rats, Harrison [15] has studied the acquisition and maintenance of a discrimination between the fixed positions of two sound sources. Stimuli were pulsed 70 dB broad-band (4-40 kHz) noise bursts, presented randomly at either source. Responses at the sounding source were reinforced. No agerelated differences were observed in either acquisition or response latency. While the asymptotic percentage of correct responses was lower (p<0.05) for the older animals than for either the 12 month animals or the 3 month animals studied earlier [14,16], all animals performed at better than a 90%-correct level, and the differences between the age groups were attributed to age-related threshold decrements [ 15]. It therefore appears that position discriminations of the type reported [15] can be maintained without serious impairment in old animals even in the presence of changes such as those described in the present report. Whether or not the anatomical changes occurring with age in the MTB will be reflected in other aspects of localization behavior remains to be investigated. It may be of interest to note that establishment and maintenance of a position discrimination of the type just described has been recently found to be disturbed in animals with unilateral incudectomy [7], an experimental procedure which reduces sound intensity at the cochlea on the operated side. Subsequent work (Burlile, Feldman, Harrison, and Craig, unpublished) has shown, however, that if the animals are bilaterally incudectomized, the position discrimination is not affected. These behavioral results emphasize the importance of left-right stimulus balance in the auditory periphery. It is possible that a similar principle is also important with regard to central structures involved in localization behaviors. If so, certain anatomical aging changes which happen to develop with bilateral symmetry in structures such as the MTB may influ-
PRINCIPAL CELLS ..+-
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STELLATE CELLS 75 50 25
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FIG. 7. The effect of age on the estimated total number of principal, elongate, and stellate cells in the rat MTB. The values plotted are means of the individual animal values at each age. The vertical lines represent___ 1 SEM. See "Fable2 for analysis of variance and Scheff6 tests.
ence only specialized aspects of localization behavior such as the minimum discriminable angle.
FIG. 8. The appearancc of MTB neurons in plastic embedded 2/xm sections o f a 3 month (a) and a 27 month (b) ~at. MTB ncurons at both ages exhibit evidence of pigment deposits tat'rows), but the heavier deposits are seen in the older n e u r o n s Note that thc profiles of several neurons in section (b) arc frec of pigment, Toluidine blue stain, ×873.
NEURON
LOSS IN THE RAT SUPERIOR OLIVE
195
In v i e w o f t h e p r e s u m e d s i g n i f i c a n c e o f t h e M T B in loc a l i z a t i o n , t h e e x t e n t to w h i c h a g e - r e l a t e d c h a n g e s d e v e l o p in bilaterally s y m m e t r i c a l f a s h i o n m a y b e a r e l e v a n t issue. S u c h d e t e r m i n a t i o n w a s n o t a p a r t o f t h e p r e s e n t study. If, h o w e v e r , t h e d e g r e e o f left-right s y m m e t r y o f t h e a s c e n d i n g a u d i t o r y p a t h w a y s is a critical d e t e r m i n a n t o f b e h a v i o r a l loc a l i z a t i o n (see d i s c u s s i o n a b o v e ) , a n a t o m i c a l s t u d i e s o f loss symmetry may be of considerable importance.
ACKNOWLEDGEMENTS W e thank Carol Craig for technical assistance, Shirley Kelley and Mary Alba for the typing, and Dr. J. Michael Harrison for his comments on the manuscript. W e also thank Dr. Herbert Kayne and Lynda Rose for help with statistics.This study was supported by Program Project Grant AG00001, and Research Career Development Award AG00016 to M.L.F. from the National Institutes of Health.
