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A morphometric study of the pallid mutant mouse inner ear
Original Contributions Am ] Otalaryngol 4 2fii-272, 1983
A Morphometric Study of the Pallid Mutant Mouse Inner Ear DENNIS R. TRUNE, PH.D., AND DAVIDJ. LIM, M.D. Mice homozygous for the mutant gene pallid (pa/pa) often lack otoconia in some or all of their maculae and are used to study the influences of gravity receptor hypostimulation on vestibular-related behaviors. Since the value of this animal model is based on the assumption that the vestibular sensorineural elements are normal, a morphometric analysis was done on the inner ear of these otoconia-deficient mice to see whether sensorineural structures are also affected by the pallid gene. In pallid mice lacking all otoconia, the sensory epithelia of the utricle, saccule, and semicircular canal cristae were the same size as in their heterozygous (pa/+) controls. Although the superior and inferior divisions of the vestibular ganglion of the pallid mice were smaller than normal, the first-order neurons within these divisions were normal in size, number, and density. However, the superior divisions in both groups had larger neurons than did the inferior divisions. Within the pallid cochlea, first-order auditory neurons within the spiral ganglion were smaller than normal, but the scala media was larger. Since the significant vestibular influences of the pallid gene are limited primarily to the otoconia, behavioral abnormalities reported for these otoconia-deficient mice are apparently due only to gravity receptor hypostimulation. (Key words: Pallid mouse; Cochlea; Vestibule; Otoconia; Morphometry.)
Lyon1,2first described the absence of otoconia in the inner ears of mice homozygous for the recessive, mutant gene pallid (pa). Although all homozygotes (pa/pa) have the pale coat color and u n p i g m e n t e d eye, the penetrance of the otoconial effect is incomplete and can range, within the same animal, from no otoconia in any of the gravity receptors to normal otoconia in some or all receptors. Also, the pa gene is considered pleiotropic, since otoconial loss is never observed without the lack of pigmentation, and it is apparently responsible for both the otoconia and pigment defects. Mutants in which otoconia deficiency is a predominant characteristic have been observed among a variety of species, most of which also have an associated pigment defect. These include the mutant mice mocha, 3 tiltedhead, 4 and lethal-milk, 5 as well as the ocular albino rabbit, gray-loco chukar partridge, pastel mink, 6 octopus, cuttlefish, and squid. 7 Recently, otoconia deficiency was also observed in hu-
mans, 8 although a genetic linkage has not been established. Therefore, this animal model makes a useful vehicle for gaining new insight into the otoconia-deficient syndrome. The lack of otoconial development in the mutant mouse is correlated with a decrease in the synthesis of the macular sulfomucopolysaccharides that apparently are necessary for the initiation of crystal growth. Q Increasing manganese concentrations in the diets of pregnant pallid females prevents this otoconial defect in their offspring, and, conversely, removal of manganese from the diets of pregnant normal mice prevents otoconial formation in their young, thus mimicking the effects of the mutant gene. I~ The exact role of manganese in the development of normal otoconia has not been established, but it is suggested that normal biosynthesis of mucopolysaccharides in otoconia requires manganese.14.15 This lack of otoconia in some or all of the gravity receptors leads to a variety of vestibular behavioral problems. These include head tilting, ataxia, circling, disorientation in water, and absence of righting reflexes. 1,11,~3.1BOtoconial loss in pallid mice also has been correlated with reduced spatial orientation and increased emotionality. 17 Previous investigators have observed only temporary endolymphatic hydrops during gestatioal,9,1B and immature melanin granules
Received March 22, 1983, from the Otological Research Laboratories, Department of Otolaryngology,The Ohio State University College of Medicine, Columbus, Ohio. Accepted for publication April 7, 1983. Supported by National Aeronautics and Space Administrationgrant NSG-2220 (to D.J.L.) and National Institutes of Health grant NINCDS-NRSA5 F32 NSO6211 (to D.R.T.). Address correspondenceand reprint requests to Dr. Trune: Otological Research Laboratories,4331 UniversityHospitals Clinic, 456 Clinic Drive, Columbus, OH 43210.
261
SENSOKINEURAL STRUCTURES IN PALLID MOUSE INNER EAR
within otherwise nomnal vestibular melanocytes in the m u t a n t adults, 13 Aside from the etoconial loss, f e w morphologic abnormalities have been observed in the pallid inner ear. Because these morpholagic studies suggest this genetic defect is limited to the otocania, the pallid mouse has b e c o m e an important animal m o d e l with which to s t u d y the effects of gravity receptor hypostiraulation on the d e v e l o p m e n t and function of the brain. 1B,~7,~9,20However, before the changes seen in these central studies can be properly interpreted, it is important to rule out other genetic influences besides otoconial deficiency on the d e v e l o p m e n t of the pallid vestibular periphery. To this end, we u n d e r t o o k a morphometric inv e s t i g a t i o n of all the p e r i p h e r a l auditory and vestibular sensorineural structures. Once this ass e s s m e n t is m a d e of the pallid vestibular per i p h e r y , our b r a i n s t e m s t u d i e s can be more properly interpreted w i t h regard to the role of gravity receptor sensation in brain development and function, M A T E R I A L S AND METHODS
American Journal
of Otolaryngalogy 262
Our pallid mutant colony at The Ohio State University w a s originally derived from the University of Cincinnati [Dr. L.C. Erway's laboratory). The pa gene is maintained in the C57BL/ 6J strain of Mus musculus. Phenotypically normal heterozygous ( p c / + ) females were mated with pallid (pa/pa) males to provide us with litters that i n c l u d e d both pallid mutants and heterozygous controls. Since the pa gene displays inc o m p l e t e p e n e t r a n c e w i t h regard to the otoconial effect, there are varying degrees of atoc o n i a l d e v e l o p m e n t in the p a l l i d offspring. Therefore, w e selected only those mutants with complete penetrance, i.e., bilateral absence of all otoconia, since the maximal influence of the pa mutation should be most apparent in this condition. Since previous studies have demonstrated that mice w i t h no otoconia are disoriented in water and c a n n o t right t h e m s e l v e s in a vertical drop, 4,11,~3 w e t e s t e d t h e s w i m m i n g and airrighting abilities of the pallid mutants to identify those with all gravity receptors deficient of otoconia. Heterazygous control animals were also tested to indicate normal behavior in these tests. All behavioral tests were conducted w h e n the mice were b e t w e e n 45 a n d 60 days of age. Eight p a l l i d m u t a n t s t h a t c o u l d n o t s w i m or right themselves and eight heterozygous controls were thus selected for the m a r p h o m e t r i c analyses.
Histology The 16 mice, when 60 days old, were anesthetized and intracardially perfused with 0.1 ml of 1.0 per cent sodium nitrite, 5 ml of saline, and 75 to 100 ml of fixative [1 per cent paraformaldehyde and 1.25 per cent glutaraldehyde in 0.1M sodium cacodylate buffer, p H 7.2). After decapitation, the lower jaws and soft tissues were removed and the skulls were stored in fixative for one to two years until histologic processing was initiated. The brain and periotic bones were removed from each skull and rinsed one hour in buffer. The brains were embedded in celloidin and analyzed in another portion of the study. ~6The perieric bones s decalcified in 4.5 per cent EDTA (two to three weeks) and rinsed in buffer again before d e h y d r a t i o n for e m b e d d i n g in glycol methacrylate. The ears were then serially sectioned in the longitudinal plane at 5 p.m on a rotary microtome with glass knives. The individual sections were sequentially mounted on a l b u m i n i z e d slides, stained w i t h Gill's triple strength hematoxylin and eosin-phloxine, 2~ and coverslipped.
Morphometry The various sensorineural structures of the vestibule and cochlea were measured to determine whether the mutant gene has any developmental influence on the inner ear in addition to the otoconial loss. Although the cochlea and hearing have not been examined in the pallid mice, the auditory organs are included in this study since genetic malformations of the ear often affect both vestibule and cochlea. 22 The slides were viewed with a light microscope fitted with a drawing tube, and the various structures were drawn to quantify and compare their dimensions in the pallid and heterozygous control mice. Each drawing w a s traced on a digitizer-computer, and its area was measured. In the cases of serially drawn structures, the area measurements for each structure were s u m m e d and multiplied by section thickness and number of intervening sections to calculate its total volume. GRAVITY RECEPTOR MACULAE. The sensory epi t h e l i u m of each utricle and saccule was viewed at a magnification of 200 x and outlined in every fourth section throughout the extent of the macula. In the control animals the otoconial mass was outlined as well. The areal sums were mult i p l i e d b y 5 (section t h i c k n e s s ) and 4 (inter-
T R U N E AND L[M
vening sections) to calculate the total volume for each utricle, saccule, and their otoconial masses (if present). SEMICIRCULAR CANAL CRISTAE. The sensory epithelium for each of the semicircular canal cristae (lateral, superior, and posterior) was also drawn for each animal. Because these structures are s o m e w h a t smaller than the maculae, they were drawn at a magnification of 320 x. These were also taken from e v e r y fourth section throughout the ear and were digitized, and the total volume for each was calculated in the same manner as described for the maculae. VESTIBULAR GANGLION.
