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An investigation into the physical basis of the pirouette mutation in the house mouse (Mus musculus) Walden, Adelene Mary 1951

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AN INVESTIGATION INTO THE PHYSICAL BASIS OF THE PIROUETTE MUTATION IN THE HOUSE MOUSE (MUS MUSCU1US)  ADELENE MARY WA1DEN  A THESIS SUBMITTED IN PARTIAL FULFILMENT Off THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n the Department of B i o l o g y and Botany.  We accept t h i s t h e s i s as conforming to the standard r e q u i r e d from candidates f o r the degree o f MASTER OF ARTS.  Members o f t h e Department o f B i o l o g y and Botany  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1951.  1^5/5/ Iff/  fif  -1Abstract. An  I n v e s t i g a t i o n i n t o the P h y s i c a l B a s i s o f the P i r o u e t t e M u t a t i o n In the House Mouse (Mus musculuV).  f h e object o f t h i s  i n v e s t i g a t i o n was to study the  abnormal behavior produced by the p i r o u e t t e mutation i n the house mouse (Mus musculus) and to determine i t s p h y s i c a l basis. The  p i r o u e t t e mutation f i r s t  appeared i n 1943 and was  d e s c r i b e d by Wolley and D i c k i e (1945) who gave I t the g e n e t i c symbol p i .  T h i s r e c e s s i v e mutation i s l o c a t e d on  the t h i r d chromosome o f the house mouse. Mice homozygous f o r t h i s mutation show mixed c i r c l i n g , head shaking and deafness.  The abnormal movements  commence f o u r t e e n days a f t e r b i r t h and these mice are e v i d e n t l y deaf throughout t h e i r e n t i r e  lives*  S i m i l a r abnormal movements and deafness have been produced not o n l y by other mutations i n mice but a l s o In other  animals I n c l u d i n g man.  A survey o f the l i t e r a t u r e  r e v e a l e d many p o s s i b l e sources o f abnormalles which c o u l d produce the a t y p i c a l behavior o f the p i r o u e t t e mouse and these r e p o r t e d sources o f a b n o r m a l i t i e s w i t h the f o l l o w i n g r e s u l t s :  were  Investigated  -2-  1. 2. 3. 4* 5. 6.  Muscles of the head, neck and thorax were normal* Liver gross and h i s t o l o g i c a l anatomy was normal. Brain * gross anatomy was normal. Skull - gross anatomy was normal. Eighth nerve - gross and h i s t o l o g i c a l study revealed no tumors. Bar - gross anatomy and "blood supply normal. .» h i s t o l o g i c a l studies showed degenerative changes within the s p i r a l ganglion, s t r i a vascularis and organ of Corti within the cochlea.  The degenerative  changes within the cochlea of  ;  pirouette mice are similar to those reported In waltzing and shaker-1 mice but their time of onset and sequence o f degenerative  changes are d i f f e r e n t *  In the pirouette mouse  the extensive loss of nerve c e l l s within the s p i r a l ganglion l a t e r followed by degenerative  changes In the s t r i a vas-  c u l a r i s and i n the organ of Corti suggests that the cause of these changes l i e s outside the cochlea. the degenerative  The fact that  changes of a l l structures are found f i r s t  and most extensively In the basal whirl and later i n the middle and upper whirls of the cochlea also supports the contention that the change i s external to the cochlea and gradually involves the whole organ* Similar changes are known to be produced by tumors, osseous compression or severing of the eighth nerve or leslgans •within the brain*  Since i n the pirouette mouse no tumors were  present and since the nerve was intact, there remains only the p o s s i b i l i t y , of osseous compression or lesions within the central nervous system causing t h i s abnormality.  Acknowledgements. May  I take t h i s opportunity to express my sincere  thanks to the following people, whose interest and help have made the preparation of t h i s thesis possible. especially indebted to Mrs. R.  I am  Brink, Assistant  Professor, by whom the problem was f i r s t suggested and under whose d i r e c t i o n the work was carried on.  Her valTsable  advice and assistance when the problem seemed most d i f f i c u l t i s deeply appreciated. My thanks are due to Dr. A.H. Hutchinson, Head of the Department of Biology and Botany, for his suggestions and encouragement during the preparation of the microscope s l i d e s and for his aid in photography. To Dr. W. Gibson, lecturer, of the Department of Physcology,  for his advice and assistance i n interpreting  the neurological changes which were observed within the inner ear. To Dr. V . C , Brink, Associate Professor of the Department of Agronomy, for his many u s e f u l suggestions. I am indebted to the President's Committee on Research for a grant from the Board o f Governors i n 19481950, which made the preparation of t h i s thesis possible.  TABLE OF CONTENTS.  I  II  III  Page 11:  Introduction A. O r i g i n o f Pirouette  1  B. Statement of the Problem  1  Description of the Mutation  2  A. Genetic Description o f Pirouette  2  B. Effect o f the Mutation  2  Review of the L i t e r a t u r e  3  A. E f f e c t o f Mutations on Behavior  3  B. Ghorie Mutations i n the House Mouse  4  1. Waltzer  4  2. Shaker-1  8  3. Shaker-2  10  4. Hertwig's shaker  10  5 * Shaker-short  11  6* figet  13  7* J i t t e r y  14  8. E r e l s l e r  15  9. a r i t i n t waddler  16  v  10. Jerker 11. P a l l i d 12. Comparison o f mutations  17 _  18 18  Ci Choric Mutations  i n Other Animals  1. Rat a. Waltzer  '23 i 22  b. Wobbly  22  2. Peromyscus  23  --  l  Waltzer 3. Rabbit  23 24  a. Waltzer  24  b. Shaking Paralysis  24  o. Ataxia  24  4. Guinea-pig  26  a. Waltzing  26  b. Congenital palsy  26  D. Choric Abnormalities i n Man IV  Page . 22  Materials and Methods  27 34  A. Origin of the Mice  34  B. Care of Mice  34  1. Cages  34  2. Food  36  3. Records  35  G. Preparation of M a t e r i a l  36  1. K i l l i n g and f i x i n g  36  2. H i s t o l o g i c a l techniques  36  a. 5?he l i v e r  36  b. The ear  36  v"  Comparison of the Behavior of Normal and Pirouette Mioe  Page >38 l  VI  A. Adult Mice  38  B. Development of Abnormalities  39  C. Comparison of Normal and Mutant Mice  40  Investigation of the Possible Causes for the Abnormal Behavior o f the Pirouette Mouse.  48  A. Pathological Infection  48  B i Muscular Abnormalities  49  G* The Liver  49  D. 3?he Brain  50  1. Tumors  51  IP. ^he Bar  52  1. The structure and function of the normal ear.  53  a. The middle ear  53  b. The inner ear  56  1 S t a t i c Labyrinth i i The Cochlea Oi Post-natal development of the cochlea of a normal mouse. 2. Structure o f the ear of the pirouette mouse  56 60 67 73  a. Position of the ear vessicle  73  b. The middle ear  74  o. The inner ear  74  Page 1 The s t a t i c L a b y r i n t h i i The c o c h l e a d. P o s t - n a t a l development o f the c o c h l e a o f a p i r o u e t t e mouse S. S k u l l  75 76  80  87  VII  Discussion  89  VIII  Conclusion  96  Summary  98  IX X XI XII  Biblography  100  Plates  111  Appendix  114  An Investigation into the Physical Basis of the Pirouette Mutation i n the House Mouse ( Mus fftusculus). I Introduction A. Origin o f Pirouette In 1943 a mutation appeared i n a s t r a i n of mice at the Roscoe B. Jackson Memorial Laboratory, Bar Harbor.,Maine, which caused deafness, abnormal head shaking and c i r c l i n g movements similar to those produoed by the waltzer mutation, Ehis new mutation, pirouette, was named and described by G. W, Woolley and M.M. Dickie (1945) who gave i t the genetic symbol p i . eo 0 oo B.oStatement of the Problem. The objeot o f t h i s investigation i s to study the abnormal behaviour produoed by the pirouette mutation i n thehouse mouse (Mus muaculus) and to determine i t s physical basis. oo 0 oo  II Description of the Mutation. i  A , Genetic description of pirouette,. The inheritance of pirouette (pi) was by <J. W. Woolley and M.M.  Dickie (1946), who  investigated  established the  faot that i t was a re*oessive mutation located on the t h i r d chromosome o f the house mouse.  Linkage tests showed that  pirouette was located 7,2 map u n i t s from dominant viable spotting (wv), 36*5 map units from hairless (hr) and was linked with piebald ( s ) . Thus the genes on chromosome three are arranged as follows. s T:  hr §  pi 3B75  wv 7,£  B. E f f e c t of the mutation. Mice homozygous for t h i s recessive mutation show mixed c i r c l i n g , head shaking and deafness,  Woolley and  Dickie (1945) tested the hearing of these mutant mioe and reported that they are deaf throughout l i f e .  They also  found that mice homozygous for the pirouette mutations are unable to swim and do not become dizzy when rotated. oo 0 oo  - 3 III  Review of the Literature  A. Mutations A f f e c t i n g Behaviour. Uhe t r i a d of symptoms exhibited by the pirouette mice have been produced by other mutations In mice and also by mutations i n many other animals including man.  These  chorio mutations appear to a l t e r the developmental reactions oocuring within the animal i n such a way as to produce changes i n i t s struotnlre and to modify i t s physiology. Some of these mutant effects may be telly s l i g h t and thus d i f f i c u l t to detect or they may produoe changes that are e a s i l y recognized. J.P. Soott (1942) and C.E. Eeeler (1942) noticed that mutations a f f e c t i n g coat colour i n mice also produoe s l i g h t changes i n the behaviour pattern.  Thus mice  with grey coats are found to be wilder than those having black pelages.  While studying autogenetio seizures i n mice,  C. H a l l (1947) observed that the severity of the convulsions was to some degree affected by the coat colour of the mice since black mice were less susceptable than d i l u t e brown mice. It has been suggested by K e l l e r (1947) that mutations a f f e c t ing coat colour also produce s l i g h t behaviour changes by p l e i t r o p l c action*  Henoe, a mutationnay produce i t s main  e f f e c t on some d e f i n i t e part or structure of the body and at the same time influence the behaviour of the animal to a minor degree.  Other mutations suoh as pirouette, produce  t h e i r main e f f e c t on the behaviour of the animal.  - 4 B. Choric Mutations i n the House Mouse. Other mutations have occurred i n the house mouse which produce choric "behaviour, similar to that of pirouette. The following mutations: Japanese waltzer ( v ) , shaker-1 ( s h - l ) , #haker~2 (sh-i2), shaker-short ( s t ) , f i g e t ( f i ) , jerker ( j e ) , j i t t e r y ( j i ) , E r e l s l e r (k) and p a l l i d (pa) a l l produce peculiar movements of the head and body, incessant a c t i v i t y and often deafness.  The physical basis of some of these  mutations has been determined while the cause of others remains unknown. 1. Waltzer The Japanese waltzer i s the oldest and most commonly known of these behaviour mutations.  According to  Keeler (1931) the waltzing mutation appeared i n China as early as 80 B.C. in Mus baotrianus and was Europe during the year 1890.  introduced into  These mice were bred with the  European house mouse, Mus muscuius.  Yerks (1907) stated  that the dancing mice can be distinguished from normal mioe by their innate habit of running around describing greater or smaller c i r c l e s or whirling around on the same spot with incredible r a p i d i t y .  These mice also show a c h a r a c t e r i s t i c  head shaking movement and are deaf.  Yerks (1907) found that  the r e s t l e s s , jerky movements and general e x o i t a b i l i t y appear in these mice during the tenth to the fourteenth day after birth.  The ears of the waltzers open between the twelfth to  the fourteenth day hut only i n a few oases did the mice react to sound between the thirteenth and seventeenth days. Grmneberg  (1943) suggests that ... there i s some v a r i a t i o n  in the time of onset of deafness, so that, i n most animals i t i s almost oomplete by the time an auditory response can f i r s t be evoked i n the normal mouse" Yerks (1907) noted that some of the adult waltzers were mixed o i r c l e r s while others always appeared to c i r c l e i n one d i r e c t i o n only. Was  He also reported that the  waltzer  able to run up and down i n c l i n e s or v e r t i c a l rods,  cross  narrow bridges and maintain i t s balance; i t s equilibrium, however, was not as good as that o f the normal mouse. was  It  also noted that these mutant mice never jumped when  frightened as did normal mice.  Waltzing mice were unable to  swim when placed i n water and da not become dizzy when rotated. Gruneberg  (1943) stated that t;v<- Alexander and  E r e i d l (1900) found that although waltzers did not become dizzy when rotated i n a oyclastat, they became dizzy after galvanic stimulation of the head as do normal mice. Genetic studies have Bh9"wn that the mutant gene for waltzer i s looated on the tenth chromosome of the house mouse. When homozygous t h i s recessive mutation produoes abnormalities whioh cause the mouse to run i n c i r c l e s , w h i r l , show a r e s t l e s s , jerky, v e r t i c a l movement of the head and become  deaf.  Many theories have been developed to explain the  physical basis of t h i s mutation.  Yerks (1907) reported that  Saint Loup stated the abnormal movements o f the waltzers were due to nervous defect while Landais believed t h i s defect was a b i l a t e r a l abnormality i n the b r a i n .  Yerks (1907)  stated that Hawitz (1899) reconstructed the ear of the waltzer i n wax from microsoope s l i d e s and found that the anterior v e r t i c a l canal was normal but the horizontal canals were crippled or fused together, the u t r i c u l u s was warped and communicated by a wide opening to the sacoulus.  Yerks  (1907) also noted that Pense (1901) found no s t r u c t u r a l difference i n the ears of the waltzer and those of the . normal mouse.  He suggested that the sensory nerve supply  to the ear was responsible for the deafness and abnormal behaviour of the mouse and that there might be s t r u c t u r a l defects within the cerebellum.  Yerks (1907) reported that  Baginsky i n 1902 also found the Inner ear of the waltzer to be normal and that Alexander and E r e i d a l (1900) (quoted from Yerks, 1907) stated that: Structural p e c u l i a r i t i e s o f the waltzer are the r e s u l t o f primary changes...The facts of behaviour are almost c e r t a i n l y due to pathological changes...in the nerves, ganglia and e s p e c i a l l y i n the peripheral nerve endings of the ear of the mouse.  