REFERENCES 1. Abercrombie, M. Estimation of nuclear population from microtome sections. Anal. Rec. 94: 239-247, 1946. 2. Bodian, D. A new method for staining nerve fibers and nerve endings in mounted paraffin sections. Anat. Rec. 65: 89-97, 1936. 3. Bodian, D. The staining of paraffin sections of nervous tissues with activated protargol. The role of fixatives. Anat. Rec. 69: 153--162, 1937. 4. Bondareff, W., C. Q. Mountjoy and M. Roth. Selective loss of neurones of origin of adrenergic projection to cerebral cortex (nucleus locus coeruleus) in senile dementia. Lancet 8223: 783784, 1981. 5. Boselia, A.-W. A., S. M. Hashem and Y. H. Badawy. Volumetric studies on the red nucleus of the rat at different ages. Aeta Anat. 91: 175-180~ 1975._ 6. Brizzee, K. R., J. M. Ordy and B. Kaack. Early appearance and regional differences in intraneuronal and extraneuronal lipofuscin accumulation with age in the brain of a nonhuman primate (Macaca mulatta). J. Gerontol. 29: 366--381. 7. Burlile, C. J., M. L. Feldman and J. M. Harrison. Auditory position discrimination in unilaterally incudectomized rats. Abstracts of Eastern Psychological Association, 1982. 8. Elverland, H. H. Ascending and intrinsic projections of the superior olivary complex in the cat. Expl Brain Res. 32:117134, 1978. 9. Feldman, M. L. Synaptic loss with advanced age in the cochlear nucleus. Neuroscience 7: 567, 1982. 10. Feldman, M. L., C. Craig and E. Keithley. Differential age pigment accumulation in type I and type II ganglion cells of rat cochlea. Abstracts of the American Aging Association, 1981. 11. Feldman, M. L. and D. W. Vaughan. Changes in the auditory pathway with age. In: Special Senses in Aging, edited by S. S. Han and D. H. Coons. Ann Arbor: Univ. of Michigan Institute of Gerontology, 1979, pp. 143-162. 12. Glendenning, K. K., J. K. Brunso-Bechtold, G. C. Thompson and R. B. Masterton. Ascending auditory afferents to the nuclei of the lateral lemniscus. J. comp. Neurol. 197: 673-703, 1981. 13. Goldman, G. and P. D. Coleman. Neuron numbers in locus coeruleus do not change with age in Fisher 344 rat. Neurobiol. Aging 2: 33-36, 1981. 14. Harrison, J. M. The control of responding by sounds: Unusual effect of reinforcement. J. exp. Analysis Behar. 32: 167-181, 1979. 15. Harrison, J. M. Effects of age on acquisition and maintenance of a location discrimination in rats. Expl Aging Res. 7: 467-476, 1981. 16. Harrison, J. M., P. Downey, M. Segal and M. Howe. Control of responding by the location of auditory stimuli: Rapid acquisition in rat and monkey. J. exp. Analysis Behav. 15: 373-386, 1971. 17. Harrison, J. M. and M. L. Feldman. Anatomical aspects of the cochlear nucleus and superior olivary complex. In: Contributions to Sensor)., Physiology, Vol. 4, edited by W. D. Neff. New York: Academic Press, 1970, pp. 95-142. 18. Harrison, J. M. and R. Irving. Ascending connections of the anterior ventral cochlear nucleus in the rat. J. comp. Neurol. 126: 51-64, 1966. 19. Harrison, J. M. and R. Irving. Visual and non-visual auditory systems in mammals. Science 154: 738-743, 1966.