Several measurements
were made of the vestibular (Scarpa's] ganglion since this is the first-order neuron between the vestibular sensory structures and the brain. At a magnification of 200 • the outlines of the superior and inferior divisions of each ganglion were drawn from every eighth section to determine the overall ganglion size, As with the maculae and cristae, the area of each drawing was measured and the volume of each division within each ganglion was calculated (in this case, multiplied by 8 since they were drawn from every eighth section). In order to quantify the number and sizes of the neurons within the ganglion, the same sections were v i e w e d u n d e r 320• and every neuron with a visible nucleolus was drawn. These drawings were digitized, and the size and number of neurons for each drawing were stored on cassette tape for the comparison of cell sizes and numbers in the two groups of animals. The total number of ganglion neurons in each division was derived b y multiplying the measured number by 8. SCALA MEDIA. The scala media was one of the cochlear structures m e a s u r e d to determine whether the mutant genome has any influence on the auditory portion of the inner ear. The mouse cochlea has approximately 1.5 turns, so three cross sections of the membranous labyrinth are seen when it is viewed in the mid-mediolar section. From the mid-modiolar section of each normal and pallid cochlea, the endolymphatic portion of each half-turn was traced and digitized to obtain the area of the scala media from each half-turn (basal, middle, and apical). SPIRAL GANGLION. To determine the influence of the mutant gene on the auditory neural elements, each spiral ganglion associated with the middle half-turns (in the scala media analysis) was drawn to lOO x magnification (oil immersion). Spiral ganglion neurons with visible nucleoli were drawn, and these drawings were dig-
itized to o b t a i n the data on n e u r o n size a n d number for each animal. Since only one section was used from each ear, it was n o t possible to calculate the total n u m b e r of spiral ganglion neurons within each ear. However, a comparison of the size and number of cells at that particular location within the cochlea was possible.
Statistical Analysis All morphometric data for the two groups were statistically compared to determine whether the mutant gene h a d any effect on the various sensorineural elements of the inner ear. Unless otherwise stated, the final calculations for right and left sides were averaged within each animal to remove within-animal variability as a factor in the c a l c u l a t i o n of the overall v a r i a n c e . This created a slightly more conservative analysis than would occur if the two sides were treated as ind e p e n d e n t observations. Since sectioning or mounting problems occasionally disrupted the collection of serial sections for a particular segment of the ear, the quantitative analyses often had to be performed on fewer than the eight animals, or 16 ears, in each of the two groups. Therefore, averaging w i t h i n each animal also prevented u n e q u a l contributions to the group mean and variance by animals from which both ears were used and those providing data from only one ear. These means were then subjected to t tests to test the hypothesis that there were no significant differences b e t w e e n the t w o groups. A probability value of 5.0 per cent was chosen as the level at w h i c h the null hypothesis w o u l d be a c c e p t e d or rejected. A chi-square analysis was performed on the vestibular ganglion cell-size data to determine whether neurons within the superior and inferior divisions were similar, and on the spiral ganglion cell size data to determine w h e t h e r the pallid n e u r o n s were within the normal size ranges. RESULTS
The q u a l i t y of tissue e m b e d d e d in glycol methacrylate is considerably better than that embedded in paraffin or celloidin, This watersoluble plastic eliminates organic solvents from the dehydration procedure and thereby minimizes shrinkage artifacts. This plastic also permits thinner sectioning than is possible with such other m e d i a as celloidin or paraffin. This, in conjunction w i t h the i m p r o v e d cellular preserve-
Volume 4 Number 4 July 1983
263
SENSORINEURALSTRUCTURESIN PALLIDMOUSE INNEREAR
lOOPm
Figure 1. Saccular maculae from the normal (top) and pallid [bottom) mice. Although the otoconia (O) are absent in the pallid animals, the sensory epithelium (SE] and nerve fibers (NF) appear normal (hematoxylinand eosin-phloxine, x 240). tion, e n h a n c e s the r e s o l u t i o n of tissue detail (Figs. 1 through 5). Thus, this embedding med i u m is p a r t i c u l a r l y s u i t e d for m o r p h o m e t r i c studies as well as for descriptive studies of normal a n d pathologic material. American JournaS of Oto[aryngology
264
Vestibule Aside from the absence of otoconia in the maculae, there were no abnormalities observed under
light microscopy in the pallid vestibule (Figs. 1 through 4}. All other sensory epithelia and neural structures within the vestibule appeared normal. The various sensory structures were in their normal relationships to each other, with no abn o r m a l i t y of the m e m b r a n o u s labyrinth. Hair cells and supporting cells were normal in all the sensory epithelia, including sensory cilia (Figs. 2 and 3). The quantitative analysis (Fig. 6) re-
TRUNE AND LIM
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Figure 2, High-magnificationviews of the normal (left) and pallid (right) utxicularmaeulae, which demonstrate the normalappearing sensory epithelium (SE] and nerve fibers (NF) in the pallid macula in spite of absence of otocolfia(O) (hematoxytin and eosin-phloxine, • vealed the maculae of the utricle and saccule and the cristae of the semicircular canals to be of normal size in the mutants (P > 0.05). Since the otoconial mass was absent in the mutants' ears, no statistical comparison between the two g r o u p s was m a d e on this structure, and its volume is not presented graphically. However, in normal animals, the utricular otoconial layer has a measured volume of 0.00125 m m 3 (standard deviation, 0.00032 ram3), and the saccular
Figure 3. The superior semicircularcanal cristafroma pallid animal, The sensory epithelium (SE) and nerve fibers (NF) are normal in appearance [hematoxylinand eoxin-phlexine, • 440).
otoconial layer is 0.00139 m m 3 (standard deviation, 0.00033 mm3). Thus, the otoconial layer has a volume of approximately one-third its corresponding sensory epithelium.
Vestibular Ganglion The vestibular (Scarpa's} ganglion was qualitatively similar in the two groups (Fig. 4). The inferior a n d superior divisions of the ganglion were apparent in the pallid mice and demarcated by the normal orientation of the afferent and efferent processes of their neurons. The Nissl substance within the somas of these cells stained comparably in the two groups, and the various cellular elements within the ganglia appeared to be distributed normally. Fibers from the superior division of the ganglion innervate the cristae of the lateral and superior semicircular canals and the macula o:[ the utricle. A small portion of the saccule is also innervated by fibers from the superior division. The inferior division of the ganglion contains cell bodies of those neurons that innervate the crista of the posterior semicircular canal and the remainder of the saccular macula. However, since it was not possible to identify the specific somas within each division that innervate the various sensory structures of the vestibule, the two divisions were not broken down by end organ in this portion of the analysis. The m e a n total volumes for e a c h d i v i s i o n within the ganglia of the two groups are shown
Volume 4 Number 4 J u l y 1983
265
SENSORINEURAL STRUCTURES IN PALLID MOUSE INNER EAR
Figure 4, The inferior (IVG) and superior (SVG) divisions of the normal (top left) and pallid (top right) vestibular ganglia. The dashed line approximates the boundary between the two divisions, Higher-rnagnification views of the normal (bottom left} and pallid (bottom right} inferior divisions demonstrate their qualitative similarities (hematoxylin and eosin-phloxine; top left and top right, x 200; bottom left and bottom right, x 440), Americnn Journal ot~
Otoloryngology
266
in F i g u r e 7. In b o t h g r o u p s of a n i m a l s , the sup e r i o r d i v i s i o n was a p p r o x i m a t e l y t w i c e the size of the inferior d i v i s i o n . I n the p a l l i d animals, b o t h s u p e r i o r a n d i n f e r i o r divisions w e r e signif-
icantly smaller than n o r m a l animals (P < O n c e it h a d h e e n sions of the ganglion
t h e i r c o u n t e r p a r t s in t h e 0.05). d e t e r m i n e d t h a t t h e diviw e r e smaller in the pallid
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Volume
4
Number July
4 1983 267
SENSORINEURAL STRUCTURES IN PALLID MOUSE INNER EAR 5.0-
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Figure 6. The volumes of the various vestibular sensory epithelia in the normal (pal+) and pallid (pu/pa) animals. No significant differences (P > 0.05] were found in comparisons of normal and pallid superior, lateral, or posterior semicircular canal cristae, utricular maculae, or saccular maculae. The sample size for the cristae was seven normal mice [12 ears] and seven pallid mice (13 ears). The sample size for the maculae was eight normal mice [13 ears} and eight pallid mice (15 ears).