They also believed that deafness was due to a defective ooohlea and lack of dizziness was due to diminuatlon of the nerve fibers which supply the canal organs plus a degeneration o f the ganglion s p i r a l s . Gruneberg (1943) states that K i s h i (1902) found a reduction i n the size of the s t r i a vascularis cochlea and a s l i g h t diminuatlon of the hair c e l l s i n the waltzer's organ of C o r t i .  Acoording to Gruneberg (1943), Tan Lennep's  (1910) r e s u l t s indicated that: •••• shortly after b i r t h , the s t r i a vascularis cochlea undergoes a process of degeneration; as t h i s organ i s reputed to secrete the endolymph necessary f o r the n u t r i t i o n o f C o r t i s organ (which lacks blood vessels o f i t s own) the degeneration of that organ and the degeneration of the ganglion s p l r a l e i n Rosinthal's canal, i s regarded as secondary to. the changes i n the s t r i a vascularis; no anomalies were found i n the pars superior of the labyrinth. ?  Gates (1926) stated that i n 1911 Morgan supported the findings of Alexander and Z r e i d l (1900) by producing a r t i f i c i a l waltzers by i n j e c t i n g acetyl-atoxyl into normal mice*  These  mice waltzed l i k e the mutant animals and remained abnormal for the remainder of their l i v e s .  The drug whieh caused  these permanent changes i n the mice i s known to cause degeneration of certain nerve fibers i n man.  Thus, i f the  drug affects mice i n the same manner, i t would appear that the waltzing mutation can be explained by either a degener-  ation or an arrest i n development  o f the nerve terminals.  Gates (19E6) also reports that P. Ehriok (1911) produoed a r t i f i c i a l waltzers by using a drug arsacetin which i s known to affect the nerves and that Ealper (1913) found degeneration of the nerve fibers i n the acoustic tracts of the  waltzer*s b r a i n . Euiper believed that t h i s degeneration  Is the primary factor and that the degeneration of the s t r i a vascularis cochlea i s secondary. Prom the above discussion of the r e s u l t s obtained by the various research workers, i t i s evident that the fundamental cause of t h i s mutation i s controversial. The above c o n f l i c t i n g statements lead one to conclude that the waltzing mice are normal at b i r t h and later a degeneration of the acoustic t r a c t s of the brain and o f the structures and nerves In the organ of Oprti are p a r t l y , I f not wholly, responsible for the behaviour seen i n adult Japanese waltzing mice. 2. Shaker - 1 Shaker-1 produces a similar nervous, rapid up and down head movement but c i r c l i n g i s not as common as i n the waltzing mice. the  When c i r c l i n g i s present i n the shaker-1  animals turn more frequently in a counter-clockwise than  In a clockwise d i r e c t i o n .  Shaker-1 mice are able to hear  u n t i l they are three to s i x months o l d . The deafness and  ohorie head movement are the most noticeable part of the abnormal syndrome produced by t h i s mutation.  The  shaker-1  mutation i s completely recessive and has been located on the f i r s t chromosome.  Lord and Sates (1929) found that mice  which are hetrozygous for both the shaker-1 recessive gene and waltzing, show normal behaviour but have a tendency to become deaf.  These two mutations occurlng together seem to  exert a cumulative e f f e c t which results In deafness at the age o f s i x months. from his study of the development of the shaker*! mice, Gruneberg (1943) found that the organ of Oorti i s normal twelve days after b i r t h .  Degenerative changes within  the organ of Corti set in after the vas s p i r a l e has d i s appeared.  The vas s p i r a l e nourishes the organ of Corti during  the embryonic and early post-natal l i f e of the mouse.  After  t h i s vessel atrophies the organ of C o r t i depends f o r i t s n u t r i t i o n e n t i r e l y on the endolymph^ which is secreted by the s t r i a v a s c u l a r i s .  The s t r i a vascularis i s vist-bly  abnormal in the shaker-1 mice and evidently f a i l s to f u l f i l l i t s secretory functions adequately.  Hence Gruneberg con-  cluded that degenerative ohanges whioh occur i n the organ of C o r t i pf these mutant mice are caused by malnutrition and the changes within the ganglion s p i r a l e are probably secondary.  Zlmmermann (1935) found atrophy of the corpus striatoma i n the brain of the shaker-1 mouse accompanied a s l i g h t hydrocephalus internus.  by  These findings resemble  the pathalogical changes which oocur i n Huntington's chorea in man.  These changes explain the nervous head movements  and lack of muscle co-ordination but t h e i r connection with those which occur i n the inner ear s t i l l remains obscure* 3. Shaker-2 Shaker-2 i s a mutation which resembles shaker-1 so olosely that they are only able to be distinguished by the linkage studies.  The shaker-1 mutation occurs on the f i r s t  chromosome while the gene for shaker-2 i s located on the seventh chromosome. Mice heterozygous for both these mutations show normal behaviour and hearing. The pathological changes produced by t h i s mutation are as yet unknown. 4. Hertwig's Shaker Hertwig (1942) produced shaker mice by x-radlatlon. This abnormal change produces i t s e f f e c t on the homozygotes when they are ten days old, causing them to r o l l over on t h e i r backs, shake t h e i r heads, drag their hind legs and sometimes circle.  These abnormal movements are almost completely  lost during the second month.  Hertwig s mice were also found 1  to be deaf, unable to swim and did not become dizzy when rotated.  - 11 5. Shaker Short Another ehorio mutation, shaker-short ( s t ) produces deafness, c i r c l i n g , ataxia o f the head and muscular  inco-  ordination similar to that found among waltzing mice,  This  recessive mutation also r e s u l t s i n a shortening o f the t a i l j and at b i r t h the mice show one or two hernias near the middle of the p a r i e t o o c c i p i t a l suture. The abnormal behaviour i n these mice has been observed as early as five days after birth.  Similar to the other mutations shaker-short produces  deafness i n i t s homozygotes. Bpnnevie (1936) made an embryonic study o f the shaker-short mioe and reported that at eight days ah abnormal compression o f the brain i s evident. After the medullary tube closes a coalescence between the epidermis and the dorsal median l i n e o f the myelencephalon ocourts.  The r o o f o f the  l a t t e r i s narrow due to a compression of the b r a i n .  The  choroidal pleicus remains rudimentary and the foramen magendie i s absent.  During the l a s t days of embryonic  ment the r o o f o f the myienoephalon  develop-  and cerebellum i s broken  by the abnormal tension within the cranium.  The pieces of r  the roof, the blood and spinal f l u i d form the hernia which is present at b i r t h .  I f t h i s disturbance i s severe the  embryos are s t i l l - b o r n .  The surviving animals show only the  dorsal blood blebs which are gradually reabsorbed.  Bonnevie  - 12 (1936) stated that an abnormality of the inner ear accounts for the disturbance of equilibrium and the deafness which occur In these mice.  The inner ear of the shaker-short mice  is abnormal and forms an oval, l a t e r a l l y compressed v e s i c l e without any endolymphatic  appendage, semicircular canals  or d i v i s i o n into separate chambers.  These abnormalities are  caused by changes in the posterior part of the head and brain, surrounding the ear v e s i c l e s .  Bonnevie (1936) found that  in normal embryos the myelencephalon i s retarded and consequently the surrounding tissues including the ear vesleles become abortive. Thus, she concluded that the primary effect of the shaker-short mutation i s to change the  developmental  rate of parts of the brain i n suoh a manner as to also a f f e c t the inner ear. Gruneberg (1947) reported that the primary effect of the shaker-short mutation i s a disturbance of the eotoderm which forms a t r a n s i t o r y connection between the skin and the dorsal olosure region o f the brain.  This Interferes with the  formation of the plexus choroideus o f the fourth v e n t r i c l e , while the plexus of the l a t e r a l v e n t r i c l e s also remain rudimentary, no foramen of Magendie i s formed.  As the r u d i -  mentary plexuses f a i l to produce a sufflcienoy of cerebros p i n a l f l u i d , the whole ventricular system i s considerably narrowed down. This narrowness of the brain case r e s u l t s i n an abnormally high pressure within the brain cavity and a  - 13 reduced pressure on the ear v e s i c l e s which as a consequence develop abnormally.  Thus, the shaker-i-short mutation acts  d i r e c t l y on the extoderm i n the region of the dorsal "brain closure.  The above discussion explains the deafness in these  shaker-short mice, and one may assume that the abnormal compression of the brain and the effect of the pressure to which i t Is subjected is responsible for abnormal changes within the brain i t s e l f whioh produce the a t y p i c a l behaviour of  these mice.  The effect of the short t a i l has not yet been  investigated. 6. Elget Figet mutation oocured i n 1941. Mice homozygous for t h i s recessive faotor show side to side head movements, c i r c l i n g in one d i r e c t i o n and deafness. These mice are hypersensitive to sounds at three weeks of age.  This period is  followed by gradual loss of hearing which ends in deafness when the mice are three t o four months o l d . Like the  waltzers  the figets are unable to swim and Immediately lose a l l sense of d i r e c t i o n when placed in water. affects the eyes of the homozygote.  The figet mutation also After the eyes are open  a discharge from the oonjuotima occurs and the eyelids tend to s t i c k together and permanently close the eyes.  - 14 The morphological e f f e c t o f t h i s mutation has not been determined.  The phenotypic e f f e c t of the mutation  d i f f e r s from the other members o f t h i s group i n that the head is shaken from side to side rather than i n a dorso-ventral direction.  The physical basis for this mutation i s unknown. 7. J i t t e r y J i t t e r y i s a reoessive juvenile l e t h a l mutation  which i s characterized by muscular incoordination and tetany. These mice when twelve days o f age tend to lean to one side while running.  At fourteen days .Jittery mice f a l l on one  side when they attempt to run and cannot r e a d i l y r i g h t themselves.  A few days l a t t e r S n e l l (1945) observed tetany  involving the fore legs.  These mice died when thirty-two  days o l d . De Ome (1945) found that when a j i t t e r y mouse was p a r a b i o t i e a l l y joined to a normal s i b i t could l i v e for about f i f t y - o n e days.  In j i t t e r y mice the anterior p i t u i t a r y shows  increased c e l l u l a r i t y and engorged blood v e s s e l s .  Implanta-  t i o n o f normal p i t u i t a r i e s was found to be without e f f e c t . In l a t e r stages the thyroid gland of the j i t t e r y mouse became hypoactive and involution o f the thymus ooours i n the more advanced stages.  I t i s also recorded that the blood oalclum  is normal but a vaouolar degeneration i s seen i n the motor c e l l s of the lumber region of the s p i n a l cord.  J i t t e r y d i f f e r s from  - 15 previously mentioned mutations i n that i t i s a juvenile lethal.  The l e t h a l action of the mutation a p p B a r s to he  through an upset of the hormones secreted by the p i t u i t a r y , thyroid and thymus glands.  In the other mutations a f f e c t i n g  the musculature and sense of balance, the main abnormalities appear to be i n the development  of the ear and the b r a i n .  8. E r e i s l e r The mice homozygous for the E r e i s l e r mutation are affected at the age of ten days.  At t h i s time they are seen  to crawl i n c i r c l e s , t i r e r a p i d i l y and when fatigued f a l l into a r i g i d p o s i t i o n on t h e i r backs or s i d e s .  The adults  show deafness, dance and occasionally toss their heads. The E r e i s l e r mice shake t h e i r heads less frequently but c i r c l e more often than do the shaker-1 mice.  Hertwig (1942)  found that i n nine day old E r e i s l e r embryos the ear v e s i c l e s are displaced l a t e r a l l y so that they are separated from the neural tube by a layer of mesenchyme.  Since the ear vesicles  appear to develop under the inductive influences of the neural tube the abnormalies of the labyrinth may be due to the displacement of the ear v e s i c l e s .  In older embryos  ductus and sacculus endolymphatics are absent and the semic i r c u l a r canals are incomplete.  The saoculus and u t r i c u l u s  are incompletely separated, the cochlea i s abnormal and i t s ganglia are absent.  The absence of the endolymphatic duct  r e s u l t s i n increased pressure of the endolymph whieh causes a oyst to evaginate into the subarachnoid space.  After b i r t h  these cysts continue to grow and interfere with neighbouring parts of the b r a i n .  The abnormal configuration of the  labyrinth leads to changes within the os petrous . The fossa acuata which normally harbours the flocculus c e r e b e l l i i s absent and consequently t h i s structure of the brain i s not formed.  Thus the main effect o f the E r e i s l e r  mutation i s produced by changes within the ear o f the mouse whioh In turn produoe abnormalities within the brain of the mutant animal. 9. V a r l t i n t Wadaier. T&e dominant v a r i t i n t waddler mutation (va) produces a variously t i n t e d coat and causes the mice to have a duck l i k e walk.  Mice having t h i s mutation are nervous,  r e s t l e s s and deaf.  These mice also show choric head move-  ments and mixed c i r c l i n g fourteen days after b i r t h .  The  choreic tendency i s more pronounced in homozygous v a r l t i n t waddler mice.  At fourteen days old these animals are ex-  tremely excitable and deaf.  I f suddenly disturbed they r o l l  over and over then leap into the a i r several times and f i n a l l y end with a convulsive s t i f f e n i n g of the body.  The heterozygote  is not as excitable as the homozygote but i s in constant motion unless asleep. Mice which are homozygous for t h i s  - 17 mutation are unable to swim while the mice which are heterozygous for v a r i t i n t waddler are able to swim i n o i r c l e s . Both types of mice show head-shaking similar to that seen i n shaker-1 mice.  They waddle when walking, sometimes c i r c l e ,  and do not become dizzy when rotated. Since t h i s mutation affects both the integument and nervous system Qloudman (1945) believed that the disturbances of hearing, equilibrium and pigment formation might conceivably be r e l a t e d in t h e i r early embryonic o r i g i n *  He  suggests that since pigment arises from the neural crest a single predisposing factor might operate quite early i n embryonio development to affect both the integument and nervous system.  