20. Harrison, J. M. and W. B. Wart. A study of the cochlear nuclei and ascending auditory pathways of the medulla. J. comp. Neurol. 119: 341-378, 1962. 21. Irving, R. and J. M. Harrison. The superior olivary complex and audition: A comparative study. J. comp. Neurol. 130: 77-86, 1967. 22. Jean-Baptiste, M. and D. K. Morest. Transneuronal changes of synaptic endings and nuclear chromatin in the trapezoid body following cochlear ablations in cats. J. comp. Neurol. 162: I I 1-134, 1975. 23. Keithley, E. M. and M. L. Feldman. Hair cell counts in an age-graded series of rat cochleas. Hearing Res., in press. 24. Keithley, E. M. and M. L. Feldman. Spiral ganglion cell counts in an age-graded series of rat cochleas. J. comp. Neurol. 188: 429-442, 1979. 25. Konigsmark, B. W. and E. A. Murphy. Volume of the ventral cochlear nucleus in man: its relationship to neuronal population and age. J. Neuropath. exp. Neurol. 31: 304-316, 1972. 26. Mann, D. M. A. and P. O. Yates. The effect of ageing in the pigmented nerve cells of the human locus coeruleus and substantia nigra. Acta Neuropath. 47: 93-97, 1979. 27. Moatamed, F. Cell frequencies in the human inferior olivary nuclear complex. J. comp. Neurol. 128:109-116, 1966. 28. Monagle, R. D. and H. Brody. The effects of age upon the main nucleus of the inferior olive in the human. J. comp. Neurol. 155: 61-66, 1974. 29. Morest, D. K. The collateral system of the medial nucleus of the trapezoid body of the cat, its neuronal architecture and relation to the olivo-cochlear bundle. Brain Res. 9: 288-311, 1968. 30. Peters, A. and T. M. Walsh. A study of the organization of apical dendrites in the somatic sensory cortex of the rat. J. comp. Neurol. 144: 253-268, 1972. 31. Powell, T. P. S. and S. D. Erulkar. Transneuronal cell degeneration in the auditory relay nuclei of the cat. J. Anat. 96: 249-268. 32. Stotler, W. A. An experimental study of the cells and connections of the superior olivary complex of the cat. J. comp. Neurol. 98: 401-431, 1953. 33. Tomlinson, B. E., D. Irving and G. Blessed. Cell loss in the locus coeruleus in senile dementia of Alzheimer type. 3. Neurol. Sci. 49: 419-428, 1981. 34. Van Buskirk, C. The seventh nerve complex. J. comp. Neurol. 82: 303-333, 1945. 35. Vijayashankar, N. and H. Brody. Aging in the human brainstem. A study of the nucleus of the trochlear nerve. Acta Anat. 99: 169-172, 1977. 36. Vijayashankar, N. and H. Brody. A study of aging in the human abducens nucleus. J. comp. Neurol. 173: 433--438, 1977. 37. Vijayashankar, N. and H. Brody. A quantitative study of the pigmented neurons in the nuclei locus coeruleus and subcoeruleus in man as related to aging. J. Neuropath. exp. Neurol. 38: 490-497, 1979. 38. Wree, A., H. Braak, A. Schleicher and K. Zilles. Biomathematical analysis of the neuronal loss in aging human brain of both sexes, demonstrated in pigment preparations of the pars cerebellaris loci coerulei. Anat. Embryol. 160:105-119. 1981.
Aging in the Rat Medial Nucleus of the Trapezoid Body I. Light Microscopy MICHAEL
A. C A S E Y A N D M A R T I N L. F E L D M A N
D e p a r t m e n t o f A n a t o m y , Boston University School o f Medicine, Boston, M A 02118 R e c e i v e d 21 J u l y 1982 CASEY, M. A. AND M. L. FELDMAN. Aging in the rat medial nucleus of the trapezoid body. 1. Light microscopy. NEUROBIOL. AGING 3(3) 187-195, 1982.--The medial nucleus of the trapezoid body (MTB), a cell group in the superior olivary complex, was examined in an age-graded series of rats for neuron loss, changes in the giant synaptic endings (chalices of Held) on MTB neurons, and accumulation of age pigment. Neuron counts were done on protargol-stained paraffin sections of MTBs from a series of 17 rats aged 2-3, 6, 18, and 24 months. Between 2-3 and 24 months a 34% decrease in the mean number of MTB neurons was observed. Significant loss Ip <0.05) was first evident in the early portion of the life span, between 2-3 and 6 months. In thionin-stained sections, there was no change with aging in the proportions of three MTB neuron types: principal cells (~82%), elongate cells (~15%), and stellate cells (~3%). In young adult rats, 25-:26% of all MTB neurons were associated with identifiable chalices of Held in protargol stained sections. This ratio did not vary significantly with aging. Age pigment accumulation in the MTB was examined in 2 p.m Araldite sections stained with toluidine blue. Age pigment deposits were larger and more numerous in the MTBs of old animals, but not as extensive as has been described previously in many other parts of the nervous system. This study is the first to report neuron loss in an animal brainstem nucleus. Aging Neuron loss Superior olive Brainstem aging Medial nucleus of the trapezoid body Aging auditory system