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VESTIBULAR SENSORY STRUCTURE
American
Journal of Otolaryngology 268
animals, an effort was m a d e to isolate the specific n e u r o n a l changes w i t h i n the ganglion that could be c o n t r i b u t i n g to this v o l u m e change. T h e r e f o r e , m e a s u r e m e n t s were m a d e of the number, size, and d e n s i t y of the ganglion cells. The results of file cell number analysis are shown in Figure 7. The data s h o w that there were no significant differences between the two groups in the n u m b e r of neurons in the divisions of the vestibular ganglion (P > 0.05). Since ganglion volume and cell number data were available, cell d e n s i t y for the superior and inferior divisions was calculated to determine whether neuropil loss was a factor contributing to the volume reduction of the pallid vestibular ganglion. These values are s h o w n in Table 1. However, the statistical tests performed on these data f a i l e d to d e m o n s t r a t e any d e n s i t y differences between the two groups (P > 0.05). The results of the cell size analysis are shown in Figure 8. These data were obtained from the same sections as the ganglion volume measurements. Since the neuronal size data were stored on tape, we could create bins of specific size categories, group the n e u r o n s within these bins, and then compare either the absolute number of neurons or the percentage of total neurons within these bins between the two groups. The absolute numbers of neurons d r a w n for each group were not equal, so comparison of the total number of cells in each bin was not valid, owing to unequal sample sizes. Therefore, w e chose to compare the percentage of total neurons that occur in each size group. Thus, if the neurons are smaller in one group, that group will have relatively more neurons in the smaller size categories, i.e., the curve will shift to the left. As one can see from the distribution curves in Figure 8, there were no differences between the pallid a n d normal animals in their proportions
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Figure 7. The volume (top] of the normal (pa/+) and pallid (pa/pa) vestibular ganglia divisions and the number of neurons (bottom} within each of these divisions. Although the divisions of the pallid ganglion are significantly smaller than normal [P < 0.05), the number of neurons within them is the same as in the controls (P > 0.05]. The sample size for the volume measurements was eight normal mice (12 ears) and seven pallid mice (superior, 12 ears; inferior, 13 ears}, The sample size for the neuron number data was seven normal mice (10 ears) and seven pallid mice (superior, 12 ears; inferior, 13 ears). P, probability; S.D., standard deviation,
TRUNE AND LIM
TABLE 1. Density of Neurons Within the Vestibular Ganglia of Pallid and Normal Mice DIVISION
OF
DENSITY +-- S . D . ( E / N ] *
GANGLION
Normal
Pallid
PROBABILITY*
Superior Inferior
1.76 • 0.18 (1017) 1.80 • 0.12 (10/7)
1.78 • 0.12 (1117} 1.87 -- 0.19 (12/7]
P > 0.80 P > 0.40
ABBREVIATIONS:N, sample size (number of animals); E, number of ears; S.D., 1.0 standard deviation. * Density in cells/10,000 p,m3. -~Probability determined by t test. of ceils in the various size categories. The curves for each division a p p e a r to overlap completely, indicating that the n e u r o n s in the pallid vestibular ganglion w e r e the same size as those in the control animals. H o w e v e r , n e u r o n a l size comparisons in the two divisions revealed larger neurons in the superior division of b o t h groups t h a n in the inferior d i v i s i o n (P < 0,05). This is demonstrated by a chi-square analysis (Table 2) of
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Figure 8. The percentage of superior (top) and inferior (bottom) division neurons within the various size categories for normal (pa/+) and pallid (pa/po) vestibular ganglia. Since the pallid and normal curves within each division overlap completely, the size distribution of pallid ganglion neurons appears to be normal. The normal data were derived from 6,382 superior division neurons and 3,239 inferior division neurons (eight mice, 12 ears). The pallid data represent 4,564 superior division neurons (seven mice, 12 ears) and 2,627 inferior division neurons (seven mice, 13 ears).
their proportions smaller and larger t h a n 245 t~m2 and e x p l a i n s w h y the s u p e r i o r d i v i s i o n curves are shifted to the right relative to the inferior division curves.
Cochlea Qualitatively the b o n y a n d m e m b r a n o u s labyrinths a p p e a r e d n o r m a l in the p a l l i d animals. The b o n y labyrinth was o r i e n t e d p r o p e r l y , and the c o c h l e a had the usual t u r n and a half, T h e r e were no abnormalities of a n y of the scatae, and they all w e r e in p r o p e r r e l a t i o n to each other, The a u d i t o r y s e n s o r i n e u r a l e l e m e n t s a p p e a r e d normal t h r o u g h o u t the c o c h l e a (Fig. 5). T h e hair cells a n d the v a r i o u s s u p p o r t i n g c e i l s w e r e normal in all turns of the organ of Corti. S e n s o r y cilia w e r e a p p a r e n t at the apical e n d s o2 t h e hair cells, a n d often the cilia of t h e o u t e r hair cells w e r e a p p o s e d to the r e c t o r i a l m e m b r a n e . T h e nerve fibers w i t h i n the t u n n e l of Corti w e r e apparent e v e n at the light m i c r o s c o p i c level. T h e spiral ganglion n e u r o n s in R o s e n t h a l ' s canal a n d the a u d i t o r y n e r v e w i t h i n t h e m o d i o l u s appeared normal. SCALA MEDIA. The m e a s u r e m e n t s of the scala m e d i a f o r each h a l f - t u r n in the m i d - m o d i o l a r section are s h o w n in Figure 9. T h e pallid animals h a d significantly larger scalae m e d i a e i n the basal (P < 0.001) and m i d d l e (P < 0.01) halfturns t h a n the h e t e r o z y g o u s controls. Since there did not a p p e a r to be any d i s t e n t i o n of Reissner's m e m b r a n e into the scala vestibuli, the larger scala m e d i a size c o u l d not be attributed to endolymphatic hydrops. The qualitatively normal appearance of the scala media in the basal turn i n d i c a t e d s i m p l y that it was larger in the mutants. T h e m e a s u r e m e n t s for t h e apical port i o n of t h e c o c h l e a s h o w e d no d i f f e r e n c e bet w e e n the two groups (P > 0.05). SPINAL GANGLION. T h e r e s u l t s of the spiral ganglion analysis are s h o w n in F i g u r e 10. The neurons were separated into bins in the same m a n n e r as for t h e vestibular ganglion n e u r o n s ; the distributions of the various cell sizes for the two g r o u p s are shown. In this case, t h e curve for
Volume 4 Number 4 July 1983 269
SENSORINEURALSTRUCTURESIN PALLIDMOUSEINNEREAR TABLE 2. Chi-square Analysis of the Percentage of Superior and Inferior Division Neurons Smaller and Larger than 245 ~m 2. GENOTYPE
pa/+
NEURON SIZE GROUP (I~Mz)
Superior
Inferior
X2
PROBABILITY
~245.0 >245.0
52.3 47.7 100.0
62.3
4.26
P < 0.05
37.7 100.0
~245.0 >245.0
54.6 45.4 I00.0
64.5 35,5 I00.0
4.28
P < 0.05
TOTAL
patpa TOTAL
PERCENTAGEOF TOTAL
* The data were derived from 6,382 superior division neurons and 3,239 inferior division neurons from normal animals (eight mice, 12 ears) a n d from 4,564 superior division neurons (seven mice, 12 ears) and 2,627 inferior division neurons (seven mice, 13 ears) from p a l l i d animals.
the pallid animals is shifted slightly to the left, w h i c h indicates that there are more neurons in the smaller-size classes i n the pallid ears, i.e., the spiral ganglion cells are smaller than normal. In order to test this size difference statistically, a chi-square analysis was performed on the proportion of cells less t h a n and greater than 125 ~m 2, the size where the two curves cross in Figure 10. The design and results of the chi-square test are s h o w n in Table 3. W h e n 125 ~m 2 is used as the dividing point, the proportions of cells below and above this size differ between the two groups (P < 0.05). The same results are obtained if any size between a23.5 and 127 ~m 2 is used. This indicates that the pallid animals have smaller spiral ganglion neurons t h a n normal. A t test was also r u n on the n u m b e r of spiral ganglion ceils in this m i d d l e half-turn of the mid-modiolar section. T h e r e was no s i g n i f i c a n t d i f f e r e n c e between the two groups in the n u m b e r of spiral ganglion cells (P > 0.05); however, this Comparison was based on o n l y one section for each ear.
spiral ganglion were slightly smaller than normal, as d e t e r m i n e d by the m e a s u r e m e n t of n e u r o n sizes from one section in each ear. Since we have not performed a thorough analysis of the pallid cochlea, it is not possible to judge what degree of disparity exists in spiral ganglion neuron sizes, if any. However, we tentatively conclude that there is some cochlear, as well as vestibular, inv o l v e m e n t w i t h this m u t a n t gene. This is common in a variety of mutant mice that have inner ear involvement. 2z Temporary endolymphatic hydrops has been reported during the development of the pallid inner ear, but not in the adult. 9,ms Based on our morphometric analysis of the mid-modiolar sections, the endolymphatic space of the scala media
8~ *I 7.0
~
ak
[] palpa P .r
,,
** P
O
DISCUSSION
American Journal of Otolaryngology 270
Qualitatively, the o n l y abnormality apparent in the pallid inner ear was the absence of otoconia in the utricle a n d saccule, confirming earlier reports. ',~a,l" However, the quantitative portion of this study did reveal some subtle changes. In addition to the absence of otoconia, the volume of the vestibular ganglion was smaller, as were the cochlear spiral ganglion neurons. Since the sensory epithelia of the utricular and saccular maculae, as well as the semicircular canal cristae, were of normal size in the pallid animals, we conclude that neither the lack of otoconia nor the m u t a n t gene had any significant deleterious trophic influence on them. The first-order a u d i t o r y n e u r o n s w i t h i n the
7
rr
6.0
~
J.
"~0.001
1.0 S.D.
5.0
4.0
5.0
BASAL J/~' TURN
M I DOLE V~, TURN
APICAL ' I/2 TURN
HEMI-TURN OF COCHLEA
Figure 9. The average scala media cross-sectional area for the three hemi-turns in the normal ( p a / + ) and pallid (pa/pa) cochleas, This e n d e l y m p h a t i c space was significantly larger in the pallid basal and middle hemi-turns (P < 0.05), whereas the apical region measurements were the same for both groups (P > 0,05). The sample size was eight normal mice (15 ears) and eight pallid mice (16 ears). P, probability; S.D., standard deviation.