However, nothing i s known of the physical  basis o f t h i s mutation. 10. Jerker Jerker i s a recessive mutation which produces abnormal behaviour that can only be distinguished from waltzer by genetic studies.  Jerker (je) has been found to  be located on chromosome four and i s thus linked with ruby eye and s i l v e r .  No investigation has been oonducted on the  physical basis o f t h i s mutation.  18 11. P a l l i d . Tfie p a l l i d mutation (pa), also referred to as pink eye-2i produces abnormal head movements.  Head weaving  occurs i n some of these mutant mice and further investigation revealed that the affected mice showed continual nystagmus• The head weaving i s presumably a compensation for the nystagmus. The physical cause of this mutation i s unknown. 12. Comparison o f the Mutations. The above mutations are similar In that they a l l produce abnormal behaviour i n the house mouse.  They cause  nervous, excitable movements which keep the mice i n oonstant motion when they are awake*  These mutations, with perhaps  the exception o f j i t t e r y , cause deafness i n the adult mice* In some cases (shaker-short and E r e i s l e r ) the ear v e s i c l e s have been founa to be abnormal, while i n others (shaker-1 and waltzer) only certain structures within the cochlea are affeotea.  Some o f these mutations (waltzer, shaker-1,  shaker-short ana K r e l s l e r ) proauee aegeneratlon or des t r u c t i o n of the nerve traets in the b r a i n .  These changes  within the central nervous system are apparently responsible for the abnormal movements o f the b o a y .  A comparison of the  symptoms produeea by these mutations with their physical changes i s given i n table 1.  * 19  -  SABLE I SYMPTOMS  MUTATION WALTZER  (v) Chromosome 10  SHAKER-1 (sh) (Chromosome-l)  SHAKER- 2  (sh-2)  PHYSICAL CHANGES  C i r c l i n g , head s h a k i n g , deafness, restlessness, e x c i t a b i l i t y . Head shaking at 17 days deaf at 14 days, circle in either d i r e c t i o n . Do not become dizzy-when r o t a t e d . Cannot swim.  Abnormalities i n sensory nerve supply to the e a r . P a t h o l o g i c a l change i n the p e r i p h e r a l nerve endings o f the e a r . Deafness due t o d e f e c t ive c o c h l e a and l a c k o f d i z z i n e s s due t o d i m i n u a t i o n o f nerve f i b e r s i n the canal organs. Degeneration o f the g a n g l i o n s p i r a l e . Degeneration o f nerve fibers i n acoustic t r a c t o f the b r a i n .  Head shaking i n a r a p i d up and down movement i s the main e f f e c t . Few c i r c l e . Deaf at 3 to 6 months. Do not become d i z z y when r o t a t e d . Cannot swim.  In the organ o f C o r t i the abnormal s t r i a v a s c u l a r i s f a i l s to s e c r e t e enough e n d o l * ymph whioh r e s u l t s i n degeneration o f the former s t r u c t u r e and a l s o leads to the deg e n e r a t i o n o f the ganglion s p i r a l e . Muscular i n c o o r d i n a t i o n i s a r e s u l t o f an atrophy o f the corpus s t r i a t u m i n the b r a i n .  Same as  unknown.  i n Shaker-1  (CHROMOSOME 7)  SHAKER SHORT (st) (Chromosome?)  Deafness, c i r c l i n g , headshaking (up and down), and muscular incoordination, s h o r t e n i n g o f the t a i l and 2 c e r e b r a l hernias on the p a r i e t o o c c i p i t a l suture.  E i g h t day embryos show an abnormal compression o f the b r a i n . Choroid plexus i s rudimentary, foremen Magendle i s absent. Thus, drainage o f the b r a i n i s hampered and pressure develops,  so * MUTATION  SYMPTOMS  which causes the brain roof to break and hernias are formed. The inner ear remains an oval compressed vessicle lacking semicircular canals. These abnormalities are due to the retarded growth of the myelencephalon,  SHaker-Short continued  PI GET  (fir;.)  JITTERY  (It)  (Chromosome  -10)  PHYSICAL CHANGES  The symptoms are side to side head movements circling in one direction, deafness, unable to swim, do not become dizzy when rotated. Mioe three weeks old are hypersensitive to sounds then gradually become aeaf when 5-4 months old. Eyelias also tend to fuse.  unknown  A juvenile lethal mutation producing muscular incooraination ana tetany, When 12 days o l a mice tend to lean to one siae while moving. 14 aay ola mice f a l l ana have a i f f i e u l t y in righting themselves. Tentany in the fore-limbs. They ale when 32 aays ola*  Mice have abnormal anterior pituitary, thyroia ana thymus glanas. Also show a Segeneration of the motor oells of the lumber region of the spinal oolumn.  - 21  MUTATION  -  SYMPTOMS  PHYSICAL CHANGES  10 day o l d mioe crawl i n o i r l e e s , (Chromosome 5) t i r e r a p i d l y and f a l l into a r i g i d position on either t h e i r hacks or sides. Adults are deaf, c i r c l e and ocoasiona l l y show head shaking.  9 day embryos show an ear vessiele which i s displaced l a t e r a l l y and i s separated from the neural tube by mesenchyme. Abnormali t i e s of the l a b r i n t h develop, Abnormal pressure o f the endolymph produces cysts in the subarachnoid space whieh grow and interfere with the surrounding brain t i s s u e . The folcculus c e r e b e l l i i s absent.  VARITINT WADD1ER  Duck-like walk* some c i r c l i n g and head shaking, nervousness and r e s t l e s s . Mice are also deaf. The pigment of the skin i s effected and produces a variously t i n t e d coat •  Disturbance in the very early development of the neural crest material.  PIROUETTE (pill) (Chromosome 3)  mixed c i r c l i n g , head shaking, deaf, do not become dizzy.cannot swirn^, Young have a crab-like walk..  unknown  JEREER (le) (Chromosome 4)  mixed c i r c l i n g , head shaking, deafness, r e s t lessness, and excitability  unknown  PALLID (pa) (Chromosome  pink eye, headweaving and nystagmus•  headweaving due to nystagmus, cause of nystagmus unknown.  EREISLER (krl?)  (V T;) a  (Chromosome ?)  (5)  * 22 •? C  #  Chorie Mutations i n Other Animals, 1. Rat  A. Waltzer Waltzing resembling that seen i n the mouse has been observed in the r a t , ( R a t t u s n o r v e g i c u s ) . the waltzing mutation i n the r a t causes the animal to c i r c l e and shake i t s head but i t s hearing i s unimpaired.  This mutation acts  as a recessive gene which i s influenced by modifying factors which may favour or i n h i b i t i t s expression.  In the r a t  waltzing i s linked with albino, red-eyed yellow and pink* eyed yellow, B. Wobbly Castle, Zing and Daniels (1941) have described another mutation i n the r a t whieh they have named wobbly. Animals affected by t h i s simple reeessive mutation show a peculiar, locomotion which i s f i r s t notioeable when the eyes / open at about fourteen days*  The movements o f these animals  are jerky and the young animals walk a step at a time with a jerk of the body as each step i s taken.  These movements  suggested an infection o f the inner^ear but Castle, King and Daniels (1941) state that h i s t o l o g i c a l sections d i d not reveal any pathological condition within t h i s structure.  - 23 i 2. Peromyscus. Waltz lng. Waltzing has occur eel i n the Peromyscus similar to that which occurs i n the mouse and the r a t ,  Gruneberg (1947)  stated that waltzing Peromyscus tend to run i n c i r c l e s , often ao r a p i d l y t h a t they seem to be r o t a t i n g on t h e i r l e g s . The same mouse may rotate either right or l e f t .  Affected  mice may waltz with or without external s t i m u l i .  Being  placed i n a strange cage, the Jangling o f keys, etc. w i l l , stimulate a waltzer to o i r c l e ,  This recessive mutation varies  in the degree o f i t s expression according to the species, Thus, the head tremor i s most pronounced i n Peromyscus ermatus (catus mouse) but these individuals may not waltz.  The exprer  ssion o f t h i s mutation also varies within a subspecies.  Thus,  Peromyscus maniculatus a r t i s i n a e and P*m. b a i r d i i d i f f e r i n that the former show deafness except when very young, but the l a t t e r respond to sound at a l l ages.  When a cross was made  between waltzing mice o f these sub-species the hybrids obtained appeared normal, indicating that the waltzing mutations are caused by different hereditary f a c t o r s .  The  cause of t h i s abnormal behaviour i n Peromyscus has not been investigated .  84 3. Rabbit, A, Waltzer Waltzing rabbits have also been recorded by Gruneberg (1947),  The behavior o f these animals closely  resembles the condition seen in the house mouse.  In the  waltzing r a b b i t the vestibular and cochlea apparatus i s apparently normal, B, Shaking P a r a l y s i s . Another mutation produces shaking paralysis i n the rabbit*  Gruneberg (1947) states that the affected animals  when 1G days o l d develop a fine tremor which only ceases when they are at r e s t , i n the hind limbs.  f l a c c i d paralysis gradually develops  Gruneberg (1947) states that pathological  study showed a "degeneration o f nerve o e l l s i n the basal ganglia of the brain; the neo striatum i s poor i n b i g c e l l s and there i s a great s c a r c i t y of ganglion e e l l s In the pallidum."  These changes are apparently responsible for the  abnormalities produoed by t h i s mutation. C, Ataxia. Another mutation r e s u l t i n g i n a degenerative disorder Involving the brain stem o f the rabbit i s ataxia. This recessive mutation produces i t s abnormalities when the rabbit i s 58-86 days old*  It f i r s t expresses i t s e l f as a  -  25  clumsy g a i t , caused by the I n a b i l i t y of the legs to support the  body.  Later a coarse tremor of the head and neck develop,  accompanied by a l a t e r a l nystagmus. Gruneberg (1947) reports the occurrence o f degenerative  changes within the nervous systems of these mutant  animals.  The f i f t h nerve and i t s naol£«? show the f i r s t signs  of degeneration. Later changes were found in the cochlear fibers and t h e i r nuol&4 and to some extent i n the vestibular nerve.  Within the brain degenerative ohanges occurred i n the  trapezoid body, the l a t e r a l lemniscus and the vestibulocerebellar f a s i c u l u s .  Gruneberg (1947) states that  theBe  changes i n the brain and nerves account for the abnormal behavior produced by t h i s mutation since "changes i n the vestibular system and the medial lemniscus probably leads to loss o f normal sensation from the feet and causes postoral diffioultiesi" the  v  Involvement of the oerebellum accounts for  tremor o f head and neck, combined cerebellar and  vestibular lesions r e s u l t i n nystagmus•  - 26  4, Guinea-pig A. Waltzing. Waltzing i n the guineafpig i s produced hy a simple recessive f a c t o r .  The movements seen i n these animals  resemble those of the waltzing house mouse hut with a r e duction o f the circus movements. G i r d i n g i s predominantly in one d i r e c t i o n , hut the animals can reverse the movements. Head shaking i s common and the animals are deaf.  When these  animals are rotated they do not show nystagmus or compensatory head movements.  I f the waltzer can see when i t i s dropped  It w i l l land on i t s feet hut when blindfolded i t w i l l land on Its baek.  Gruneberg  (1947) states that the structure of the  cochlea i s these animals i s normal u n t i l they are ten days old*  A f t e r t h i s time degenerative changes ocour within the  stfta vascularis cochlea, the organ o f C o r t i and the ganglion spirals.  The vestibular part o f the ear i s apparently normal.  The c i r c l i n g and head shaking movements probably originate i n the central nervous  system.  B. Congenital Palsy. Congenital palsy i n the guinea-pig resembles j i t t e r y i n the house mouse and shaking paralysis i n the r a b b i t . It i s caused by a recessive juvenile l e t h a l mutation whieh produces a jerky p a r a l y t i c walk and a hypersensitivity to auditory stimulus. This mutation k i l l s the animal during the f i r s t week by producing hypoplasia o f the parathroid glands*  -27  -  Ohorlo Abnormalities in Man. Similar abnormal movements have been produced in man either by disease or by genetic aotion.  Some of ther>.  abnormalities which resemble those seen in the pirouette mouse, are given in Table II.  Destruction of the vestibular  labyrinth in man or its sensor)receptors within the brain results In a marked body sway preventing the maintenance of an erect and stable posture.  As the patient learns to com-  pensate, the body sway is lessened. Vertigo is produced by several abnormal conditions: an increase in the pressure of the endolymph of the inner ear; tumors of the csrebellopontine angle; inflammation of the geniculate ganglion; or degeneration of the superior cerebellar peduncles.  Cerebellopontine angle tumors also  produces tinnitus which is followed by gradual deafness* Menleres syndrome is characterized by attacks of vertigo and nystagmus accompanied by rotation of the body.  In more  advanoed cases tinnitus and gradual deafness occur. This syndrome Is produced by a dilation of the endolymph system, with a resultant increase of pressure within the inner ear which causes it to become extremely sensitive to slight changes of pressure producing disturbed functions of the labyrlnthene end organs.  -  28  Tonie-elonio spasms of the neck muscles occur In t o r t i c o l l i s , labyrlnthene t o r t i c o l l i s and central t o r t i c o l l i s • Torticollis  i s produced by mechanical misalignment and changes  in the c e r v i c a l region of the s p i n a l cord. t o r t i c o l l i s , as i t s name suggests,  Labyrinthine  i s due to i r r i t a t i o n  of  the semicircular canals on one side of the head while ceatral torticollis ganglia.  i s the r e s u l t of organic changes of the basal  Thus, the same symptoms can be produced by three  d i s t i n c t pathological changes. Abnormal movements of the trunk and limbs occur in L i t t l e s f  disease* Preldreiok's disease, Huntington's chorea,  Hereditary chorea, Wilson's disease, Multiple s c l e r o s i s * St. V i t u s ' dance, Parkinson's  disease and dystonia musculorum.  The congenital s t i f f n e s s of the limbs and spastic adduction of the thighs which r e s u l t s in the cross legged walk c h a r a c t e r i s t i c of L i t t l e ' s  disease has been found to be  caused by atrophy or s c l e r o s i s of the l a t e r a l columns of the s p i n a l cord.  Hereditary l e s i o n of the posterior columns and  pyramidal t r a c t s of the s p i n a l cord and degeneration of the cerebellar tracts and cerebellum r e s u l t in the ataxia, nystagmus, head tremor and asynergy of the arms seen in Preidreick's disease. The heredity jerky movements of the entire body produced by Huntington's chorea are the r e s u l t of d i s -  - 29 -  Integration o f the motor o e l l s within the basal ganglia.  