T H E medial nucleus of the trapezoid body (MTB) is the largest cell group, in terms of cell number, of the superior olivary complex in rats [21]. As shown in Fig. 1, it is situated in the vential medulla at the level of exit of the facial nerve. The principal source of afferents to the MTB is the projection from the contralateral ventral cochlear nucleus (VCN) via the trapezoid body [17, 18, 19, 20, 32]. The major auditory projection of the MTB is to the ipsilateral lateral superior olive [8, 17, 20, 32] and to the nuclei of the lateral lemniscus [12]. The MTB thus occupies a key position in the classical ascending auditory pathway. While no specific function has clearly been attributed to the MTB, it is believed to play a role in the localization of auditory stimuli by relaying input from the contralateral VCN to the ipsilateral lateral superior olive [2 I]. Since recent studies have shown that peripheral auditory structures degenerate with aging in rats [9, 10, 23, 24], this study was.. initiated to determine whether similar effects occur in central portions of the system. The MTBs of an age-graded series of rats were examined for neuron loss, changes in the giant synaptic endings (chalices of Held) on rat MTB neurons [17], and age pigment accumulation. This is the first aging study of the MTB, and indeed one of the first to address the question of neuron loss in an animal brainstem nucleus. METHOD Male
Sprague-Dawley-derived
albino
rats
C o p y r i g h t © 1982 A N K H O
from
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Age pigment
Rats
Charles River Breeding Laboratories (Wilmington, MA) were studied. The rats were divided into four age groups: 2-3 months, 6 months, 18 months, and 24 months. The rats over 12 months of age were retired breeders, and were maintained by Charles River in a separate aging colony. All animals were reared with food and water available ad lib, and no antibiotics were administered. Under deep chloral hydrate anesthesia (36% aqueous, 0.1 cc/100 g body weight), each rat was tracheotomized and artificially respirated with a mixture of 95% 0., and 5% COz via an endotracheal cannula. Following thoracotomy the aorta was cannulated via the left ventricle and the animal was perfused with Bodian's flush and fixative [3] or with aldehydes [30]. Brainstems from the Bodian-fixed animals were removed, processed routinely, and embedded in paraffin. The tissue blocks were cut in the transverse plane at a thickness of 16 /xm. Every fourth section was mounted and Bodian-stained with protargol [2]. Additional sets of paraffin sections were mounted and Nissl-stained with thionin for study of cell shapes and Nissl patterns. The aldehyde-fixed brainstems were cut into small blocks, post-fixed in buffered osmium tetroxide, dehydrated in ethanols and propylene oxide, and embedded in Araldite. Tissue embedded in Araldite was cut at 2 /xm and stained with a mixture of toluidine blue and pyronin B. Neuron counts for each of 17 animals were done on all mounted protargol-stained sections containing the MTB. The nuclei of neurons in the MTB on one side of each
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FIG. 1. Ventral medulla, in cross-section at the level of the superior olivary complex, of a young adult rat. The boundary of the mcdtai nucleus of the trapezoid body (MTB) is indicated by the interrupted line. BA, basilar artery; LSO, tatcrat superior ~livary nucleus; P't pyramidal tract; TB, fibers of the trapezoid body; VII. exiting fibers of the facial nerve. Protargol stain, + 55
brainstem were counted at a magnification of 400 × with the aid of a grid reticle. Since every fourth section was mounted, the total number of MTB neurons was estimated by multiplying the actual number of nuclei counted by a factor of 4. The correction method of Abercrombie [I] was used. As each neuron was counted, it and the surrounding neuropil were examined for the presence of an identifiable chalice of Held (see Fig. 2). Thus, the percentage of MTB neurons with identifiable chalices and the estimated total number of identifiable chalices in the MTB were determined. It is important to emphasize that, due to plane of section considerations, the obtained chalice counts underrepresent the number of chalices actually present, and are consequently relative rather than absolute values. For this reason. the designation "identifiable chalice" has been used in this study. It is assumed, based on the observations made (see R E S U L T S ) , that the identifiability of chalices in the present material does not change with age. One-way analysis of variance (ANOVA) was used to test for age-dependent effects in the data. When A N O V A s were significant (p<0.05), Scheff6 multiple comparison tests were done a posteriori to detect significant (p<0.05) differences between individual age groups. RESULTS