TRUNE AND LIM
was slightly larger in the basal turn (basal and middle half-turns). However, since there was no a c c o m p a n y i n g d i s t e n t i o n of Reissner's membrane, it is unlikely that the larger scalar size is due to a true endolymphatic hydrops; rather, it m a y be just structurally larger. Therefore, we tentatively suggest that a true phenotypic structural modification m a y occur in the auditory system of this mutant mouse. Although our volumetric measurements rev e a l e d the pallid v e s t i b u l a r ganglion to be smaller, our cytomorphometric analysis failed to demonstrate any change in its neuronal numbers, sizes, or densities. The underlying reason(s) for the smaller vestibular ganglion size is not clear; however, several possibilities exist. It may be due to 1) a reduction in the vestibular nerve neuropil that lies around and within the ganglion; 2) reduced glia; 3) fewer connective tissue elements within the ganglion; or 4) vascular hypotrophy that w o u l d decrease ganglion volume but not affect its neural structures. A more thorough analysis of the ganglion is certainly necessary before this matter can be settled, but at this point there is good reason to suspect some trophic neural involvement with the mutant gene. Even though the present morphometric analyses failed to demonstrate any significant differences between the sizes of the remaining vestibular sensorineural elements, a number of the comparisons p r o v i d e d probability levels only slightly above 5.0 per cent. This was especially true of the lateral semicircular canal crista, saccular macula, and the number of neurons in both divisions of the vestibular ganglion. Since the
20z ~,
15
-- p a l +
o - -.-o
polpo
~o
5
s
?
)% i
<75
7585
95105
i
i
115125
i
i
135145
NEURONAL
i
i
155IIS5
AREA
176185
I=J5205
215- >225
225
(/zm 2)
Figure 10. The percentage of spiral ganglion neurons within the various size categories for normal (pa/+] and pallid (pc~ pc) mice. The pallid curve is shifted to the left and indicates more neurons than normal in the smaller size categories. The sample size was 1,021 neurons from eight normal mice (16 ears) and 1,077 neurons from eight pallid mice (16 ears}.
TABLE 3. Chi-square Analysis of the Percentage of Spiral Ganglion Neurons Smaller and Larger Than 125 p,m 2. NEURON SIZE GROUP (t.~M2} ~<125.0 >125,0 TOTAL
PERC6NTAGEOF TOTAL
Normal Pallid 42,0 58.0
51.7 48.3
10O.O
100.0
X2 3,86
PROBABILITY P ~ 0.05
* The data were derived ~rom 1,021 n e u r o n s from eight normal mice (16 ears} and from 1,077 n e u r o n s from eight pallid mice [16 ears}.
probability was greater than 5.0 per cent, these structures in the pallid animals were judged statistically as normal in size. However, one cannot rule out the possibility that a statistical difference in size may emerge if a larger sample size is used. Even if future analyses of these structures demonstrate these trends to be true phenotypic abnormalities, the size differences would be so slight that a real functional difference would be doubtful. The pallid mice exhibit such behavioral deficits as head tilting, disorientation in water, a b s e n t or i m p a i r e d r i g h t i n g r e f l e x e s , and ataxia. ~,4,'1,~8Since the size differences of the sensorineural elements in the pallid inner ear are relatively minor, as mentioned above, the dominant feature of the pallid mutation is the lack of otoconia in the gravity receptors. Therefore, the dysfunction of the pallid inner ear is closely linked to this otoconial agenesis and, ultimately, to gravity receptor h y p o s t i m u l a t i o n . It is apparent that the brain stem structures that rely on gravity receptor input are not receiving proper stimulation and that a condition of vestibular deprivation exists. Merphologic studies of the deprived s e c o n d - o r d e r n e u r o n s h a v e b e e n conducted to determine the p o s t s y n a p t i c consequences of hypostimulation. Clark et el. 19 found cells less dense in nucleus y and medial vestibular nucleus of the otoconia-deficient mutants, and Trune and Lira .6 found some vestibular nuclei to be smaller than normal. These results were interpreted as being due to the lack of proper presynaptic stimulation during the critical period of vestibular nuclear d e v e l o p m e n t . Other investigators have examined the influence of such deprivation on higher brain functions. Although studies of hippocampal electrophysiology did not demonstrate measurable differences in the pallid animals, 2~studies of spatial orientation and emotionality did reveal significant differences in these otoconia-deficient mutants. 17 All of these studies demonstrating significant
Volume 4 Number 4 July 1983 27!
SENSORINEURAL STRUCTURES IN PALLID MOUSE INNER EAR differences in the m u t a n t s c o n c l u d e that s u c h deficits are the direct r e s u l t of g r a v i t y r e c e p t o r h y p o s t i m u l a t i o n d u r i n g d e v e l o p m e n t . T h e definite c o r r e l a t i o n of s u c h deficits w i t h the lack of o t o e o n i a certainly i m p l i e s that s u c h a c o n c l u sion is valid, O n the o t h e r h a n d , t h e m u t a n t gene c o u l d be affecting t h e central n e r v o u s s y s t e m s of t h e s e a n i m a l s directly, a n d a n y l a c k of v e s t i b u l a r i n p u t during d e v e l o p m e n t m a y be s e c o n d a r y and of l e s s e r s i g n i f i c a n c e , Creel 23 h a s r e c e n t l y rev i e w e d the b i o c h e m i c a l a n d n e u r a l a b n o r m a l i ties t h a t are often a s s o c i a t e d w i t h a l b i n o a n d hyp o p i g m e n t e d animals, T h e effects of a p a r t i c u l a r m u t a n t gone w i t h p i g m e n t a t i o n abnormalities are c o n s i d e r a b l y m o r e diffuse since so m a n y m e t a h o l i c p a t h w a y s in t h e b o d y d e p e n d o n p i g m e n t related biochemistry. These potential influences m u s t c e r t a i n l y be i n v e s t i g a t e d before the b e h a v ioral a n d central m o r p h o l o g i c a b n o r m a l i t i e s in the p a l l i d a n i m a l s are i n t e r p r e t e d as b e i n g s o l e l y the r e s u l t of gravity r e c e p t o r h y p o s t i m u l a t i o n . H o w e v e r , properly designed investigations, using the p a l l i d s a n d p r o p e r c o n t r o l s to s e p a r a t e genetic, dietary, and developmental influences, s h o u l d p r o v i d e us w i t h s i g n i f i c a n t i n s i g h t s into the r o l e of gravity r e c e p t o r s t i m u l a t i o n in b r a i n development and maintenance.
6. 7.
8. 9.
10. 11. 12. 13. 14. 15. 16.
Acknowledgments.
The authors thank Roberta Arbaugh, Anabel Ocasio, and Atha Ralston for preparation of the tissue; Scott Gordon for developing the software for data c o l l e c t i o n and analysis; James Douthitt for photographic assistance; Nancy Sally for preparation of the illustrative material; and Katherine A d a m s o n for assistance in the preparation of the manuscript.
17.
18. 19.
References 1. Lyon MF: Hereditary absence of otoliths in the house mouse. J Physiol 114'.410-418, 1951 2. Lyon MF: Absence of otoliths in the mouse: an effect of the pallid mutant. J Goner 51:63s-650, 1953 3. Lyon MF, Meredith R: Muted, a new mutant affecting coat colour and otoiiths of the mouse, and its position in linkage group XIV. Goner Res 14:163-166, 1969 4. Lira ]3], Erway LC, Clark DL: Tilted-head mice with genetic otoconial anomaly: behavioural and morphological correlates, in Hood JD (ed.}: Vestibular Mechanisms in Health and Disease. London, Academic Press, 1978, pp 195-206 5. Erway LC, Piletz JE, Ganschow RE: Lethal-milk mutant
American Journol of OtoJaryngolagy
272
20.