The  pathological changes produced by hereditary chorea which only affects the muscles of the face and The  arms  i s as yet unknown.  inherited b i l a t e r a l athetoid movements of hands,  armB  and legs which increase on voluntary movements are seen i n Wilson's disease.  The pathological changes produced by t h i s  disease are a hobnail c i r r h o s i s o f the l i v e r and changes within the l e n t i c u l a r n u c l e i .  Degeneration of multiple  s c l e r o s i s r e s u l t s i n a spastic condition of the affected muscles and often produces a tremor of the head and upper extremities.  In St. V i t u s  1  dance tempory lesions In the  s u b c o r t i c a l regions produce coarse tremors which a r e lost during sleep.  Parkinson's disease r e s u l t s i n a rhythmical  tremor o f the r e s t i n g limb which i s lost during voluntary motion. Dystonia muscularum causes abnormal movements which closely resemble those seen i n the pirouette mouse • Patients having t h i s disease show i r r e g u l a r , involuntaryj clonio contortions of the trunk and proximal muscles o f the extremities.  These symptoms a r e l o s t when r e s t i n g but r e -  occur with voluntary motion.  The abnormal movements are  most pronounced when the patient i s walking and as he proceeds the body i s bent and twisted forward and sideways. These abnormalities  are caused by a progressive  degeneration  -  of the l e n t i c u l a r nucleus.  30  -  Thus, In the above diseases the  main pathological changes are found i n the l e n t i c u l a r n u c l e i of the b r a i n ,  The following table contains a summary of the  nervous diseases i n man whieh produce abnormal movements similar to those seen In the pirouette mouse*  - 31  -  TABLE I I DISEASES OF THE NERVOUS SYSTEM OP MAN  PRODUOINS ABNORMALITIES  SIMILAR TO THOSE PRODUCED BY THE PIROUETTE MUTATION DISEASE  SYNDROME  PATHOLOGY  Little's disease  Atrophy or s c l e r o s i s Congenital s t i f f n e s s o f the l a t e r a l columns of the limbs, crosslegged walk due to of the s p i n a l cord. spastic adduction of the thighs. Arks less involved than the legs.  Friedreich's disease  Hereditary ataxia nystagmus, head tremor and."nodding, asynergy of "both arms •  Lesion of the posterior columns and pyramidal t r a c t s of the s p i n a l cord and degeneration o f the cerebellar t r a c t s and cerebellum.  Huntington's disease  Hereditary, jerky, irregular movements of the entire body. Disturbed g a i t ; head, shoulders and arms twisted from side to side as patient walks. Dementia dev e lops gr adually •  Degenerative changes i n motor c e l l s of caudate nuoleus and the putman of the corpus striatum and of some pyrimidal cells.  Hereditary chorea  Only affects the muscles of the face and arms.  Wilson's disease (progressive lenticular degeneration)  Inherited b i l a t e r a l athetoid movements of the hands, arms, legs and face, emotional disturbance. Twistings are rhythmic and Inorease on voluntary movement. Limbs s p a s t i c .  undetermined  Hobnail c i r r h o s i s o f the l i v e r and changes in the l e n t i c u l a r nuolei.  - 38 SYNDROME  DISEASE  Symptoms varied, spastic conditions predominate. Ataxic tremor of the head and upper extremities and nystagmus occasionally occur.  Multiple sclerosis  ^PATHOLOGY Degeneration of mult i p l e areas of the brain mainly involving the motor centers.  St. V i t u s Infectious disease dance producing coarse (acute chorea)muscular twitchings, incoordination of muscles, ataxia which are lost during sleep.  Temporary l e s i o n i n subcortical region, perhaps the caudate nucleus •  Parkinson's disease (Paralysis agitans)  Rhythmical tremor o f the r e s t i n g limb.  Organic a f f e c t i o n o f the central nervous system.  Gerebellopont ine angle tumor  Tinnitus and vertigo followed by gradual deafness.  Tumor on cerebellopontine angle.  Torticollis;  A tonic-olonic spasm o f the neck muscles.  Occurs i n Pott's disease produced by c e r v i c a l changes and mechanical misalignment.  Labyrinthine Torticollis;  As above  Due to i r r i t a t i o n o f the semioircular canals on one side o f the head.  Central Torticollis  As above  Follows organic changes in the region of the basal ganglion*  1  33 DISEASE  SYNDROME  PATHOLOGY  Dystonia museularum (Tortlpelvis)  Abnormal constant s h i f t i n g tonus o f the muscles characterized by i r r e g u l a r , Involuntary, clonic contortions o f the trunk and proximal muscles o f the ext r e m i t i e s . Symptoms are l o s t when r e s t ing but reoccur with voluntary movement. Abnormality most pronounced i n walking when the body i s bent and twisted forward and sideways.  Produoed by progressive degeneration i n the l e n t i c u l a r nucleus* Pyramidal t r a c t s are normal. Reflexes sensation and mentality are normal. Muscles do not atrophy.  Meniere's syndrome  Characterized by attacks of vertigo and nystagmus. In advanced oases i t becomes associated with t i n n i t u s and increasing d i f f i c u l t y i n hearing. A sensation of r o t a t i o n of the body accompanies the attacks.  Gross d i l a t i o n o f the endolymph system and p a r t i c u l a r l y the saccule, which occupies nearly the entire v e s t i b u l e , The d i l a t e d soala media of the cochlea d i s places the Re is snap's membrane to the wall of the soala vestibule and thus o b l i t e r a t e s the perilymph space. This increase i n pressure causes the inner ear to be extremely sensitive and s l i g h t .changes i n pressure r e s u l t s i n disturbances i n the function of the labyrinthine end organs.  - 34 TV Materials and Methods A. O r i g i n of the Mice In January, 1949, two* house mioe homozygous for pirouette mutation were obtained from the Hosooe B* Jackson Memorial laboratory at Bar Harbour, Maine.  The stock b u i l t  up from these o r i g i n a l mice has been used i n t h i s Investigation into the physical basis of the pir.ouette mutation; B. Care o f Mice: 1. Cages Plywood cages measuring 24"~£l2  lt  divided by a central  p a r t i t i o n into two compartments were used to house the mice. A semicircular hole out i n the base of the p a r t i t i o n enabled the mice to pass from one side of the box to the other.  The  only opening through which l i g h t and a i r could enter the cage was by a window 3^X4"' covered with wire mesh.  Two wooden l i d s  f i t t e d over the top of the box allowed easy aooess for feeding and cleaning.  Twice weekly the mice were put into clean oages,  s o i l e d boxes were emptied, washed and thoroughly being used again.  4Med before  Water dishes were r e f i l l e d d a i l y , while  food containers were washed when the mice were moved into clean cages. The floor o f the oage was covered with dry peat moss. A Jar of water and a p e t r i d i s h of food were placed below the window and a small amount of cotton batting was put i n the other side o f the box.  Bight to ten mice were kept i n the cages  * 35  that were used for maintaining the stock.  Only two mice were  placed i n the breeding cages and the female was often isolated while she was r a i s i n g her young.  This method of housing kept  the mice warm and protected them from d r a f t s .  The room i n which  the cages were kept was heated during the winter months to prevent the mice from beooming c h i l l e d .  This method of care  and feeding proved to be s a t i s f a c t o r y u n t i l the room was r e quired for other purposes.  The mice were then removed to the  animal husbandry laboratory where they continued to receive s i m i l a r care. 3. Pood The mioe were given a balanced diet by feeding them fox cheokers or dog meal p e l l e t s .  In addition they were  frequently fed; grass, clover, groundsel and dandelions.  A  p l e n t i f u l supply of food and water was available at a l l times. 3. Records The ears of the mioe were marked by a series of holes and notches to f a c i l i t a t e I d e n t i f i c a t i o n .  The system of  numbering suggested by G.B. S n e l l was used In order to keep adequate records o f the mice*  By t h i s method the mice can be  numbered from one to ninety-nine. A record of each mouse was kept on a 4Z5* l i b r a r y card.  Its number, sex, mutations, date of b i r t h and death  were recorded on the card*  Observations, on the behavior o f the  mouse were also noted as well as matlngs and the number of litters  produced.  - 36 C* Preparation of M a t e r i a l . 1* K i l l i n g and Fixing* The mioe used for gross and h i s t o l o g i c a l studies were k i l l e d with ether. dissected  The parts required were immediately  out and fixed i n 10% saline formaldehyde E. H i s t o l o g i c a l techniques. 4.The l i v e r . Pieoes o f l i v e r , fixed i n formaldehyde, were  embedded i n p a r a f f i n .  Seotions 10^ t h i c k were stained with  either alum haematoxylin and eosin or Feulgen's nuclea* s t a i n and l i g h t green. b* The ear After f i x a t i o n in formaldehyde the inner ears of the mice were d e c a l j o i f l e d with either a 5$ solution o f n i t r i c acid i n 70$ alcohol or a 5$ aqueous solution of formic acid.  The l a t t e r method was found to be preferable  since  i t d i d not adversely effect the stains which were employed* The  ears were stained either before or after embedding i n  paraffin.  After the tissues were dehydrated i t was found  advisable to clear them with n-amyl acetate before embedding in p a r a f f i n since other olearing agents such as xylene and toluene made the tissues b r i t t l e and d i f f i c u l t to section. After, embedding i n p a r a f f i n the ears were cut into 15 * t h i c k and mounted on s l i d e s with egg albumen. 1  sections  - 37  -  S e v e r a l s t a i n s were employed to show the s t r u c t u r e s w i t h i n the ear. employed as a g e n e r a l s t a i n .  Alum haematoxylin and  March's osmic a c i d s t a i n was  was  connective  used to study changes  w i t h i n the mylen sheaths o f the nerve f i b e r s . s i l v e r s t a i n was  e o s i n were  Mallary's t r i p l e s t a i n  employed f o r a study o f the blood v e s s e l s and tissue.  various  Cajal*s  used on the normal nervous t i s s u e w i t h i n  the cochlea o f the mouse.  ~ 38  •  ¥ Comparison of the Behavior o f Normal and Pirouette Mice. A detailed comparison of the pirouette mice and normal mice was undertaken in order to determine the age at which the abnormalities appear and t h e i r e f f e c t on the behavior of the mutant mice.  Dickie and Woolley (1945-1946)  have described the general e f f e c t of t h i s imitation and have determined i t s p o s i t i o n on the t h i r d chromosome o f the house mouse• A . Adult Mice. When awake the pirouette mouse Is i n constant motion; nodding i t s head, moving r e s t l e s s l y around i t s cage and often c i r c l i n g .  The mutant mouse shows a head tremor  which increases to a spastic jerky head nodding as the mouse becomes more a c t i v e . shoulders  The head i s tossed back over the  and is often drawn s l i g h t l y to one side so that  the mouse appears to be constantly s n i f f i n g the a i r . I f the mouse i s held in the hands one can f e e l the trembling r muscles of the neck.  This tremor gradually increases u n t i l  the head i s suddenly p u l l e d back.  The neck muscles then  r e l a x before the tremor begins again.  Thus the head nodding  i s apparently due to involuntary tonio-olonic spasms of the neck muscles.  Pirouette mice are able to move along straight l i n e s as do normal mice; however, the mutant mouse frequently whirls or runs i n a c i r c l e .  The c i r c l i n g movement occurs more  often among the younger mice, and i s usually preceded by violent head shaking.  The mouse takes a few steps backwards  and then either whirls i n the same spot or describes a larger circle.  The c i r c l i n g lasts for varying lengths of time and  may be either clockwise  or anticlockwise.  Thus, the mixed  c i r c l i n g movement appears to be caused by an incoordination of fore and hind  limbs.  The auditory responses of both the normal and pirouette mice were tested by whistling, jangling of keys and banging of t i n l i d s .  When s u b j e c t e d t o t h e above s t i m u l i the  normal mice reacted i n s e v e r a l ways.  They usually respond  to whistling by p r i c k i n g up their ears and remaining motionless.  When tested w i t h louder n o i s e s they respond e i t h e r by  s t a r t i n g v i o l e n t l y and darting away or by remaining p e r f e c t l y quiet and often trembling.  The pirouette mouse did not r e r  spond to any of the above noises and i f i t was asleep the noises did not awaken i t . This evidence agrees with the r e port o f Woolley and Pickle (1945) that pirouette mice are always deaf. B. Development of abnormalities. A developmental study of the pirouette mouse revealed the age at whieh the above abnormalities occur* mouse i s indistinguishable  The mutant  from the normal animal u n t i l i t i s  * 40 ten days o l d . At t h i s age, a normal mouse tends to walk i n a straight line and seldom shows the c i r c l i n g movement; while a pirouette mouse of the same age moves more frequently in c i r c l e s and i s less steady on i t s feet than the normal mouse.  The equilibrium of the mutant mouse i s poor and i t  frequently loses i t s balance and r o l l s over on i t s back; When twelve days o l d the pirouette mouse takes longer to r i g h t i t s e l f than does the normal mouse. A fourteen day old pirouette mouse can be distinguished from the normal mouse since the l a t t e r then responds to sound while the former remains deaf. A t t h i s age the mutant mouse begins to show head shaking which increases i n i n t e n s i t y u n t i l the mouse is approximately eighteen days o l d . During t h i s period o f growth the c i r c l i n g movements occur very frequently.  This  t r i a r d o f symptoms gradually increases i n i n t e n s i t y u n t i l maturity i s reached j after which time the c i r c l i n g movements occur less frequently.  The development of the pirouette  mouse i s compared i n greater d e t a i l with that of the normal mouse i n the table on page 44. c. Comparison of Normal and Mutant Mice. A further comparison of the mutant and normal mouse of the same age (fourteen days) revealed that the pirouette mouse i s more active than the normal.  It i s constantly i n  motion and shows incessant head shaking which increases when  - 41 i t becomes e x c i t e d .  