Qualitative comparison of the thionin-stained MTB sec-
tions from 3 and 24 month old rats (Fig. 3) revealed an apparent loss of neurons over this age range. Quantitatively, this impression was confirmed: the number of neurons in the MTB of Sprague-Dawley rats is reduced with aging (Fig. 4). The observed neuron loss was statistically significant (p <0.05) by 6 months of age and an additional 20% reduction in the mean number of neurons was observed in 18 month old animals. At 24 months, the mean ( - S E M ) number of MTB neurons (1776+_110) was lower than the mean at 18 months, (1945+_127), but the difference as determined with the Scheffe method was not statistically significant. A 34% decrease in the mean number of MTB neurons was observed between 2-3 and 24 months of age (2700+_48 vs. 1776___110; Fig. 4). As was the case with MTB neurons, the number of identifiable chalices of Held steadily declined with age (Fig. 4). Although the decrease at 6 months was not significant (1159+_30 vs. 1033+_57) according to the Scheff6 method, that seen at 18 months was (1159_+30 vs. 799_.+48, p<0,05)+ The mean number of identifiable chalices in 24 month old rats (739__.40) was lower than but not significantly different from that in 18 month old rats (799+_48). At each of the four ages studied, 25-26% of the cells counted in protargolstained sections exhibited chalices. Thus there was no age effect on the percentage of MTB neurons associated with identifiable chalices. No chalices were observed in isolation
NEURON
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FIG. 2. (a) Chalices of Held (arrows) in the M T B , x960; (b) two principal n e u r o n s of the MTB, neither showing evidence o f a chalice of Held in the plane of focus illustrated, × 1040; (c) the s a m e two n e u r o n s illustrated in (b) but at a different plane o f focus showing chalices of Held, × 1040. Protargol stain.
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in the neuropil, i.e., not associated with neuronal cell bodies. In addition, no age-related change was seen in either the form of the chalices (size, number of branches, degree of envelopment of the postsynaptic soma) or their staining properties. Similar to the findings reported in cats [29], three MTB neuron types based on shape were identified in the present study. These cell types were most readily differentiated in the thionin-stained material. The most abundant type of neuron, the principal cell, was rounded or slightly ovoid with an eccentric nucleus (Fig. 5). Principal cells accounted for about 82% of all neurons in the rat MTB (Fig. 6 and Table 1). About 16% of all MTB neurons were elongate or spindleshaped (Figs. 5, 6 and Table 1). Large stellate cells (Fig. 5) made up about 2.5% of the MTB neuronal population (Fig. 6 and Table 1). These proportions of principal, elongate, and stellate cells were relatively consistent along the rostrocaudal extent of the MTB (Fig. 6) and did not vary systematically with age (Table 1). The Nissl patterns of the three cell types were not strikingly different. Both clumped and diffuse forms of Nissl substance were present in MTB neurons. No age-related variations in Nissl patterns were observed• While aging did not significantly alter the ratio of the three neuron types in the MTB, the estimated total number of principal and elongate cells did diminish with age (Fig. 7). The numbers of principal and elongate cells decreased significantly by 18 months of age (Table 2 and Fig. 7). Between 2-3 and 24 months, principal cells decreased by 32'% and elongate cells by 47%. The number of stellate cells, however did not change significantly with age (Fig. 7 and Table 2).
FIG. 4. Effect of age on the total number of neurons and identifiable chalices of Held in the rat MTB. Data points represent the means of N rats. Error bars represent_+l SEM. Analysis of variance (neurons): F(3,13)=21.823, p<0.001. Significant (,o<0.05) Scheff~ tests: 2-3 vs. 6 months, 2-3 vs. 18 months, 2-3 vs. 24 months, 6 vs. 18 months, and 6 vs. 24 months. Analysis of variance (chalices of Held): F(3,13)=24.527, p<0.001. Significant (p<0•05) Scheff~ tests: 2-3 vs. 18 months, 2-3 vs. 24 months, 6 vs. 18 months, and 6 vs. 24 months.