mice: zinc deficiency and otolith defects. Abstract of Second Midwinter Research Meeting of the Association for Research in Otolaryngology, 1979, p 24 Erway LC, Mitchell SE: Prevention of otolith defect in pastel mink by manganese supplementation. J Herod 64:111--119, 1973 Colmers WF, Hixon RF, Hanlon RT, eta[: "Spinner" cephalopods: defects of statolith formation in an invertebrate model of the vestibular system. Abstract of Sixth Midwinter Research Meeting of the Association for Research in Otolaryngology, 1983, pp 87-88 Wright CG, Hubbard DG, Graham JW: Absence of otoconia in a human infant. Ann Otol Rhinol Laryngol 88:779-763, 1979 Shrader RE, Erway LC, Hurley LS: Mucopolysaccharida synthesis in the developing inner ear of manganesedeficient and pallid mutant mice. Teratology 8:257266, 1973 Erway L, Hurley LS, Fraser A: Neurological defect: manganese in phenocopy and prevention of a genetic abnormality of inner ear, Science 152:1766-1768, 1966 Erway L, Hurley LS, Fraser AS: Congenital ataxia and etolith defects due to manganese deficiency in mice. J Nutr 100:643-654, 1970 Erway LC, Fraser AS, Hurley LS: Prevention of congenital otolith defect in pallid mutant mice by manganese supplementation. Genetics 67:97-108, 1971 Lira DJ, Erway LC: Influence of manganese on genetically defective otolith: a behavioral and morphological study. Ann OEolRhinol Laryngol 83:565-581, 1974 Shrader RE, Everson GJ: Anomalous development of otolithe associated with postural defects in manganesedeficient guinea pigs. J Nutr 91:453-460, 1967 Hurley LS: Teratogenic aspects of manganese, zinc, and copper nutrition. Physio[ Rev 61:249-295, 1981 Trune DR, Lira DI: The behavior and vestibular nuclear morphology of otoconia-deficient pallid mutant mice, I Neurogenet, in press Douglas R1, Clark GM, Erway LC, et ah Effects of genetic vestibular defects on behavior related to spatial orientation and emotionality. J Comp Physiol Psychol 93:467-489, 1979 Lyon MF: The developmental origin of hereditary absence of otoliths in mice. J Embryol Exp Morphol 3:230-241, 1955 Clark CM, Douglas RL Erway LC, et el: Vestibular nuclei: neuronal loss in mice with otoconial agenesis and evidence of right-left asymmetry, in Gualtierotti T (ed): The Vestibular System: Function and Morphology. New York, Springer-Verlag, 1981, pp 101-119 Frederickson CJ, Frederickson MH, Lewis C, et ah Hippocampal EEQ in normal mice and in mice with congenital vestibular defects. Behav Neural Biol 34:121131, 1982
21. Lim B, Liew CT, Craig JR: Plastic-embedded thin sections for light microscopy. Lab Med 12:145-149, 1981 22. Deol MS: Inherited diseases of the inner ear in man in the light of studies on the mouse. ] Med Goner 5:137158, 1968 23, Creel D: Inappropriate use of albino animals as models in research. Pharmacol Biochem Behav 12:969-977, 1980
A Morphometric Study of the Pallid Mutant Mouse Inner Ear DENNIS R. TRUNE, PH.D., AND DAVIDJ. LIM, M.D. Mice homozygous for the mutant gene pallid (pa/pa) often lack otoconia in some or all of their maculae and are used to study the influences of gravity receptor hypostimulation on vestibular-related behaviors. Since the value of this animal model is based on the assumption that the vestibular sensorineural elements are normal, a morphometric analysis was done on the inner ear of these otoconia-deficient mice to see whether sensorineural structures are also affected by the pallid gene. In pallid mice lacking all otoconia, the sensory epithelia of the utricle, saccule, and semicircular canal cristae were the same size as in their heterozygous (pa/+) controls. Although the superior and inferior divisions of the vestibular ganglion of the pallid mice were smaller than normal, the first-order neurons within these divisions were normal in size, number, and density. However, the superior divisions in both groups had larger neurons than did the inferior divisions. Within the pallid cochlea, first-order auditory neurons within the spiral ganglion were smaller than normal, but the scala media was larger. Since the significant vestibular influences of the pallid gene are limited primarily to the otoconia, behavioral abnormalities reported for these otoconia-deficient mice are apparently due only to gravity receptor hypostimulation. (Key words: Pallid mouse; Cochlea; Vestibule; Otoconia; Morphometry.)
Lyon1,2first described the absence of otoconia in the inner ears of mice homozygous for the recessive, mutant gene pallid (pa). Although all homozygotes (pa/pa) have the pale coat color and u n p i g m e n t e d eye, the penetrance of the otoconial effect is incomplete and can range, within the same animal, from no otoconia in any of the gravity receptors to normal otoconia in some or all receptors. Also, the pa gene is considered pleiotropic, since otoconial loss is never observed without the lack of pigmentation, and it is apparently responsible for both the otoconia and pigment defects. Mutants in which otoconia deficiency is a predominant characteristic have been observed among a variety of species, most of which also have an associated pigment defect. These include the mutant mice mocha, 3 tiltedhead, 4 and lethal-milk, 5 as well as the ocular albino rabbit, gray-loco chukar partridge, pastel mink, 6 octopus, cuttlefish, and squid. 7 Recently, otoconia deficiency was also observed in hu-
mans, 8 although a genetic linkage has not been established. Therefore, this animal model makes a useful vehicle for gaining new insight into the otoconia-deficient syndrome. The lack of otoconial development in the mutant mouse is correlated with a decrease in the synthesis of the macular sulfomucopolysaccharides that apparently are necessary for the initiation of crystal growth. Q Increasing manganese concentrations in the diets of pregnant pallid females prevents this otoconial defect in their offspring, and, conversely, removal of manganese from the diets of pregnant normal mice prevents otoconial formation in their young, thus mimicking the effects of the mutant gene. I~ The exact role of manganese in the development of normal otoconia has not been established, but it is suggested that normal biosynthesis of mucopolysaccharides in otoconia requires manganese.14.15 This lack of otoconia in some or all of the gravity receptors leads to a variety of vestibular behavioral problems. These include head tilting, ataxia, circling, disorientation in water, and absence of righting reflexes. 1,11,~3.1BOtoconial loss in pallid mice also has been correlated with reduced spatial orientation and increased emotionality. 17 Previous investigators have observed only temporary endolymphatic hydrops during gestatioal,9,1B and immature melanin granules
Received March 22, 1983, from the Otological Research Laboratories, Department of Otolaryngology,The Ohio State University College of Medicine, Columbus, Ohio. Accepted for publication April 7, 1983. Supported by National Aeronautics and Space Administrationgrant NSG-2220 (to D.J.L.) and National Institutes of Health grant NINCDS-NRSA5 F32 NSO6211 (to D.R.T.). Address correspondenceand reprint requests to Dr. Trune: Otological Research Laboratories,4331 UniversityHospitals Clinic, 456 Clinic Drive, Columbus, OH 43210.
261
SENSOKINEURAL STRUCTURES IN PALLID MOUSE INNER EAR
within otherwise nomnal vestibular melanocytes in the m u t a n t adults, 13 Aside from the etoconial loss, f e w morphologic abnormalities have been observed in the pallid inner ear. Because these morpholagic studies suggest this genetic defect is limited to the otocania, the pallid mouse has b e c o m e an important animal m o d e l with which to s t u d y the effects of gravity receptor hypostiraulation on the d e v e l o p m e n t and function of the brain. 1B,~7,~9,20However, before the changes seen in these central studies can be properly interpreted, it is important to rule out other genetic influences besides otoconial deficiency on the d e v e l o p m e n t of the pallid vestibular periphery. To this end, we u n d e r t o o k a morphometric inv e s t i g a t i o n of all the p e r i p h e r a l auditory and vestibular sensorineural structures. Once this ass e s s m e n t is m a d e of the pallid vestibular per i p h e r y , our b r a i n s t e m s t u d i e s can be more properly interpreted w i t h regard to the role of gravity receptor sensation in brain development and function, M A T E R I A L S AND METHODS
American Journal
of Otolaryngalogy 262
Our pallid mutant colony at The Ohio State University w a s originally derived from the University of Cincinnati [Dr. L.C. Erway's laboratory). The pa gene is maintained in the C57BL/ 6J strain of Mus musculus. Phenotypically normal heterozygous ( p c / + ) females were mated with pallid (pa/pa) males to provide us with litters that i n c l u d e d both pallid mutants and heterozygous controls. Since the pa gene displays inc o m p l e t e p e n e t r a n c e w i t h regard to the otoconial effect, there are varying degrees of atoc o n i a l d e v e l o p m e n t in the p a l l i d offspring. Therefore, w e selected only those mutants with complete penetrance, i.e., bilateral absence of all otoconia, since the maximal influence of the pa mutation should be most apparent in this condition. Since previous studies have demonstrated that mice w i t h no otoconia are disoriented in water and c a n n o t right t h e m s e l v e s in a vertical drop, 4,11,~3 w e t e s t e d t h e s w i m m i n g and airrighting abilities of the pallid mutants to identify those with all gravity receptors deficient of otoconia. Heterazygous control animals were also tested to indicate normal behavior in these tests. All behavioral tests were conducted w h e n the mice were b e t w e e n 45 a n d 60 days of age. Eight p a l l i d m u t a n t s t h a t c o u l d n o t s w i m or right themselves and eight heterozygous controls were thus selected for the m a r p h o m e t r i c analyses.
Histology The 16 mice, when 60 days old, were anesthetized and intracardially perfused with 0.1 ml of 1.0 per cent sodium nitrite, 5 ml of saline, and 75 to 100 ml of fixative [1 per cent paraformaldehyde and 1.25 per cent glutaraldehyde in 0.1M sodium cacodylate buffer, p H 7.2). After decapitation, the lower jaws and soft tissues were removed and the skulls were stored in fixative for one to two years until histologic processing was initiated. The brain and periotic bones were removed from each skull and rinsed one hour in buffer. The brains were embedded in celloidin and analyzed in another portion of the study. ~6The perieric bones s decalcified in 4.5 per cent EDTA (two to three weeks) and rinsed in buffer again before d e h y d r a t i o n for e m b e d d i n g in glycol methacrylate. The ears were then serially sectioned in the longitudinal plane at 5 p.m on a rotary microtome with glass knives. The individual sections were sequentially mounted on a l b u m i n i z e d slides, stained w i t h Gill's triple strength hematoxylin and eosin-phloxine, 2~ and coverslipped.