The p i r o u e t t e mouse does not respond  t o sounds b u t , , l i k e the normal mouse, shows response to a i r c u r r e n t s , changes i n t h e i n t e n s i t y o f l i g h t  and v i b r a t i o n s .  I t was noted that when a shadow was suddenly cast over the p i r o u e t t e and the normal mouse both responded by l e a p i n g i n t o the a i r and d a r t i n g away.  In t h i s respeot  p i r o u e t t e and the normal mouse d i f f e r  both the_  from the w a l t z i n g  mouse which never jumps. The  constant  activity  o f the p i r o u e t t e mouse can  be observed when i t i s p l a c e d on a t a b l e . around and appears to be e x p l o r i n g  The animal moves  i t s new environment.  It  runs a i m l e s s l y back and f o r t h on the t a b l e and w i l l  often  oircle.  I f i t comes i n contact w i t h a w a l l i t w i l l  attempt  to climb  i t . The normal mouse under the same c o n d i t i o n s i s  l e s s a c t i v e and moves c a u t i o u s l y about, o f t e n s t o p p i n g t o s n i f f the a i r or t o l i s t e n . The  locomotor a b i l i t i e s o f the p i r o u e t t e and normal  mice were t e s t e d t o determine the e f f e c t o f the mutation on the general behavior o f the mouse.  I t has a l r e a d y been r e -  p o r t e d that mice homozygous f o r t h i s mutation show head shaking, c i r c l i n g and deafness.  Adult  mutant mice are unable  to swim as was noted by D i c k i e and Woolley (1946). observation  Further  r e v e a l s t h a t normal adult mice when p l a c e d i n  water a r e able to swim d i r e c t l y to the edge o f the container and  climb  out o f the water.  I f a normal mouse i s submerged  -  42  i t r i s e s r a p i d l y t o the s u r f a c e  -  and commences swimming.  However, when an adult p i r o u e t t e mouse i s placed i t reacts  i n e i t h e r o f two ways.  i n water  I t may l o s e a l l sense o f  d i r e c t i o n and p o s i t i o n and t w i s t and t u r n under the water, i n which case i t w i l l q u i c k l y drown, or i t may manage t o swim to the s i d e o f t h e c o n t a i n e r .  |h the l a t t e r case i t  was found that i f the mouse became submerged i t l o s t a l l sense o f d i r e c t i o n and was unable to r e g a i n the s u r f a c e . A d d i t i o n a l tests indicated that p i r o u e t t e mouse was c a r e f u l l y p l a c e d  i f the adult  i n the water so t h a t  i t s head remained dry i t was u s u a l l y able to swim, but i f i t once l o s t  i t s balance i t was unable t o r e g a i n  of d i r e c t i o n .  i t s sense  I t was a l s o observed t h a t the p i r o u e t t e  mouse became f a t i g u e d r a p i d l y and i t s movements were not well coordinated.  The d i f f e r e n c e i n swimming movements was  n o t e d as e a r l y as the e i g t h day. A t t h i s age the p i r o u e t t e holds i t s hind  legs more s t i f f l y than does the normal mouse.  A f u r t h e r comparison o f the swimming movements o f t h e normal and p i r o u e t t i n g mice i s contained i n the t a b l e  i n the  following section. The vestigated.  r i g h t i n g r e f l e x e s o f these mice were i n I t was found t h a t when the normal mouse was  dropped i t always landed on i t s f e e t w h i l e the p i r o u e t t e mouse f r e q u e n t l y  landed on i t s back or s i d e .  Thus the  e q u i l i b r i u m o f these mice i s a l s o e f f e c t e d by the p i r o u e t t e mutation*  The mice were f u r t h e r t e s t e d by p l a c i n g them on  - 43 the wooden handle of the dissecting needle which was held horizontally.  The wild type mice were able to balance on the  s t i c k , and when i t was slowly revolved they were able to maintain t h e i r upright p o s i t i o n .  The pirouette mice were able  to balance on the s t i c k bat eould not maintain an upright position when the s t i c k was revolved. The mutant mouse clung t i g h t l y to the s t i c k at f i r s t but t i r e d r a p i d l y and f i n a l l y i t l e t go with i t s fore feet and hung momentarily and then f e l l to the table below.  In their study of the pirouette  mouse Woolley and Dickie (1946) discovered that the mutant mouse did not beoome dizzy when rotated. The main behavior changes produced by t h i s mutation are o f the same intensity i n both the males and females. The pirouette males are more aggressive than the wild type males. This tendenoy to fight r e s u l t s i n a higher mortality among them than among the wild type males.  Therefore, i t has been  found necessary to isolate the mutant males which were to be kept for breeding.  The female pirouette mice are less  aggressive than the wild type females. This comparison of pirouette and normal mice indicates the abnormalities produced by t h i s mutation and the time of their occurrence.  Deafness, c i r c l i n g , and head shaking are the  main distinguishing c h a r a c t e r i s t i c s of the pirouette mutation. The iKitant mice do not become dizzy when rotated, are unable to swim as easily and become fatigued more r a p i d l y than do normal mice.  Thus the changes produced by- t h i s mutation have  a manifold e f f e c t on the behavior of the pirouette mouse.  _ 44 _ TABLE I I I . AGE IN DAYS.  NORMAL MOUSE  PIROUETTE MOUSE.  1.  Although the mouse becomes fatigued r a p i d l y i t i s able to move i n c i r c l e s and show the normal righting reflexes. The c i r c l i n g appears to be due to incoordination o f the hind legs. When placed i n water the mouse makes swimming movements with Its fore legs but Is unable to keep i t s head above the water.  The behavior and r e actions o f the mutant mouse are indistinguishable from that o f the normal animal.  2.  The mouse i s able t o hold i t s head up as i t travels i n c i r c l e s Swimming Is the same as at 1 day.  Indistinguishable from the normal mouse.  3.  The mouse usually moves In c i r c l e s , however, i t can t r a v e l along a straight l i n e . The legs are more eo* ordinated when the mouse i s i n water than at 2 days.  Indistinguishable from the normal mouse.  4.  The mouse i s more active and i s able to t r a v e l either i n c i r c l e s or along a straight l i n e . A l though i t s t i l l swims In c i r c l e s , the hind limbs are used more often. The mouse i s able to hold i t s head above water  The movements are the same as seen i n the normal animal. In the majority o f the pirouette mice the d i s t a l end of the pinna of the ear i s s t i l l bent down.  - 45  AGE IH DAYS. 4.  -  ijieRMAl MOUSE  PIROUETTE MOUSE.  r  while swimming. The d i s t a l pinna of the ear which was bent down over the external auditory meatus i s now f r e e . Movements as above. Mouse i s more a c t i v e .  Movements as i n normal mouse. The d i s t a l end of the pinna has become f r e e .  6.  As at 5 days.  Same as normal mouse.  7.  C i r c l i n g movements s t i l l oecur and the coordination o f the fore-limbs i s s t i l l better than that o f the hind limbs.  Same as normal mouse.  8.  Mouse usually moves in c i r c l e s but when disturbed I t w i l l progress straight forward. The mouse swims with the head held high above the water. At t h i s age the sexes can be easily distinguished,  Movements similar to those of the normal mouse except when swimming. In the water the mutant mouse tends to hold i t s hind legs more s t i f f l y .  Locomotion as above. When dropped the mouse always lands on i t s feet.  Locomotion on land as in normal mouse. In water the pirouette mouse shows marked s t i f f n e s s and incoordination of the hind legs and one fore leg is used more frequently  -46AGE IN DAYS  NORMAL MOUSE  (PIROUETTE MOUSE.  'than the other. The mutant mouse swims i n c i r c l e s with i t s nose under water. When dropped i t does not always land on i t s feet.  9.'.  10.  The mouse c i r c l e s or walks i n a straight line and is able to ewim. Teats are v i s i b l e on the females.  Mouse moves i n circles; and i s unsteady on i t s feet. It swims with i t s head under water and i t s body tends to lean to one side. The mutant mouse becomes fatigued more r a p i d l y than the normal animal.  11.  Locomotion as above. When dropped i t lands on i t s f e e t .  Mouse i s unsteady on i t s feet and moves i n c i r c l e s . When dropped i t lands more often on i t s back or side than on i t s f e e t . Swims as above*  12.  Behavior as at 11 days.  Movements on land are the same as at 11 days. Swimming movements are less coordinated than above.  13.  Auditory meatus opens and the mouse responds to sound by making quick jerky movements. Eyes beginning to open.  Auditory meatus opens but mouse does not appear to be able to hear.  AGE IH DAYS  NORMAL MOUSE  MUTANT MOUSE  The mouse no longer walks i n c i r c l e s and is able to dart around r a p i d l y and swims e a s i l y . Its eyes are p a r t l y open and i t responds to sounds.  The mouse i s less steady bn i t s feet than the normal animal. It i s very active and c i r c l e s either to the r i g h t or l e f t . A s l i g h t head tremor i s v i s i b l e when the mouse i s walking. It loses i t s orientat i o n when placed i n water and r o l l s and twists below the surfaoe. Although the mouse does not respond to sound the ears are open. The eyes are p a r t l y open.  15.  Byes open, active, darts around the cage. Responds to sounds.  Eyes open, very active, c i r c l e r a p i d l y . It i s deaf. Head shaking more pronounced.  16.  As above  As above.  17.  As above  More active than the normal mouse, constantl y i n motion, frequent c i r c l i n g and violent head shaking.  18.  Able to feed and care for i t s e l f .  As above. How t r y i n g to feed i t s e l f .  19.  Mouse Is active, responds to sounds, swim£ e t c . It acts l i k e the adult normal mouse.  Mouse i s able to care for i t s e l f but i s s t i l l more active than adult pirouette mouse. Some animals show swimming movements but when they lose t h e i r balance they drown•  - 48  -  V f "Invest igat ion of the Possible Pauses for the Abnormal Behavior of the Pirouette Mouse* A review of the l i t e r a t u r e revealed many d i f f e r e n t physical causes for deafness and choreic behavior i n various animals.  Since nothing was  known of the physical basis of  the pirouette mutation, i t was  deemed advisable to investigate  a l l sources of abnormalities known to give r i s e to behavior similar to that found i n the pirouette mouse. A. Pathological Infection. It i s known that patholdgieal Infection of the inner ear w i l l produce deafness and. disturbances equilibrium in man(Jones, 1918),  of  The abnormalities  of these  mioe are evidently not due to i n f e c t i o n since mioe heterozygous for t h i s mutation, although r a i s e d among homozygous pirouette s l b s , w i l l not show any abnormalities.  Genetic  studies of the pirouette mutation, made by Woolley and Dickie (1945), indicate that the deafness and abnormal behavior seen in the mice is produced by some unknown inherited factor.  Thus pathological i n f e c t i o n as a cause of the  abnormal behavior is eliminated.  - 49 B. Muscular  _ Abnormalities;  The p o s s i b i l i t y that the abnormal movements of the pirouette mice were produced by the presence of muscular defects was'L-Investigated.  A study and comparison of the  gross muscular anatomy of the adult normal and pirouette mouse was made.  The origins and insertions as well as the  development of the muscles were noted.  This study of the  musculature did not reveal any s i g n i f i c a n t differences between the normal and the pirouette mice*  In a l l cases  the muscles of the mutant mice were found to be well developed* C. The L i v e r . The movements produced by the pirouette mutation are s i m i l a r to those produced by Wilson's disease in  man.  This disease caused a rhythmic twisting of the arms and legs which increases with voluntary movement. Associated with t h i s disease are pathological changes within the l i v e r , causing i t to become lumpy and discoloured.  This abnormal  condition i s r e f e r r e d to as "hobnail" c i r r h o s i s of the  liver  ( C e c i l , 1944). The pirouette mice show abnormal twisting which increases with voluntary movement.  Since this condition  resembles that of Wilson's disease i n man,  the l i v e r s of  <  -  50  *  normal and pirouette mice were compared.  Gross examination  revealed no difference between the l i v e r s of the normal and the pirouette mice.  Pieces of l i v e r tissue from both  pirouette mice and normal mice were prepared for h i s t o l o g i c a l examination as described on page 36.  Uo v a r i a t i o n be-  tween the l i v e r s of the normal and the mutant mice was observed.  From these r e s u l t s i t i s apparent that the  pirouette mutation, unlike Wilson's disease, has no v i s i b l e pathological effect on the l i v e r . D. The Brain. The abnormal movements exibited by the pirouette mouse suggest possible changes within the b r a i n .  A gross  comparison of brains of normal and pirouette mice was undertaken to determine whether there was an abnormal compression of the brain as reported in shaker-short; (Bonnevie 1936). cysts within the subarachnoid  space and absence of the  flocculus c e r e b e l l i as found i n E r e i s l e r : (Her twig, 1942 ). or y  hydrocephalus interims as i n shaker-1 (Gruenberg,1943).  A  study of the brain of the pirouette mouse d i d not reveal any of the above abnormalities.  The gross anatomy of the  pirouette brain appears to be i d e n t i c a l to that of the normal animal.  However, i t i s possible that a h i s t o l o g i c a l study  ustfing 'special staining techniques might reveal h i s t o l o g i c a l abnormalities.  - 51 B. Tumors * Abnormal movements and deafness similar to those produced by the pirouette mutation are known t o occur in man* Dr. Gibson suggested that the presence of tumors on the eighth nerve could be responsible for the abnormal movements and deafness which occurs i n the pirouette mouse since a similar syndrome i s found to be produced by von Recklinghausen^ disease (neurofibromatosis) i n man.  This disease i s pro-  duced by a dominant mutation which results i n the formation of tumors.  Gardner and Frazier (1931) report the inheritance  of b i l a t e r a l acoustic tumors on the eighth c r a n i a l nerve. The pressure exerted by these tumors causes degeneration of both the auditory and vestibular nerves r e s u l t i n g i n deafness and abnormal movements. The p o s s i b i l i t y of the formation of tumors upon the eighth c r a n i a l nerve of the pirouette mouse was i n vestigated.  The eighth nerve was followed from the ear to  the brain of the mouse but in no case was there any indication of a tumor.  