Systematic differences in cell body and nuclear size as a function of age were not apparent in qualitative observation of the present material• This notion was confirmed quantitatively by measuring somal and nuclear areas, using a Zeiss MOP-3 analyzer, in the MTBs of two young (2-3 month) and two aged (24 month) animals. For each animal, approximately 60 principal cells and their nuclei were measured• The means (_+standard deviation) for somal area were 163.7_+33.1 /zmz and 169.9_+28.5/~m 2 in the young animals, and 160.8_+30.1/zm2 and 171.8-+38.9/xm2 in the old animals. The mean nuclear areas obtained were 63.2-_ + 11.4/zm 2 and 66.2-+ 12.1/~m 2 in the young animals and 68.6-. + 14.2/xm 2 and 64.2-+ 10.5/~m 2 in the old animals. Age pigment was observed in some MTB neurons of both young and old rats (Fig. 8). When present, it appeared in the light microscope as dense granules of various sizes which in young animals were usually dispersed within the cytoplasm. Heavy clumped accumulations of age pigment were observed more often in old rats (Fig. 8). Heavy deposits in glial cells in the MTB were present only in old rats. In general, an age-related increase in neuronal pigment was observed, but it was not as dramatic as that seen in, for example, the spiral ganglion [10]. DISCUSSION Relatively few studies have dealt with age-related neuron loss in the nuclei of the brainstem. In humans the facial nucleus [34], trochlear nucleus [35], abducens nucleus [36], ventral cochlear nucleus [25], and the inferior olivary complex [27,28] reportedly do not lose neurons with age. The
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locus coeruleus undergoes neuron loss in humans [4, 26, 33, 37, 38], but not in rats [13]. Similarly, the red nucleus in rats does not lose neurons with age [5]. The present study has shown that the MTB in Charles River Sprague-Dawley albino rats undergoes substantial neuron loss with age. Comparable data in humans is lacking, owing chiefly to uncertainties about the identification of MTB neurons in human brainstem. The present study provides the first evidence of significant neuron loss in a non-human brainstem nucleus. Since only two previous studies have addressed the question of neuron loss in an animal brainstem nucleus [5,13], it re-. mains an open question whether loss of neurons with age occurs in other parts of the animal brainstem. Aging in the MTB may be affected by age-related degeneration of other parts of the auditory system. Hair cells and spiral ganglion cells in the rat cochlea are progressively lost throughout the lifespan of rats [23,24], and the auditory nerve, which contains the central processes of spiral ganglion cells, displays nerve fiber degeneration with aging [11]. Quantitative ultrastructural studies of neurons in the rat VCN have revealed loss of synaptic endings from axons originating in the spiral ganglion, indicating a loss of periph-
eral input to the VCN with age [9]. In addition to this reduction in input from the periphery, synapses originating from other sources are also reduced with age [9]. Since the major input to the MTB is from the contralateral VCN [17, 18, 20, 32] a decreased input to the MTB from the partially deafferented VCN is possible in aged animals. Meaningful evaluation of this possibility will of course require considerable additional information about the transneuronal effects of age-related decreases in auditory nerve input on the postsynaptic VCN cells and their axons, in addition to information about other aging changes. The question of cell loss in the aging rat VCN, for example, still remains unresolved. Even though observation of VCN neurons in old animals has revealed cytological alterations [1 I], these have not been studied systematically enough to permit predictions about their consequences in the MTB. While it is possible to speculate about such consequences on the basis of lesion studies in non-aged animals (e.g., [22,31]), it is important to recognize that the findings from such studies may not be applicable to the naturally occurring changes encountered in old animals. Aging changes differ in several respects from the effects of lesions in young animals. They are usually less
192
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TABt.E 1 THE EFFECTOF AGE ON THE PERCENTAGESOF NEURON TYPES 1N THE MTB Mean (SEM) Percentage Age (months)
No. Rats
Principal Cells
5 3 4 5
2-3 6 18 24
8t.68 83.13 83.88 84.16
F(3,12)* = p=
(0.97) (1.56) (0.64) (I.16)
1.086 >0.25 INS)
Elongate Cells 15.78 14.33 13.28 13.08
(1.19) (1.53) (0.52) (I.01)
1.331 >0.25 (NS)
St el]aIc Cells 2.55(0.26) 2.53(0.39) 2.85(0.27) 2.76 (0.29) 0.79 >0.75 (NS)
*Prior to performing the analysis of variance, the percentages of each cell type m each animal were converted to arc sines, since percentages are not normally distributed.