Morphometry The various sensorineural structures of the vestibule and cochlea were measured to determine whether the mutant gene has any developmental influence on the inner ear in addition to the otoconial loss. Although the cochlea and hearing have not been examined in the pallid mice, the auditory organs are included in this study since genetic malformations of the ear often affect both vestibule and cochlea. 22 The slides were viewed with a light microscope fitted with a drawing tube, and the various structures were drawn to quantify and compare their dimensions in the pallid and heterozygous control mice. Each drawing w a s traced on a digitizer-computer, and its area was measured. In the cases of serially drawn structures, the area measurements for each structure were s u m m e d and multiplied by section thickness and number of intervening sections to calculate its total volume. GRAVITY RECEPTOR MACULAE. The sensory epi t h e l i u m of each utricle and saccule was viewed at a magnification of 200 x and outlined in every fourth section throughout the extent of the macula. In the control animals the otoconial mass was outlined as well. The areal sums were mult i p l i e d b y 5 (section t h i c k n e s s ) and 4 (inter-
T R U N E AND L[M
vening sections) to calculate the total volume for each utricle, saccule, and their otoconial masses (if present). SEMICIRCULAR CANAL CRISTAE. The sensory epithelium for each of the semicircular canal cristae (lateral, superior, and posterior) was also drawn for each animal. Because these structures are s o m e w h a t smaller than the maculae, they were drawn at a magnification of 320 x. These were also taken from e v e r y fourth section throughout the ear and were digitized, and the total volume for each was calculated in the same manner as described for the maculae. VESTIBULAR GANGLION.
Several measurements
were made of the vestibular (Scarpa's] ganglion since this is the first-order neuron between the vestibular sensory structures and the brain. At a magnification of 200 • the outlines of the superior and inferior divisions of each ganglion were drawn from every eighth section to determine the overall ganglion size, As with the maculae and cristae, the area of each drawing was measured and the volume of each division within each ganglion was calculated (in this case, multiplied by 8 since they were drawn from every eighth section). In order to quantify the number and sizes of the neurons within the ganglion, the same sections were v i e w e d u n d e r 320• and every neuron with a visible nucleolus was drawn. These drawings were digitized, and the size and number of neurons for each drawing were stored on cassette tape for the comparison of cell sizes and numbers in the two groups of animals. The total number of ganglion neurons in each division was derived b y multiplying the measured number by 8. SCALA MEDIA. The scala media was one of the cochlear structures m e a s u r e d to determine whether the mutant genome has any influence on the auditory portion of the inner ear. The mouse cochlea has approximately 1.5 turns, so three cross sections of the membranous labyrinth are seen when it is viewed in the mid-mediolar section. From the mid-modiolar section of each normal and pallid cochlea, the endolymphatic portion of each half-turn was traced and digitized to obtain the area of the scala media from each half-turn (basal, middle, and apical). SPIRAL GANGLION. To determine the influence of the mutant gene on the auditory neural elements, each spiral ganglion associated with the middle half-turns (in the scala media analysis) was drawn to lOO x magnification (oil immersion). Spiral ganglion neurons with visible nucleoli were drawn, and these drawings were dig-
itized to o b t a i n the data on n e u r o n size a n d number for each animal. Since only one section was used from each ear, it was n o t possible to calculate the total n u m b e r of spiral ganglion neurons within each ear. However, a comparison of the size and number of cells at that particular location within the cochlea was possible.
Statistical Analysis All morphometric data for the two groups were statistically compared to determine whether the mutant gene h a d any effect on the various sensorineural elements of the inner ear. Unless otherwise stated, the final calculations for right and left sides were averaged within each animal to remove within-animal variability as a factor in the c a l c u l a t i o n of the overall v a r i a n c e . This created a slightly more conservative analysis than would occur if the two sides were treated as ind e p e n d e n t observations. Since sectioning or mounting problems occasionally disrupted the collection of serial sections for a particular segment of the ear, the quantitative analyses often had to be performed on fewer than the eight animals, or 16 ears, in each of the two groups. Therefore, averaging w i t h i n each animal also prevented u n e q u a l contributions to the group mean and variance by animals from which both ears were used and those providing data from only one ear. These means were then subjected to t tests to test the hypothesis that there were no significant differences b e t w e e n the t w o groups. A probability value of 5.0 per cent was chosen as the level at w h i c h the null hypothesis w o u l d be a c c e p t e d or rejected. A chi-square analysis was performed on the vestibular ganglion cell-size data to determine whether neurons within the superior and inferior divisions were similar, and on the spiral ganglion cell size data to determine w h e t h e r the pallid n e u r o n s were within the normal size ranges. RESULTS
The q u a l i t y of tissue e m b e d d e d in glycol methacrylate is considerably better than that embedded in paraffin or celloidin, This watersoluble plastic eliminates organic solvents from the dehydration procedure and thereby minimizes shrinkage artifacts. This plastic also permits thinner sectioning than is possible with such other m e d i a as celloidin or paraffin. This, in conjunction w i t h the i m p r o v e d cellular preserve-
Volume 4 Number 4 July 1983
263
SENSORINEURALSTRUCTURESIN PALLIDMOUSE INNEREAR
lOOPm
Figure 1. Saccular maculae from the normal (top) and pallid [bottom) mice. Although the otoconia (O) are absent in the pallid animals, the sensory epithelium (SE] and nerve fibers (NF) appear normal (hematoxylinand eosin-phloxine, x 240). tion, e n h a n c e s the r e s o l u t i o n of tissue detail (Figs. 1 through 5). Thus, this embedding med i u m is p a r t i c u l a r l y s u i t e d for m o r p h o m e t r i c studies as well as for descriptive studies of normal a n d pathologic material. American JournaS of Oto[aryngology
264
Vestibule Aside from the absence of otoconia in the maculae, there were no abnormalities observed under
light microscopy in the pallid vestibule (Figs. 1 through 4}. All other sensory epithelia and neural structures within the vestibule appeared normal. The various sensory structures were in their normal relationships to each other, with no abn o r m a l i t y of the m e m b r a n o u s labyrinth. Hair cells and supporting cells were normal in all the sensory epithelia, including sensory cilia (Figs. 2 and 3). The quantitative analysis (Fig. 6) re-
TRUNE AND LIM
50 Fm
, ;~.~, ,:~,,~,
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Figure 2, High-magnificationviews of the normal (left) and pallid (right) utxicularmaeulae, which demonstrate the normalappearing sensory epithelium (SE] and nerve fibers (NF) in the pallid macula in spite of absence of otocolfia(O) (hematoxytin and eosin-phloxine, • vealed the maculae of the utricle and saccule and the cristae of the semicircular canals to be of normal size in the mutants (P > 0.05). Since the otoconial mass was absent in the mutants' ears, no statistical comparison between the two g r o u p s was m a d e on this structure, and its volume is not presented graphically. However, in normal animals, the utricular otoconial layer has a measured volume of 0.00125 m m 3 (standard deviation, 0.00032 ram3), and the saccular
Figure 3. The superior semicircularcanal cristafroma pallid animal, The sensory epithelium (SE) and nerve fibers (NF) are normal in appearance [hematoxylinand eoxin-phlexine, • 440).
otoconial layer is 0.00139 m m 3 (standard deviation, 0.00033 mm3). Thus, the otoconial layer has a volume of approximately one-third its corresponding sensory epithelium.
Vestibular Ganglion The vestibular (Scarpa's} ganglion was qualitatively similar in the two groups (Fig. 4). The inferior a n d superior divisions of the ganglion were apparent in the pallid mice and demarcated by the normal orientation of the afferent and efferent processes of their neurons. The Nissl substance within the somas of these cells stained comparably in the two groups, and the various cellular elements within the ganglia appeared to be distributed normally. Fibers from the superior division of the ganglion innervate the cristae of the lateral and superior semicircular canals and the macula o:[ the utricle. A small portion of the saccule is also innervated by fibers from the superior division. The inferior division of the ganglion contains cell bodies of those neurons that innervate the crista of the posterior semicircular canal and the remainder of the saccular macula. However, since it was not possible to identify the specific somas within each division that innervate the various sensory structures of the vestibule, the two divisions were not broken down by end organ in this portion of the analysis. The m e a n total volumes for e a c h d i v i s i o n within the ganglia of the two groups are shown
Volume 4 Number 4 J u l y 1983
265
SENSORINEURAL STRUCTURES IN PALLID MOUSE INNER EAR
Figure 4, The inferior (IVG) and superior (SVG) divisions of the normal (top left) and pallid (top right) vestibular ganglia. The dashed line approximates the boundary between the two divisions, Higher-rnagnification views of the normal (bottom left} and pallid (bottom right} inferior divisions demonstrate their qualitative similarities (hematoxylin and eosin-phloxine; top left and top right, x 200; bottom left and bottom right, x 440), Americnn Journal ot~
Otoloryngology
266
in F i g u r e 7. In b o t h g r o u p s of a n i m a l s , the sup e r i o r d i v i s i o n was a p p r o x i m a t e l y t w i c e the size of the inferior d i v i s i o n . I n the p a l l i d animals, b o t h s u p e r i o r a n d i n f e r i o r divisions w e r e signif-
icantly smaller than n o r m a l animals (P < O n c e it h a d h e e n sions of the ganglion
t h e i r c o u n t e r p a r t s in t h e 0.05). d e t e r m i n e d t h a t t h e diviw e r e smaller in the pallid
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~ ~I~:'~"~,;"' ~.":'",~:_":~ '"!,~,., Top, a mid-madiolar view of the normal mouse cochlea. Asterisks indicate those regions m e a s u r e d to derive the Figure 5. cross-sectional area of scala media (SM). OC, organ of Corti; SG, spiral ganglion; ST, scala tympani; SV, scala v e s t i b u l i . Bottom, a higher-magnification v i e w of the spiral ganglion neurons. There were no qualitative differences b e t w e e n t h e s e and similar neurons in the pallid cochlea (hematoxylin and eosin-phloxine; top, • 240; bottom, • 560).