The nerves of the brachial plexus of the mutant  mouse were also examined to determine I f tumors were i n t e r f e r i n g with any of the nerves in such a way as to r e s u l t in abnormal movements.  The r e s u l t s of t h i s study Indicated that  although subcutaneous tumors occasionally occurred in the pirouette mice they were not responsible for the abnormal behavior.  The incidence of tumors was found to "be the same i n both the old normal and the old pirouette mice. indicated that the abnormalities  These r e s u l t s  produced by the pirouette  mutation apparentlyv«re not produced by tumors.  P. The Bar. The pirouette mutation produces deafness, head shaking and c i r c l i n g s i m i l a r to that caused by the E r e i s l e r , shaker-short,  waltzer, and shaker-1 mutations i n mice, It  is known that the E r e i s l e r and shaker-short  mutation's cause  developmental changes which r e s u l t in l a t e r a l displacement of the ear vessieles while the waltzer and shaker-1 mutations produoe changes within the organ of C o r t i of the inner ear. Since i t seemfl l i k e l y that the pirouette mutation might also produce changes i n the ear the position and structure of the ear of the wild-type  mouse was studied and compared with  that o f the pirouette mouse. Before i t was possible to determine whether the ear of the pirouette mouse showed any abnormalities, i t was necessary to make a detailed study of the structure and function of the ear of a normal mouse since no l i t e r a t u r e was available on t h i s subject.  - 53  -  1. The Structure and Function of the Normal Bar. The general structure of the ear of the normal mouse i s similar to that of any other mammal. The  outer  ear is composed of a pinna and a tuhe, the external auditory meatus*  The sound waves are scattered by the  large pinna and some of them enter the external auditory meatus which directs them from the exterior into the temporal bone of the s k u l l where they come i n contact with the tympanic membrane.  The tympanic membrane separates  the external auditory meatus from the middle ear. a. The Middle Bar. The middle ear i s located in a hollow cavity within the temporal bone.  The sound waves set the tympariio  membrane in motion and i t s vibrations are transmitted through the middle ear by three small o s s i c l e s , the  malleus,  incus and stapes which form an irregular chain across the cavity of the middle ear.  The handle of the malleus i s  pressed against the ear drum while i t s head i s attached to the incus.  The  inner surface of the incus i s convex and i t  articulates with the stapes.  The vibrations of the ear  drum cause the malleus and incus to rotate together  and  produce a rocking motion of the stapes against the oval window which transmits the vibrations to the f l u i d of the inner ear* (see figure 1.)  -54-  THE MIDDLE EAR OF THE HOUSE MOUSE.  EUSTACHIAN  TUBE  TENSOR TYM PA Nf CAVITY OF AUDITORY BULLA TYMPANIC MALLEUS  MEMBRANE  INCUS STAPES COCHLEA INTERNAL  CAROTID ARTERY  FACIAL NERVE (VII) XI5  FIGURE |  This method of transmission reduces the strength of the vibrations picked up by the ear drum to about t h i r d of their o r i g i n a l strength (Beatty, 1932).  one-  Two  muscles which are attached to the ossicles further control the strength of the vibrations which reach the inner ear. Thus, when a loud sound h i t s the ear drum the tensor typani and.the stapedius muscles, which are attached to the malleus and stapes, contract by a r e f l e x movement which lessens the strength of the vibrations s t r i k i n g the inner ear.  The vibrations produced by the sound waves may  also  be conducted through the a i r found i n the hollow oavity of the auditory b u l l a , to the round window and hence to the perilymph of the inner ear; or they may  pass d i r e c t l y  through the temporal bone to the inner ear (Stevens and Davis, 1947). The a i r pressure within the middle ear is controlled by the eustachian tube.  This structure leads from the  anterior wall of the middle ear through the temporal bone to i t s opening in the nasal passages.  The posterior wall of  the middle ear communicates with the mastoid sinuses within the temporal bone.  f  - 56b. The Inner Ear* The  general structure of the inner ear of the  normal mouse i s similar to that of man.  It i s embedded i n  the petrous region of the temporal bone and is composed o f three parts, namely, the v e s t i b u l e , semicircular  canals  and cochlea, each o f which consists of an osseous and a membranous portion.  The vestibule and semicircular  canals  contain the organs concerned with equilibrium while the cochlea contains the organ of hearing.  The vestibule l i e s  medial to the middle ear, behind It are the semicircular canals and anterior to i t l i e s the ooohlea.  In the outer v.  wall o f the osseous vestibule i s found the fenestra o v a l i s . The  inner wall oontains the small auditory foramina through  which the vestibular branch of the eighth nerve passes from the internal meatus.  The anterior wall of the vestibule i s  connected to the membranous cochlea by the canal reunions. Into the posterior wall of the vestibule open the f i v e o r i f i c e s of the three semicircular canals,  (see figures 2&3).  d. Static Labyrinth The membranous l a b y r i n t h of the inner ear i s suspended i n a f l u i d , the perilymph, and contains l i q u i d known as endolymph.  another  The membranous vestibule i s  composed of two small sac-like structures; the upper one i s  -  57  FIGURES  -  28r3 (GRAY, 1949)  -58  -  the u t r i c u l u s , and the lower larger one i s the saeculus. The l a t t e r communicates with the ductus coehlearis of the cochlea by the canal reunions and with the u t r i c l u s by a narrow passage, the ductus utriculosaccular i s .  The  v e s t i g i a l saeculus endolymphaticus arises from a small opening at the base o f the saeculus.  The five o r i f i c e s o f  the semicircular canals open into the u t r i o l u s .  The semi-  c i r c u l a r canals are named acoording to t h e i r p o s i t i o n i n the head; anterior v e r t i c a l , horizontal and posterior v e r t i c a l . ( s e e figure 4 ) . The s t a t i c organs are located within the saeculus and u t r i c u l u s and also i n swellings, the ampullae, at the base of each of the semicircular oanals.  The sense organs  of the saeculus and u t r i c u l u s are the macula.  These  structures are composed o f clumps o f hair c e l l s whose short hairs project Into a gelatinous membrane, the o t o l i t h i c membrane within which are embedded the o t o l i t h s .  The  maculae u t r i c u l i l i e s on the l a t e r a l wall o f the u t r i c l u s while the maculae sacouli occupies the medial wall o f the saeculus so that the surface o f both maculae are perpendicular to each other.  The p u l l o f gravity upon the  o t o l i t h s act as the stimulus which i s transmitted by the nerve fibers to the brain and informs the animal of the position o f i t s head.  The s t a t i c organ o f each semioircular  -  59  FIGURE  -  4  60  canal, the c r i s t a acoustioa,  -  i s composed of groups of .hair  c e l l s whose long hairs project into the endolymphatic f l u i d of the canals.  These organs are stimulated hy agitation of  the f l u i d "brought about hy head movements. The vestibular portion of the eighth nerve enters Scapa s ganglia, whioh gives r i s e to three branches. ?  The  superior branch supplies nerve f i b e r s to the c r i s t a within the ampulla of the anterior and horizontal canals and the maculae of the u t r i c u l u s ; the i n f e r i o r branch sends fibers to the maculus of the saeculus, while the posterior branch serves the c r i s t a of the posterior semicircular  canal,  (see figures 5 and 6 ) . a  The Cochlea  The osseous cochlea appears as a tube which diminishes i n diameter as i t e o i l s around a central axis, the modiolus.  A bony shelf, the osseous s p i r a l lamina,  projects from the modiolus into the tube and  imperfectly  divides i t into two passages. (Refer figure 7 ) . The d i v i s i o n i s completed by the b a s i l a r membrane which stretches from the free edge of the osseous s p i r a l lamina to the s p i r a l cochlear outer w a l l .  ligament which i s attached to the  A third passage i s formed by the membrane of  Reissner which extends from the c r i s t a s p i r a l i s to the s p i r a l ligament. These three passages thus formed within  - 61 4  3  S  r,  v  v .,  II  i„  |f  F l O . 9 - 5 . — R i p h t h u m a n m e m b r a n o u s l a b y r i n t h , remove*! from its b o n y enclosure anil viewe.1 front the a n t c r o - l a l e r a l aspect. ((!. K e t z i u s i  F l O . 92G.—The same f r o m the po.stero-me.lial aspect. 1. L a t e r a l semicircular eaual; 1'. Us a m p u l l a : 2. I'oslerior canal; 2', its ampulla. 3. Superior canal; .'I'. its a m p u l l a . 4. ( W j o i i i e i l luiib of * u p c n . . r an.I posterior canals (sinus 5. I'triele. V liecessus ulrieiili. U". Sinus utrieuli posterior. Ii D u c t u s lymphaticus. 7. C a n a l i s utriculosaccular!*. S. N e r v e to a m p u l l a of suix-rior canal 9. X e r v v to a m p u l l a »rf lateral canal". 10. Nerve to recessus utrieuli ( i n Fiir. 925. the three branches a M s a r euiijoineil) ID'. Kiiiliui: o l nerve i n recessus utriculi. 11. Kaeial nerve. 12. I.atfena cochlea-. I.I Nerve of cochlea w i t h i n spiral l a m i n a II. Ila.iil.ir  utriculi tuprrior).  membrane.  15. N e r v e  fillers  to m a c u l a of saccule. Hi N e r v e to a m p u l l a of posterior canal. 17 Saccule. is 19. C a u a l i s reuniens 20. V e s t i b u l a r e n d ol d u c t u s lib-iris. 2.1. S e c t i o n i n t e r n a l acoustic meatus (tlie w p a r a t u K i bet we., i t f o i n i s n i l apparent i n tha  Secondary membrane of t.vinpaiiuiii. of the facial ami acoustic nerves w i t h i n section),  Retzius J  (GRAY, 1948)  FIGURES  5 8 6  flnli*  .62-  APEX  GALLERY  Fir,. !>. -Sectional view of the bony cochlea (I'oirier's  Anatomy)  (GRAY, 1948)  FIGURE  7  .63  -  the cochlea are the soala v e s t i b u l i , the ductus cochlear is and the soala tympani (See figure 8 ) .  The l a t t e r i s  separated from the inner wall of the middle ear by the membrane stretched across the fenestra rotunda.  The soala  tympani Is the lower passage within the cochlea while the soala v e s t i b u l i i s the upper passage.  These two passages  communicate with each other through a small opening, the helicotrema, located at the apex of the cochlea.  The  triangular ductus cochlear is i s the central passage, i t s three walls are formed by the basilar membrane, which separates  i t from the s c a l a typani; by the membrane o f  Relssner, which separates  i t from the soala v e s t i b u l i ; and  by the s p i r a l ligament which i s attached to the walls of the cochlea.  This passage follows the s p i r a l windings o f  the coohlea and ends b l i n d l y at the apex, while at i t s base i t communicates by the canalis reunions with the sacculus. The p e r i f e r a l end organs for hearing are located in the organ of C o r t i which r e s t s on the inner edge of the b a s i l a r membrane within the ductus cochlearis. The organ o f C o r t i consists of the rods of C o r t i , hair c e l l s and supporting c e l l s .  I t i s divided into an Inner and outer  portion by a triangular tunnel formed by two rows of rods, the inner and outer p i l l a r s or rods o f C o r t i , which r e s t upon the b a s i l a r membrane.  The inner rods of C o r t i are  - 64 -  DIAGRAMMATIC  LONGITUDINAL SECTION OF THE COCHLEAR  CANAL  SCALA VESTIBULI MEMBRANE OF REISSNER STRIA VASCULARIS .DUCTUS COCHLEARIS MODIOLUS SPIRAL LIGAMENT CRISTA SPIRALIS TECTORIAL MEMBRANE -BASILAR MEMBRANE ORGAN OF CORTI SPIRAL GANGLION OSSEOUS SPIRAL LAMINA AUDITORY NERVE SCALA TYMPANI  FIGURE 8  located at the point o f attachment of the b a s i l a r membrane to the s p i r a l lamina.  On the inner side o f the inner  rods and on the outer side of the outer rods are three or four rows of smaller external hair c e l l s and several rows of supporting c e l l s .  The terminal fibers of the acoustic  nerve end In contact with these hair c e l l s , which, according to Stevens and Davis (1947) are: "the ultimate sensory c e l l s of the organ of hearing. Above the hair c e l l s o f the organ o f Corti l i e s the t e c t o r i a l membrane which arises from the c r i s t a s p i r a l e s , looated on the inner edge of the osseous s p i r a l lamina. Prom the posterior part of the c r i s t a s p i r a l i s , just behind the o r i g i n of the t e c t o r i a l membrane, arises the membrane of Reissner. The periosteum forming the outer wall o f the ductus oochlearis becomes thickened to form the s p i r a l ligament whioh i s attached to the outer edge of the b a s i l a r membrane.  The upper portion of the s p i r a l ligament, the  s t r i a v a s c u l a r i s , contains small blood vessels and c a p i l l a r y loops.  This organ secretes the endolymph which nourishes  the organ of C o r t i .  This s p i r a l ligament decreases In size  from the vestibule to the hellcatrema, whereas the t e c t o r i a l membrane increases progressively i n size from the basal to the o p l c a l end of the cochlear canal. (See figure 9 ) .  T H E ORGAN  OF CCRTI F R O M A NORMAL (227  DAYS OLD)  MOUSE  BLOOD VESSEL STRIA VASCULOSA  CRISTA  SPIRALIS  TECTORIAL MEMBRANE HENSON'S C E L L S BORDER CELLS SPIRAL LIGAMENT PHALANGEAL C E L L S - P I L L A R S OF CORTI INNER HAIR C E L L OUTER HAIR C E L L S BASILAR MEMBRANE SPIRAL LAMINA  FIGURE 9  - 67  -  The auditory nerve and ganglia which serve the organ of C o r t i are located within the s p i r a l canal of the modiolus of the osseous cochlea.  In the normal adult  animal, each bipolar nerve c e l l of the s p i r a l ganglion sends one f i b e r along the edge of the osseous s p i r a l lamina to the hair e e l l s of the organ of C o r t i , while the other branch passes along the aeotuatio nerve to the b r a i n . According to Stevens and Davis (1947) the nerve c e l l s within the s p i r a l ganglion are not evenly distributed along the b a s i l a r membrane but are more densely congregated upper portion of the b a s i l a r whirl o f the cochlea.  i n the The  auditory branch of the nerve leaves the s p i r a l canal of the modiolus and joins the vestibular branch at the base of the internal acoustie meatus to form the eighth nerve.  This  c r a n i a l nerve leaves the internal acoustic meatus and passes medially to enter the brain at the base of the i n f e r i o r cerebral peduncle.  The fibers of t h i s nerve t e r -  minate in the dorsal and v e n t r a l cochlear n u c l e i within the brain. C» Post-natal Development of the Cochlea of a formal Mouse.  :  "  In mice^ as in other mammals, the structures within the cochlea of the ear have not attained their adult form at b i r t h but a l l of them are represented.  In the day old mouse  - 68 -  the s t r i a vascularis i s composed of three layers of c e l l s . In the s u p e r f i c i a l layer of c e l l s , the nuclei l i e near the exterior of the organ and the granular cytoplasm extends inwards.  In the basal layer of c e l l s the nuclei l i e along  the inner edge o f the s t r i a vascularis and the cytoplasm projects outward to the center of the organ.  The central  layer consists of irregular c e l l s with a network of blood vessels and spaces between them* At b i r t h the organ o f Corti appears as a layer of high columnar c e l l s whioh l a t t e r d i f f e r e n t i a t e into the hair c e l l s , rods of C o r t i and their supporting c e l l s .  The  upper surface o f these c e l l s come i n contact with the t e c t o r i a l membrane while t h e i r bases are separated from the basilar membrane by supporting c e l l s . The s p i r a l ganglion, within the modiolus i s composed of closely packed o e l l s having large round vesicular nuelfck and granular cytoplasm.  The nerve fibers  within the ganglia and those serving the organ of Corti are not v i s i b l e In the day old mouse. (See figure 1 0 ) . Five days after b i r t h the cytoplasm o f the superf i c i a l c e l l s of the s t r i a vascularis i s beginning to show the s t r i a t i o n s which produced the c h a r a c t e r i s t i c s t r i a t e d appearance of the adult organ.  The c e l l membranes of the  polyhedral e p i t h e l i a l c e l l s , which are arranged haphazardly  - 70  between the s u p e r f i c i a l and basal c e l l s , are not as d i s t i n c t as i n the day old mouse. At this age the organ of C o r t i has begun to d i f f e r e n t i a t e and the hair c e l l s and rods o f C o r t i can be distinguished* The nuclfe* o f the nerve c e l l s of the s p i r a l ganglia o f the 5 day o l d mouse do not appear to be quite as large as i n the day old mouse.  In some areas t h i n f i b r i l s  can be seen passing between the c e l l s and out towards the organ of C o r t i . m the twelve day old mouse the outlines of the c e l l s forming the s t r i a vascularis have become less and t h e i r cytoplasm has become more s t r i a t e d than at 5 days of age.  Many blood c a p i l l a r i e s as well as numerous blood spaces  can be seen passing between the c e l l s of t h i s organ*  The  organ of C o r t i was broken during sectioning but the remaining rods and hair c e l l s appear to have reached t h e i r adult form. The c e l l s o f the s p i r a l ganglia were closely packed and two types o f c e l l s can be distinguished.  The large  palyhedral nerve c e l l s have large round nuel£fc surrounded by granular cytoplasm.  The nuolet o f the small neuroglia c e l l s  are dispersed between the nerve c e l l s and t h e i r outlines are indistinct.  Fibers can also be i d e n t i f i e d as they pass  from the organ of C o r t i and between the c e l l s o f the s p i r a l ganglion.  - 71 -  No v a r i a t i o n was found to occur within the inner ears of the 15, 20, 37, 55, 60, 90, 104, 154, 262 and 390 day old normal mice.  Hence, the condition of the cochlea  of the 260 day old mouse can he taken as "being t y p i c a l . (See plate 1 ) .  At this age the s t r i a vascularis is well  developed and has a c h a r a c t e r i s t i c s t r i a t e d appearance.  The  three separate c e l l layers of the organ can he distinguished and numerous blood spaces and c a p i l l a r i e s can be seen between the c e l l s . (See figure 11 and plate I I ) .  The organ of C o r t i  is well developed and the rods and hair c e l l s are d i s t i n c t , and deeply s t a i n i n g .  The s p i r a l ganglia is composed of many  closely packed nerve c e l l s which have large nuclei;.  Their  dendrites can be seen passing to the organ of C o r t i while t h e i r axons can be followed into the auditory nerve within the modiolus. nerve c e l l s ,  The supporting c e l l s , have small nuel&c.  dispersed among the  Thus these two types of  c e l l s within the s p i r a l ganglion can be r e a d i l y distinguished by the size of t h e i r nuclfep.  Several blood vessels which A  nourish this organ can be seen passing between the c e l l s of the s p i r a l g a n g l i a . (See plate I I I , IV, V  t  and V I ) .  - 73 2* The S t r u c t u r e o f t h e Bar o f t h e P i r o u e t t e Mouse A f t e r having obtained a knowledge o f the s t r u c t u r e o f the ear o f a normal mouse the sources o f p o s s i b l e abn o r m a l i t i e s w i t h i n the ear o f the p i r o u e t t e mouse were examined.  S i n c e many c h o r i c mutations  s h o r t , w a l t z e r and shaker-1,  such as E r e i s l e r ,  shaker-  are known t o produce abnormal-  i t i e s w i t h i n the ear a c a r e f u l i n v e s t i g a t i o n o f the p i r o u e t t e ear was undertaken.  In order t o determine  when  the a b n o r m a l i t i e s f i r s t occured and whether there was p r o g r e s s i v e change and degeneration the ears o f p i r o u e t t e mice Of the f o l l o w i n g ages: 1, 2, 5, 12, 15, 20, 23, 25, 30, 35, 55, 60, 85, 100, 260 and 434 days were s t u d i e d and comp a r i s o n made w i t h the ears o f normal mioe o f approximately the same ages. a . P o s i t i o n o f the Bar Y e s a i o l e . A l a t e r a l displacement o f the ear v e s s l c l e s and mal-development o f t h e i n n e r e a r has been found t o occur i n both the E r e i s l e r (Hertwig, 1944) and shaker-short (Bonnevie,  1936) mutations.  Hence, the p o s i t i o n o f the  a u d i t o r y capsule o f the p i r o u e t t e mouse was I n v e s t i g a t e d . Q T O S B  d i s s e c t i o n s o f the ears o f both newly born and a d u l t  normal and p i r o u e t t e mioe were made and t h e p o s i t i o n and development o f the ears were compared.  In a l l cases the  74 -  location of the ear and the relationship of its various components were found to he the same in both the normal and the pirouette mioe, Thus, the abnormalities produced by the Zreisler and shaker-short mutations are not apparent in mice homozygous for the pirouette mutation.  Hence, another  cause must be sought for the abnormal behavior of the pirouette mice. b. The Middle Bar: The possibility of a defect within the middle ear was considered. An imperfect tympanic membrane, fixation or malformation of the ossicles or closure of the eustachian tube results In Impaired hearing but not In complete deafness. A comparison of these parts of theemiddle ear of both normal and pirouette mioe did not reveal any visible abnormalities. Hence, the deafness produced by the prlouette mutation would appear to be the result of changes within the inner ear or the central nervous system of the mouse.  c. The Inner Bar. The gross anatomy of the inner ear of the pirouette mouse 1B identical to that of the normal animal. (Refer page 53 ).  i The S t a t i c Labyrinth Abnormalities  in the development of the vestibular  system have been recorded i n the waltzer,(Yerks 1 9 0 2 ) , shaker-short (Bonnevie 1936) mutations.  and  E r e i s l e r (Hertwig,  It i s also known that, in man,  1 9 4 4 ) _  the occurrence of  pathological changes within the vestibular system eauses disturbances  of equilibrium (Jones, 1 9 1 8 ) *  Normally when  the s t a t i c labyrinth is stimulated i t causes nystagmus as well as reaction movements of the body.  I f the end organs  of the semi-circular canals have been impaired or destroyed these responses do not occur.  Since the pirouette mouse  did not become dizzy when rotated and does not show nystagmus after r o t a t i o n i t appeared possible that the vestibular system was  defective*  Gross examination of the  osseous labyrinth, in whioh the vestibular system is located, did not reveal any abnormalities ment of the semicircular canals.  in the position or developMicroscopic  study of this  system indicated that unlike the waltzer, shaker-short and E r e i s l e r mutations, the pirouette mutation does not produce changes in the shape or position of the vestibular system. Soapa's 'ganglion and the maculae and cristas within the vestibular labyrinth of the 1 2 , 1 5 , 2 0 , 2 3 , 3 0 , 3 5 , 5 0 , 55, 60 and 100 day old pirouette mice were examined,  m all  cases the end organs of the vestibular system were well developed.  The nerves leaving these structures were traced  to Soapa s ganglion. 1  When Mallory's s t a i n was employed no  v a r i a t i o n was observed i n the branches of the vestibular nerve in normal and pirouette mice.  However, Marchi's osmic  acid s t a i n revealed some signs of degeneration within the myelin sheaths o f the vestibular nerve of a twenty day old mutant mouse.  In t h i s case the osmic acid had blackened the  majority of the myelin sheaths but many of these appeared unbroken and evenly stained and d i d not show the broken nature t y p i c a l of the degenerating sheaths.  The few de-  generating myelin sheaths were traced to the c r i s t a s and maculae. normal.  These end organs, however, appeared  completely  The Marohi method was also used in staining the  nerves of 15, 23, and 60 day old mice but none of these animals showed any signs of degeneration. Scapa's ganglion was well developed and showed no signs of degeneration i n any of the ears examined.  Cabal's  s i l v e r s t a i n was employed to study the nerve c e l l s of Seapa's ganglion i n a 60 day old pirouette mouse.  This method of  s t a i n i n g f a i l e d to reveal any abnormalities within Scapa's ganglion. i i The Cochlea. The waltzer (Yerks, 1907) and shaker-1  (Gruneberg,  Hallpike and ledoux* 1940) mutations are known to produce changes within the cochlea of the ear.  Hence t h i s region was  investigated to determine i f the pirouette mutation produced similar abnormalities. A comparison of the cochlea of normal and pirouette mice did not reveal any differences in the general location of any of the structures within the cochlea.  The size of  the scala were approximately the same and no evidence of any dilation or compression within the cochlea of the mutant mice was observed.  The position of the various  structures arising from the modiolus and spiral ligament was similar in both mutant and normal mice.  The membrane of  Reissner and the tectorial membrane of the pirouette mouse are identical to those of the normal animal. However, abnormalities do occur within the stria vascularis, the organ of Corti and the spiral ganglion of the pirouette mouse. (See figure 8). In the adult pirouette mouse (60 days) these abnormalities are very pronouneed (see plate V I I ) .  in the  basal and oentral whirls of the cochlea the cytoplasm of the cells forming the stria vascularis has lost its striated appearance.  The amount of cytoplasm has apparently been  reduced and vacuoles in the oells as well as clear spaces between them were frequently present, (see figures 12 & 13).  - 78 -  - 80 -  In the organ o f Corti the degeneration pronounoed i n the "basal whirl of the cochlea.  i s most  In t h i s region  the rods of Corti are apparently normal hut the outer hair o e l l s and supporting c e l l s have become less d i s t i n c t and are not upright as i n the normal animal. (See plate V I I I ) . The degenerative the s p i r a l ganglion.  changes are most pronounced within  In the basal whirl of the s p i r a l  ganglion the majority of the nerve c e l l s have dropped out and the structure appears to be composed mostly of t h i n f i b e r s , small scattered nuel& o f the neuroglia c e l l s and a-few blood ;  vessels.  (See figure 14 and plate IX, X^ and XI.) d. Post-natal -Development of the Cochlea o f the Pirouette Mouse. Since a h i s t o l o g i c a l study of the cochlea o f an  adult pirouette mouse revealed abnormalities within the s p i r a l ganglion, stifia vascularis and organ of Corti a developmental investigation was undertaken to determine the age at which these changes occur.  A summary o f the r e s u l t s  of t h i s investigation Is given In the appendix. This developmental study of the structures within the cochlea of the pirouette mouse revealed several significant facts.  At 1, 5, IE and 15 days after b i r t h the  structures within the eochlea of the pirouette mouse are" i d e n t i c a l to those found i n the normal animal.  Changes  it)  "  SPIRAL GANGLION' OF A PIROUETTE MOUSE (35 DAYS)  CRISTA SPIRALIS  SPIRAL  GANGLION  1 NERVE C E L L  NEUROGLIAL C E L L  BLOOD VESSEL  FIGURE 14  CD  -82  -  within the s p i r a l ganglion were f i r s t observed i n the basal whirl of the cochlea o f a 20 day o l d mutant mouse.  In t h i s  region the c e l l s o f the ganglion were not as c l o s e l y packed and the number o f nerve o e l l s had been reduced.  These de-  generative changes within the s p i r a l ganglion become more pronounced as the mouse becomes older.  The loss of the  nerve c e l l s within t h i s ganglion commenced i n the basal whirl of the cochlea and gradually progressed (See table V.)  to the apex.  The number of n u c l e i within a given area of  s p i r a l ganglia of normal and mutant mice was oounted to determine the extent of the nuclear l o s s .  A Howard mold count  disc was used i n making these counts and the number of nuclei within a single square was recorded.  In a l l specimens  n u c l e i of comparable regions were counted and the numbers averaged. occurs  These counts indicated that the greatest loss  i n the basal whirl of the cochlea and the a p i c a l whirl  is least affected (See table V ) . Counts were also made of the large and small n u c l e i within the basal whirl of the cochlea of normal and pirouette mice to determine which type o f n u c l e i was being l o s t . (Refer table VI.) The r e s u l t s of these counts indicate that there i s a progressive loss of the large n u c l e i while the small n u c l e i remain f a i r l y constant.  Cajal's s i l v e r s t a i n  indicated that the large n u c l e i were those oi the nerve c e l l s while the small n u c l e i belonged to the neuroglia c e l l s . ( p l a t e X)  - 83 -  TABLE  V .  Number o f n u c l e i In t h e b a s a l , c e n t r a l and a p i c a l w h i r l s o f the s p i r a l g a n g l i o n i n 1 square o f an o c c u l a r g r i d counter. A. Normal Mouse. BASAL WHIRL. 1 5 IE 15 SI 35 60 104 S6S 390  • CENTRAL WHIRL. * 48 46 40 42 43 41 40 42 45 48  50 45 40 49 58 56 49 54 59 53  APICAL WHIRL.. 47 48 45 40 37 36 44 43 50 47  B. P i r o u e t t e Mouse* A  1AYS 1 5 12 15 SO S3 85 30 35 55 60 100 860 434  W  BASAL  WHIRL. 49 40 42 48 44 47 45 36 37 17 34 37 39 30  CENTRAL WHIRL. 