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SECTION NUMBER FIG. 6. Percentages of principal (O), elongate (O), and stellate (A) cells throughout the rostro-caudal extent of the MTB of a 3 month old rat (top graph) and a 24 month old rat (bottom graph), Each data point represents the percentage of a given cell type observed in a given section.
severe in magnitude, they occur more gradually, and they affect cellular elements which may already be compromised by other age-dependent changes in a variety of ways. It is therefore important to avoid the uncritical assumption that MTB changes in old animals are attributable to the same causes and processes present in a young animal after, say, experimental cochlear ablation. The results of the present study indicate that aging affects the chalices of Held on MTB principal neurons. Whether chalice degeneration is a primary or independent event, is
due to possible cell changes in the VCN, or is secondary to the degeneration and loss of postsynaptic neurons is unknown. It is, however, important to note that identifiable chalices not associated with MTB neurons were not observed in the neuropil of the MTB in aged rats. Further, the tempo and extent of identifiable chalice loss parallels that of the neuronal loss (Fig. 4). These facts make it probable that neuron loss and a decrease in the number of observed chalices are related, and it is hypothesized that individual principal neurons and their chalices may disappear at approximately the same time. It is possible that, with aging, chalices undergo morphological changes, such as shrinkage or neurofilament loss, and can no longer be readily identified at the light microscope level in protargol-stained sections. If so, progressively smaller percentages of MTB neurons associated with visible chalices would probably have been observed in the old rats. Instead, these percentages were identical to those in young rats. This, coupled with a failure to qualitatively detect age-related changes in chalice form or stainability, suggests that chalice morphology does not change sufficiently to affect the comparability of the counts in young and old animals. Because principal cells account for about 82% of all neurons in the rat MTB, and a 32% decrease in the number of principal cells was observed in rats at 24 months of age, it is clear that their loss contributed the most to total neuron loss in the aged MTB. The population of elongate cells, however, was more severely affected by aging on a percentage basis, displaying a 47% loss at 24 months of age. The relatively rare stellate MTB cells did not decrease significantly with age. It is thus reasonable to conclude that, as a population, steltate ceils in the MTB are the most resistant to the effects of aging. As is true of neurons in most other parts of the nervous system, some neurons in the rat MTB accumulate cytoplasmic age pigment during the lifespan. Pigment granules were present m some MTB ceils in young adult rats. but relatively large deposits were observed only in some cells of the oldest animals. In old rats. glial cells with heavy pigment deposits were also occasionally seen. The situation thus appears to be similar to that reported for aged rhesus monkeys [6], in which relatively little pigment was observed in neurons of the medial nucleus of the superior olive (MSO) when compared with other parts of the nervous system. However, the glia in the MSO were reported to be heavy
N E U R O N LOSS IN T H E RAT SUPERIOR OLIVE
193 TABLE 2
STATISTICAL ANALYSIS OF DIFFERENTIAL NEURON LOSS IN THE MTB
Analysis of Variance Cell Type Principal Elongate Stellate Significant (p<0.05) Scheff6 Tests Principal Cells: Elongate Ceils:
% Loss* 32% 47% 29%
F(3,12)t 14.557 9.131 2.958
p <0.001 <0.005 <0.100 (NS)
2-3 vs. 18 MO 2-3 vs. 24 MO
*Losses are expressed as percent reductions observed between the youngest (2-3 months) and the oldest (24 months) ages studied. tThe analysis of variance was performed on the total cell counts, not the percentage of cell loss in the preceeding column.