Volume
4
Number July
4 1983 267
SENSORINEURAL STRUCTURES IN PALLID MOUSE INNER EAR 5.0-
[] pal+
= • 4.0" ~< %
[]
I
Figure 6. The volumes of the various vestibular sensory epithelia in the normal (pal+) and pallid (pu/pa) animals. No significant differences (P > 0.05] were found in comparisons of normal and pallid superior, lateral, or posterior semicircular canal cristae, utricular maculae, or saccular maculae. The sample size for the cristae was seven normal mice [12 ears] and seven pallid mice (13 ears). The sample size for the maculae was eight normal mice [13 ears} and eight pallid mice (15 ears).
po/po 1,0 S.D.
3.0-
=, r > 2.0-
1.0 m
Superior
Loterol
Posterior
Ulricle
Soccure
VESTIBULAR SENSORY STRUCTURE
American
Journal of Otolaryngology 268
animals, an effort was m a d e to isolate the specific n e u r o n a l changes w i t h i n the ganglion that could be c o n t r i b u t i n g to this v o l u m e change. T h e r e f o r e , m e a s u r e m e n t s were m a d e of the number, size, and d e n s i t y of the ganglion cells. The results of file cell number analysis are shown in Figure 7. The data s h o w that there were no significant differences between the two groups in the n u m b e r of neurons in the divisions of the vestibular ganglion (P > 0.05). Since ganglion volume and cell number data were available, cell d e n s i t y for the superior and inferior divisions was calculated to determine whether neuropil loss was a factor contributing to the volume reduction of the pallid vestibular ganglion. These values are s h o w n in Table 1. However, the statistical tests performed on these data f a i l e d to d e m o n s t r a t e any d e n s i t y differences between the two groups (P > 0.05). The results of the cell size analysis are shown in Figure 8. These data were obtained from the same sections as the ganglion volume measurements. Since the neuronal size data were stored on tape, we could create bins of specific size categories, group the n e u r o n s within these bins, and then compare either the absolute number of neurons or the percentage of total neurons within these bins between the two groups. The absolute numbers of neurons d r a w n for each group were not equal, so comparison of the total number of cells in each bin was not valid, owing to unequal sample sizes. Therefore, w e chose to compare the percentage of total neurons that occur in each size group. Thus, if the neurons are smaller in one group, that group will have relatively more neurons in the smaller size categories, i.e., the curve will shift to the left. As one can see from the distribution curves in Figure 8, there were no differences between the pallid a n d normal animals in their proportions
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Figure 7. The volume (top] of the normal (pa/+) and pallid (pa/pa) vestibular ganglia divisions and the number of neurons (bottom} within each of these divisions. Although the divisions of the pallid ganglion are significantly smaller than normal [P < 0.05), the number of neurons within them is the same as in the controls (P > 0.05]. The sample size for the volume measurements was eight normal mice (12 ears) and seven pallid mice (superior, 12 ears; inferior, 13 ears}, The sample size for the neuron number data was seven normal mice (10 ears) and seven pallid mice (superior, 12 ears; inferior, 13 ears). P, probability; S.D., standard deviation,
TRUNE AND LIM
TABLE 1. Density of Neurons Within the Vestibular Ganglia of Pallid and Normal Mice DIVISION
OF
DENSITY +-- S . D . ( E / N ] *
GANGLION
Normal
Pallid
PROBABILITY*
Superior Inferior
1.76 • 0.18 (1017) 1.80 • 0.12 (10/7)
1.78 • 0.12 (1117} 1.87 -- 0.19 (12/7]
P > 0.80 P > 0.40
ABBREVIATIONS:N, sample size (number of animals); E, number of ears; S.D., 1.0 standard deviation. * Density in cells/10,000 p,m3. -~Probability determined by t test. of ceils in the various size categories. The curves for each division a p p e a r to overlap completely, indicating that the n e u r o n s in the pallid vestibular ganglion w e r e the same size as those in the control animals. H o w e v e r , n e u r o n a l size comparisons in the two divisions revealed larger neurons in the superior division of b o t h groups t h a n in the inferior d i v i s i o n (P < 0,05). This is demonstrated by a chi-square analysis (Table 2) of
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Figure 8. The percentage of superior (top) and inferior (bottom) division neurons within the various size categories for normal (pa/+) and pallid (pa/po) vestibular ganglia. Since the pallid and normal curves within each division overlap completely, the size distribution of pallid ganglion neurons appears to be normal. The normal data were derived from 6,382 superior division neurons and 3,239 inferior division neurons (eight mice, 12 ears). The pallid data represent 4,564 superior division neurons (seven mice, 12 ears) and 2,627 inferior division neurons (seven mice, 13 ears).
their proportions smaller and larger t h a n 245 t~m2 and e x p l a i n s w h y the s u p e r i o r d i v i s i o n curves are shifted to the right relative to the inferior division curves.
Cochlea Qualitatively the b o n y a n d m e m b r a n o u s labyrinths a p p e a r e d n o r m a l in the p a l l i d animals. The b o n y labyrinth was o r i e n t e d p r o p e r l y , and the c o c h l e a had the usual t u r n and a half, T h e r e were no abnormalities of a n y of the scatae, and they all w e r e in p r o p e r r e l a t i o n to each other, The a u d i t o r y s e n s o r i n e u r a l e l e m e n t s a p p e a r e d normal t h r o u g h o u t the c o c h l e a (Fig. 5). T h e hair cells a n d the v a r i o u s s u p p o r t i n g c e i l s w e r e normal in all turns of the organ of Corti. S e n s o r y cilia w e r e a p p a r e n t at the apical e n d s o2 t h e hair cells, a n d often the cilia of t h e o u t e r hair cells w e r e a p p o s e d to the r e c t o r i a l m e m b r a n e . T h e nerve fibers w i t h i n the t u n n e l of Corti w e r e apparent e v e n at the light m i c r o s c o p i c level. T h e spiral ganglion n e u r o n s in R o s e n t h a l ' s canal a n d the a u d i t o r y n e r v e w i t h i n t h e m o d i o l u s appeared normal. SCALA MEDIA. The m e a s u r e m e n t s of the scala m e d i a f o r each h a l f - t u r n in the m i d - m o d i o l a r section are s h o w n in Figure 9. T h e pallid animals h a d significantly larger scalae m e d i a e i n the basal (P < 0.001) and m i d d l e (P < 0.01) halfturns t h a n the h e t e r o z y g o u s controls. Since there did not a p p e a r to be any d i s t e n t i o n of Reissner's m e m b r a n e into the scala vestibuli, the larger scala m e d i a size c o u l d not be attributed to endolymphatic hydrops. The qualitatively normal appearance of the scala media in the basal turn i n d i c a t e d s i m p l y that it was larger in the mutants. T h e m e a s u r e m e n t s for t h e apical port i o n of t h e c o c h l e a s h o w e d no d i f f e r e n c e bet w e e n the two groups (P > 0.05). SPINAL GANGLION. T h e r e s u l t s of the spiral ganglion analysis are s h o w n in F i g u r e 10. The neurons were separated into bins in the same m a n n e r as for t h e vestibular ganglion n e u r o n s ; the distributions of the various cell sizes for the two g r o u p s are shown. In this case, t h e curve for
Volume 4 Number 4 July 1983 269
SENSORINEURALSTRUCTURESIN PALLIDMOUSEINNEREAR TABLE 2. Chi-square Analysis of the Percentage of Superior and Inferior Division Neurons Smaller and Larger than 245 ~m 2. GENOTYPE
pa/+
NEURON SIZE GROUP (I~Mz)
Superior
Inferior
X2
PROBABILITY
~245.0 >245.0
52.3 47.7 100.0
62.3
4.26
P < 0.05
37.7 100.0
~245.0 >245.0
54.6 45.4 I00.0
64.5 35,5 I00.0
4.28
P < 0.05
TOTAL
patpa TOTAL
PERCENTAGEOF TOTAL
* The data were derived from 6,382 superior division neurons and 3,239 inferior division neurons from normal animals (eight mice, 12 ears) a n d from 4,564 superior division neurons (seven mice, 12 ears) and 2,627 inferior division neurons (seven mice, 13 ears) from p a l l i d animals.