accumulators of pigment [6]. Although quantitative comparative analyses were not performed in the present study, the qualitative observations during the course of this study suggest that pigment accumulation is not a strikingly prominent component of the aging picture in the rat MTB. By relaying contralateral impulses from the VCN to the LSO, the MTB presumably functions in sound localization [21]. The substantial amount of neuron loss with aging observed in the present study suggests that, in addition to other possible changes in auditory behavior, the ability to localize sounds in the azimuth, may be affected in old rats. In a behavioral comparison of 12 month and 30 month rats, Harrison [15] has studied the acquisition and maintenance of a discrimination between the fixed positions of two sound sources. Stimuli were pulsed 70 dB broad-band (4-40 kHz) noise bursts, presented randomly at either source. Responses at the sounding source were reinforced. No agerelated differences were observed in either acquisition or response latency. While the asymptotic percentage of correct responses was lower (p<0.05) for the older animals than for either the 12 month animals or the 3 month animals studied earlier [14,16], all animals performed at better than a 90%-correct level, and the differences between the age groups were attributed to age-related threshold decrements [ 15]. It therefore appears that position discriminations of the type reported [15] can be maintained without serious impairment in old animals even in the presence of changes such as those described in the present report. Whether or not the anatomical changes occurring with age in the MTB will be reflected in other aspects of localization behavior remains to be investigated. It may be of interest to note that establishment and maintenance of a position discrimination of the type just described has been recently found to be disturbed in animals with unilateral incudectomy [7], an experimental procedure which reduces sound intensity at the cochlea on the operated side. Subsequent work (Burlile, Feldman, Harrison, and Craig, unpublished) has shown, however, that if the animals are bilaterally incudectomized, the position discrimination is not affected. These behavioral results emphasize the importance of left-right stimulus balance in the auditory periphery. It is possible that a similar principle is also important with regard to central structures involved in localization behaviors. If so, certain anatomical aging changes which happen to develop with bilateral symmetry in structures such as the MTB may influ-
PRINCIPAL CELLS ..+-
2000
1000
~E Z
ELONGATE CELLS ¢.,¢) ,,....l i.e..i
400 200
Z
STELLATE CELLS 75 50 25
2-3
6
1"8 AGE (months)
24
FIG. 7. The effect of age on the estimated total number of principal, elongate, and stellate cells in the rat MTB. The values plotted are means of the individual animal values at each age. The vertical lines represent___ 1 SEM. See "Fable2 for analysis of variance and Scheff6 tests.
ence only specialized aspects of localization behavior such as the minimum discriminable angle.
FIG. 8. The appearancc of MTB neurons in plastic embedded 2/xm sections o f a 3 month (a) and a 27 month (b) ~at. MTB ncurons at both ages exhibit evidence of pigment deposits tat'rows), but the heavier deposits are seen in the older n e u r o n s Note that thc profiles of several neurons in section (b) arc frec of pigment, Toluidine blue stain, ×873.
NEURON
LOSS IN THE RAT SUPERIOR OLIVE
195
In v i e w o f t h e p r e s u m e d s i g n i f i c a n c e o f t h e M T B in loc a l i z a t i o n , t h e e x t e n t to w h i c h a g e - r e l a t e d c h a n g e s d e v e l o p in bilaterally s y m m e t r i c a l f a s h i o n m a y b e a r e l e v a n t issue. S u c h d e t e r m i n a t i o n w a s n o t a p a r t o f t h e p r e s e n t study. If, h o w e v e r , t h e d e g r e e o f left-right s y m m e t r y o f t h e a s c e n d i n g a u d i t o r y p a t h w a y s is a critical d e t e r m i n a n t o f b e h a v i o r a l loc a l i z a t i o n (see d i s c u s s i o n a b o v e ) , a n a t o m i c a l s t u d i e s o f loss symmetry may be of considerable importance.
ACKNOWLEDGEMENTS W e thank Carol Craig for technical assistance, Shirley Kelley and Mary Alba for the typing, and Dr. J. Michael Harrison for his comments on the manuscript. W e also thank Dr. Herbert Kayne and Lynda Rose for help with statistics.This study was supported by Program Project Grant AG00001, and Research Career Development Award AG00016 to M.L.F. from the National Institutes of Health.
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