the pallid animals is shifted slightly to the left, w h i c h indicates that there are more neurons in the smaller-size classes i n the pallid ears, i.e., the spiral ganglion cells are smaller than normal. In order to test this size difference statistically, a chi-square analysis was performed on the proportion of cells less t h a n and greater than 125 ~m 2, the size where the two curves cross in Figure 10. The design and results of the chi-square test are s h o w n in Table 3. W h e n 125 ~m 2 is used as the dividing point, the proportions of cells below and above this size differ between the two groups (P < 0.05). The same results are obtained if any size between a23.5 and 127 ~m 2 is used. This indicates that the pallid animals have smaller spiral ganglion neurons t h a n normal. A t test was also r u n on the n u m b e r of spiral ganglion ceils in this m i d d l e half-turn of the mid-modiolar section. T h e r e was no s i g n i f i c a n t d i f f e r e n c e between the two groups in the n u m b e r of spiral ganglion cells (P > 0.05); however, this Comparison was based on o n l y one section for each ear.
spiral ganglion were slightly smaller than normal, as d e t e r m i n e d by the m e a s u r e m e n t of n e u r o n sizes from one section in each ear. Since we have not performed a thorough analysis of the pallid cochlea, it is not possible to judge what degree of disparity exists in spiral ganglion neuron sizes, if any. However, we tentatively conclude that there is some cochlear, as well as vestibular, inv o l v e m e n t w i t h this m u t a n t gene. This is common in a variety of mutant mice that have inner ear involvement. 2z Temporary endolymphatic hydrops has been reported during the development of the pallid inner ear, but not in the adult. 9,ms Based on our morphometric analysis of the mid-modiolar sections, the endolymphatic space of the scala media
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O
DISCUSSION
American Journal of Otolaryngology 270
Qualitatively, the o n l y abnormality apparent in the pallid inner ear was the absence of otoconia in the utricle a n d saccule, confirming earlier reports. ',~a,l" However, the quantitative portion of this study did reveal some subtle changes. In addition to the absence of otoconia, the volume of the vestibular ganglion was smaller, as were the cochlear spiral ganglion neurons. Since the sensory epithelia of the utricular and saccular maculae, as well as the semicircular canal cristae, were of normal size in the pallid animals, we conclude that neither the lack of otoconia nor the m u t a n t gene had any significant deleterious trophic influence on them. The first-order a u d i t o r y n e u r o n s w i t h i n the
7
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5.0
4.0
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BASAL J/~' TURN
M I DOLE V~, TURN
APICAL ' I/2 TURN
HEMI-TURN OF COCHLEA
Figure 9. The average scala media cross-sectional area for the three hemi-turns in the normal ( p a / + ) and pallid (pa/pa) cochleas, This e n d e l y m p h a t i c space was significantly larger in the pallid basal and middle hemi-turns (P < 0.05), whereas the apical region measurements were the same for both groups (P > 0,05). The sample size was eight normal mice (15 ears) and eight pallid mice (16 ears). P, probability; S.D., standard deviation.
TRUNE AND LIM
was slightly larger in the basal turn (basal and middle half-turns). However, since there was no a c c o m p a n y i n g d i s t e n t i o n of Reissner's membrane, it is unlikely that the larger scalar size is due to a true endolymphatic hydrops; rather, it m a y be just structurally larger. Therefore, we tentatively suggest that a true phenotypic structural modification m a y occur in the auditory system of this mutant mouse. Although our volumetric measurements rev e a l e d the pallid v e s t i b u l a r ganglion to be smaller, our cytomorphometric analysis failed to demonstrate any change in its neuronal numbers, sizes, or densities. The underlying reason(s) for the smaller vestibular ganglion size is not clear; however, several possibilities exist. It may be due to 1) a reduction in the vestibular nerve neuropil that lies around and within the ganglion; 2) reduced glia; 3) fewer connective tissue elements within the ganglion; or 4) vascular hypotrophy that w o u l d decrease ganglion volume but not affect its neural structures. A more thorough analysis of the ganglion is certainly necessary before this matter can be settled, but at this point there is good reason to suspect some trophic neural involvement with the mutant gene. Even though the present morphometric analyses failed to demonstrate any significant differences between the sizes of the remaining vestibular sensorineural elements, a number of the comparisons p r o v i d e d probability levels only slightly above 5.0 per cent. This was especially true of the lateral semicircular canal crista, saccular macula, and the number of neurons in both divisions of the vestibular ganglion. Since the
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i
i
155IIS5
AREA
176185
I=J5205
215- >225
225
(/zm 2)
Figure 10. The percentage of spiral ganglion neurons within the various size categories for normal (pa/+] and pallid (pc~ pc) mice. The pallid curve is shifted to the left and indicates more neurons than normal in the smaller size categories. The sample size was 1,021 neurons from eight normal mice (16 ears) and 1,077 neurons from eight pallid mice (16 ears}.
TABLE 3. Chi-square Analysis of the Percentage of Spiral Ganglion Neurons Smaller and Larger Than 125 p,m 2. NEURON SIZE GROUP (t.~M2} ~<125.0 >125,0 TOTAL
PERC6NTAGEOF TOTAL
Normal Pallid 42,0 58.0
51.7 48.3
10O.O
100.0
X2 3,86
PROBABILITY P ~ 0.05
* The data were derived ~rom 1,021 n e u r o n s from eight normal mice (16 ears} and from 1,077 n e u r o n s from eight pallid mice [16 ears}.
probability was greater than 5.0 per cent, these structures in the pallid animals were judged statistically as normal in size. However, one cannot rule out the possibility that a statistical difference in size may emerge if a larger sample size is used. Even if future analyses of these structures demonstrate these trends to be true phenotypic abnormalities, the size differences would be so slight that a real functional difference would be doubtful. The pallid mice exhibit such behavioral deficits as head tilting, disorientation in water, a b s e n t or i m p a i r e d r i g h t i n g r e f l e x e s , and ataxia. ~,4,'1,~8Since the size differences of the sensorineural elements in the pallid inner ear are relatively minor, as mentioned above, the dominant feature of the pallid mutation is the lack of otoconia in the gravity receptors. Therefore, the dysfunction of the pallid inner ear is closely linked to this otoconial agenesis and, ultimately, to gravity receptor h y p o s t i m u l a t i o n . It is apparent that the brain stem structures that rely on gravity receptor input are not receiving proper stimulation and that a condition of vestibular deprivation exists. Merphologic studies of the deprived s e c o n d - o r d e r n e u r o n s h a v e b e e n conducted to determine the p o s t s y n a p t i c consequences of hypostimulation. Clark et el. 19 found cells less dense in nucleus y and medial vestibular nucleus of the otoconia-deficient mutants, and Trune and Lira .6 found some vestibular nuclei to be smaller than normal. These results were interpreted as being due to the lack of proper presynaptic stimulation during the critical period of vestibular nuclear d e v e l o p m e n t . Other investigators have examined the influence of such deprivation on higher brain functions. Although studies of hippocampal electrophysiology did not demonstrate measurable differences in the pallid animals, 2~studies of spatial orientation and emotionality did reveal significant differences in these otoconia-deficient mutants. 17 All of these studies demonstrating significant
Volume 4 Number 4 July 1983 27!
SENSORINEURAL STRUCTURES IN PALLID MOUSE INNER EAR differences in the m u t a n t s c o n c l u d e that s u c h deficits are the direct r e s u l t of g r a v i t y r e c e p t o r h y p o s t i m u l a t i o n d u r i n g d e v e l o p m e n t . T h e definite c o r r e l a t i o n of s u c h deficits w i t h the lack of o t o e o n i a certainly i m p l i e s that s u c h a c o n c l u sion is valid, O n the o t h e r h a n d , t h e m u t a n t gene c o u l d be affecting t h e central n e r v o u s s y s t e m s of t h e s e a n i m a l s directly, a n d a n y l a c k of v e s t i b u l a r i n p u t during d e v e l o p m e n t m a y be s e c o n d a r y and of l e s s e r s i g n i f i c a n c e , Creel 23 h a s r e c e n t l y rev i e w e d the b i o c h e m i c a l a n d n e u r a l a b n o r m a l i ties t h a t are often a s s o c i a t e d w i t h a l b i n o a n d hyp o p i g m e n t e d animals, T h e effects of a p a r t i c u l a r m u t a n t gone w i t h p i g m e n t a t i o n abnormalities are c o n s i d e r a b l y m o r e diffuse since so m a n y m e t a h o l i c p a t h w a y s in t h e b o d y d e p e n d o n p i g m e n t related biochemistry. These potential influences m u s t c e r t a i n l y be i n v e s t i g a t e d before the b e h a v ioral a n d central m o r p h o l o g i c a b n o r m a l i t i e s in the p a l l i d a n i m a l s are i n t e r p r e t e d as b e i n g s o l e l y the r e s u l t of gravity r e c e p t o r h y p o s t i m u l a t i o n . H o w e v e r , properly designed investigations, using the p a l l i d s a n d p r o p e r c o n t r o l s to s e p a r a t e genetic, dietary, and developmental influences, s h o u l d p r o v i d e us w i t h s i g n i f i c a n t i n s i g h t s into the r o l e of gravity r e c e p t o r s t i m u l a t i o n in b r a i n development and maintenance.
6. 7.
8. 9.
10. 11. 12. 13. 14. 15. 16.
Acknowledgments.
The authors thank Roberta Arbaugh, Anabel Ocasio, and Atha Ralston for preparation of the tissue; Scott Gordon for developing the software for data c o l l e c t i o n and analysis; James Douthitt for photographic assistance; Nancy Sally for preparation of the illustrative material; and Katherine A d a m s o n for assistance in the preparation of the manuscript.
17.
18. 19.
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