46 44 45 43 47 34 37 34 34 31 36 33 33 41  APICAL WHIRL. 48 41 43 39 48 42 46 35 36 37 38 41 34 39  - 84 -  TABLE VI Number o f s m a l l land l a r g e n u c l e i i n t h e b a s a l w h i r l o f the s p i r a l g a n g l i a i n 1 square o f an o c c u l a r g r i d counter. A. Normal Mouse. AGE IN DAYS  •  LAP.GS NUCLEI. . 13 19 33 34 25 21 33 34  12 15 21 35 60 104 262 390  SMALT, NUCLEI. . 33 30 25 ' 32 27 33 37 28  TOTAL 46 49 58 66 52 54 70 62  B. P i r o u e t t e Mouse. ^DAYS 12 15 20 23 25 30 35 55 60 85 100 260 434  3  LARGE NUCLEI. 13 16 14 27 12 9 7 1 8 11 ii 5 2  SMALL NUCLEI. 32 34 32 2Q 33 29 22 16 34 18 16 24 18  TOTAL 45 50 46 47 45 38 29 17 42 29 27 29 20  - 85 Changes within the s t r i a vascularis were f i r s t observed i n the basal whirl of the cochlea o f the 23 day old mutant mouse. The  cytoplasm of the s t r i a vascularis gradually l o s t i t s  normal s t r i a t e d appearance and became granular.  As the  mouse became older (35 days) there was a s l i g h t reduction i n the amount.of oytoplasm but the number of n u c l e i remained approximately the same as i n the normal animal.  This r e s u l t s  in the c e l l s of the s t r i a vascularis of the pirouette mouse not being as compactly arranged as those of the normal animal.  The blood vessels which serve this organ appear to  be normal except i n the 434 day old mutant mouse in which the entire s t r i a vascularis was reduced to a t h i n layer o f undifferentiated t i s s u e . It was not possible to trace the early progress of the degenerative  changes within the organ of C o r t i of the  mutant mouse since t h i s structure was frequently broken during the preparation of the microscope s l i d e s .  However  changes within the organ of Corti were observed i n the 55 day o l d pirouette mouse.  In the basal whirl of the cochlea  the outer hair, c e l l s and supporting c e l l s did not stand as upright as i n the normal mouse.  These changes within the  organ o f Corti were more pronounced In the 60 day old mutant mouse.  In the 85 day old mutant mouse the rods o f  Corti were normal but the hair c e l l s are less d i s t i n c t a&d  -  86 -  t h e i r nuclei, do n o t s t a i n as. deeply as i n the 60 day o l d animal. the  In the o l d e s t p i r o u e t t e mouse examined (434)days)  organ o f C o r t i w i t h i n the "basal w h i r l o f the c o c h l e a was  reduced t o a mass o f u n d i f f e r e n t i a t e d t i s s u e .  In t h e c e n t r a l  w h i r l only the rods o f C o r t i could be r e a d i l y d i s t i n g u i s h e a while at the apex the h a i r e e l l s s t i l l remained although they showea s i g n s cochlea  of aegeneration.  Thus the changes w i t h i n t h e  became p r o g r e s s i v e l y more pronounced as the age o f  the p i r o u e t t e mouse i n c r e a s e d .  (See appendix).  -87  -  G . The S k u l l . Mellanhy (1938 & 1843) observed that vitamen A deficiency i n dogs produced deafness and choric movements similar to those found i n pirouette mice.  He found that  vitamen A deficiency produced bone overgrowth which r e sulted i n increased i n t e r c r a n i a l pressure and a stretching and compression of the c r a n i a l nerves.  This osseous  pressure upon the eighth nerve produced degeneration of the s p i r a l ganglion similar to that found in the pirouette mouse. Hence, the heads of normal and pirouette mice were sectioned l o n g i t u d i n a l l y through the s a g i t t a l suture of the s k u l l so that the r e l a t i o n of the c r a n i a l bones to the brain could be observed.  Measurements were also made o f  the thickness of some of the bones (see tables VI & V I I ) This investigation of the bones of the s k u l l and their r e l a t i o n s h i p to the brain d i d not reveal any s i g n i f i c a n t differences between the normal and pirouette mice.  However, i t i s possible that s p e c i a l techniques  may reveal regions of bone overgrowth i n the foramena of the c r a n i a l nerves.  - 88 TABLE.VII Measurements o f Bones o f the S k u l l . Adult Normal Mouse. LEEISTH OF PARIETAL BONE .  89.7 85.7 90 86.5 87.5 85.f 83 77 80 82 Average.  81  DEPTH OF FRONTAL PARIETAL SUTURE .  .  .  PARIETAL BONE  77 7.5 7 9 10.5  8 3  7.7 8 . 9 8 8 6  .  .  DEPTH OP PARIETAL OCCIPITAL SUTURE -  9 8|  6 7 7 7  8  7  H  4 4 5 5 5 4 3£ 4  4*  4  4  TABLE V I I I . A d u l t P i r o u e t t e Mouse. LENGTH 6P PARIETAL . BONE _  94.5 87.7 95.5 95.5 90 77 82 80 85  DEPTH OP FRONTAL PARIETAL SUTURE -  8 7 7.7 7.7 8 9 7 7 8  Aver- -  age.86  8  DEPTH OP PARIETAL OCCIPITAL SUTURE -  9.5 9.5 8.7 7 7 6 7 8.5 7.5  :  PARIETAL BONE .  4.5 4.3 4.5 4 4.5 4 4 4.5 4.5  - 89 VII Discussion. The degenerative changes observed within the cochlea of the pirouette mouse correspond closely to those de>scrlbed by Gruneberg, Hallpike and ledoux (1940) i n the cochlea of the shaker-1 mouse.  In both these mutant animals  the structures within the cochlea were apparently normal twelve days after b i r t h .  Thus these mutations cause morbid  anatomical processes within a f u l l y developed organ.  However,  the time at which these degenerative ehanges occur within the pirouette and the shaker-1 d i f f e r .  The shaker-1 mouse  did not become deaf u n t i l It was 22-30 days o l d whereas the pirouette mouse was deaf throughout i t s entire  life.  In the cochlea of the shaker-1 mice abnormalities were f i r s t observed i n the s t r i a vascularis then In the organ of Corti and f i n a l l y within the s p i r a l ganglion.  The  degenerative changes within the s t r i a vascularis resulted in a lose of cytoplasm with a r e l a t i v e increase of the n u c l e i and vascular spaees.  In the organ o f Corti the de-  generative changes commenced i n the outer hair c e l l s and progressed Inwards toward the rods of C o r t i , u n t i l the entire organ was reduced to a small and compact h i l l o c k of undifferentiated t i s s u e .  Changes i n the density of the  ganglionic tissue were observed within the s p i r a l ganglion. This thinning of the ganglion c e l l s became more pronouneed as the mouse became older.  Gruneberg, Hallpike and Ledoux  90 (1940) suggest that changes within the s t r i a vascularis could result i n starvation of the c e l l s forming Corti*s organ and that t h i s , i n turn, would r e s u l t i n degeneration of the nerve fibers and ganglion serving t h i s organ. In the pirouette mouse changes were observed within the s p i r a l ganglion 20 days after b i r t h but the s t r i a vascularis was apparently normal and did not show signs of degeneration u n t i l the mouse was 23 days o l d . This would suggest that although the same structures degenerate in both pirouette and shaker-1 mioe these degenerative changes do not seem to be caused by the same primary factor nor do they occur i n the same order. Degeneration o f the s p i r a l ganglion, organ of C o r t i and s t r i a vascularis also occurs i n the Japanese waltzing mouse. According to Gruneberg (1943) t h i s mut a t i o n r e s u l t s i n a reduction i n the size of the s t r i a vascularis, s l i g h t degeneration o f the hair c e l l s within the organ of Corti and degeneration of the s p i r a l ganglion.  He  states that van Lennep suggests that a degeneration o f the s t r i a vascularis would r e s u l t i n changes within the organ of C o r t i and secondary degeneration within the s p i r a l ganglion On the other hand Gruneberg reports that Yasuhara (1935) found no degeneration i n the organ o f C o r t i and regards the degeneration of the s p i r a l ganglion as primary.  Thus the  exact mature, extent and sequence o f the physical changes  /  - 91 in the waltzing mouse are s t i l l contraversial.  However the  changes occuring i n the pirouette mouse are similar in sequence to those reported hy Yasuhara. Inherited ataxia i n the rabbit produces similar changes within the inner ear.  Gruneberg (1947) states that  t h i s mutation results i n changes within the cochlear nerve fibers and their nuclei and also to some extent within the vestibular nerve.  The source and nature of these abnormal  changes are as yet unknown. The waltzing mutation i n the guinea-pig produces degenerative changes within the s t r i a vascularis, organ of Gorti and l a t e r i n the s p i r a l ganglion. This mutation does not produce changes within the vestibular nerve.  Gruneberg  (1947) suggests that these ehanges are due to lesions i n the central nervous system.  Since i n pirouette mice the  degenerative changes occur f i r s t i n the s p i r a l ganglion i t suggests that the primary change may likewise be due to lesions i n the central nervous  system.  The waltzing mutation i n Peromyscus also produces degenerative changes within the s p i r a l ganglion of the cochlea.  In this case the degeneration of the cochlear  nerve was fou#d to be caused by b i l a t e r a l tumors on the eighth c r a n i a l nerve.  In man, similar degenerative changes  within the s p i r a l ganglion are produced by the presence of tumors on the eighth c r a n i a l nerve.  Since no tumors  - 92 were present on the eighth nerve in the pirouette mouse, the primary degenerative changes must have another A r t e r i o s c l e r o s i s of the labyrinth i n man  cause* is known  to produce degeneration of the s t r i a vascularis, organ of Gorti and s p i r a l ganglion.  When the "blood supply passing  through the internal auditory artery i s p a r t i a l l y or wholly cut o f f atrophy occurs within the s t r i a vascularis, organ of C o r t i , s p i r a l ganglion and nerve endings within the vestibular system.  These structures are replaced hy de-  posits of connective t i s s u e . The "blood supply to the organs of the inner ear was  imvestigated i n the pirouette mouse and was  similar to that of the normal animal.  found to he  Even after degener-  ation had occurred the affected structures had a normal blood supply.  Hence, i t is u n l i k e l y that degenerative  changes are produced by a l t e r a t i o n of the blood supply to the inner ear. In man,  otosclerosis, which i s produced by a  dominant mutation, r e s u l t s i n the deposition of soft spongy bone within the inner ear (Eyle, 1907).  These abnormal  deposits of spongy bone exert pressure upon the nerves serving the inner ear and r e s u l t i n degeneration of the eighth c r a n i a l nerve.  Similar degenerative changes were  reported by Mellanby (1938, 1943) who  observed that^in dogs,  a vitamin A deficiency produced bone overgrowth which  resulted  in a stretching and compression of the eighth nerve.  The  abnormal pressure caused the nerve fibers of the cochlear d i v i s i o n of the eighth nerve and the s p i r a l ganglion to degenerate. In severe cases a l l the nerve c e l l s within the s p i r a l ganglion were lost and a newly formed bone was observed within the mftdiolus.  Many of the nerve fibers of  the vestibular portion of the eighth nerve were destroyed but Scapa's ganglion unlike the s p i r a l ganglion, was more resistant and i n most oases remained unchanged.  Examination  of the cochlea of the pirouette mouse revealed similar degenerative changes within the s p i r a l ganglion but no abnormal deposits of spongy bone were observed within the modiolus. However, the p o s s i b i l i t y of an overgrowth of bone occurring within the foramen of the eighth nerve has not been i n * vestigated. Experimental studies have shown that severance of the eighth nerve w i l l cause degeneration of the s p i r a l ganglion although the p e r i p h e r i a l vestibular neurons show a comparative absence of degenerative changes.  Hallpike  and Rowdon-Smith (1934) report that after severance of the eighth nerve and the internal auditory artery there was a complete loss of nervous elements, sensory epithelium and membranes i n both the cochlea and the vestibular labyrinth and frequently new bone was observed.  When the nerve was  severed but the artery remained intact the rods and hair  - 94 c e l l s of Corti's organ were well preserved and the vestibular system showed s l i g h t changes although l i t t l e remained of the nerve fibers and c e l l s within the s p i r a l ganglion.  The central canal of the modiolus and the canal  of Rosenthal were occupied c h i e f l y by loose connective tissue and remnants of the nerve c e l l s . Degenerative changes similar to those produced by severance of the eighth nerve were observed within the cochlea of the pirouette mouse.  Since the s p i r a l ganglion  of the mutant mouse showed degenerative changes before any abnormalities were observed i n the s t r i a vascularis or i n the organ of C o r t i , i t seems possible that the primary change originates i n -either the eighth c r a n i a l nerve or within the central nervous  system.  - 95 -  VIII  COBTCLTJSIOH.  Mioe homozygous for the pirouette mutation show c h a r a c t e r i s t i c c i r c l i n g and head shaking movements when they are fourteen days o l d .  ffiese mioe are apparently deaf  throughout their entire l i v e s . A survey of the l i t e r a t u r e revealed many possible sources of abnormalities which could produce the atypical behavior of the pirouette mouse.  These reported sources of  abnormalities were investigated i n the pirouette mouse with the following r e s u l t s : 1.  Muscles of the head, neck and fore-limbs -- normal.  £.  l i v e r - gross and h i s t o l o g i c a l structure normal.  3.  Brain >- gross anatomy - normal.  4.  S k u l l - gross anatomy - normal.  5.  Eighth nerve •* gross and h i s t o l o g i c a l studies revealed, no tumors.  6.  Ear - gross anatomy of middle and inner ear, and blood supply normal.  7.  Oochlea n h i s t o l o g i c a l studies showed abnormalities in the following structures:  - 96 a.  S p i r a l ganglion - Loss of nerve c e l l s observed i n basal whirl of ganglion at 20 days after b i r t h . Loss of c e l l s progresses from base to apex and becomes progressively more pronounoed with Increasing age ( r e f e r chart A)  b.  S t r i a vascularis S t r i a t i o n s not as pronounced as In normal mouse at 23 days. Cytoplasm becomes more granular, c e l l s less compact, (refer ehart A ) .  c.  Organ of C o r t i Outer hair c e l l s less upright in 55 day o l d pirouette, loss of d l s t l n e t appearance. Ded i f f e r e n t i a t e s f i r s t i n basal w h i r l . At 260 days organ of Corti i n basal whirl a mass o f undifferentiated tissue (refer chart A ) .  These changes are similar to those reported i n waltzing and shaker-1 mice, but the time of onset and sequence of the degenerative  changes are d i f f e r e n t .  The extensive loss o f nerve c e l l s within the s p i r a l ganglion l a t e r followed by degenerative  changes i n the s t r i a  vascularis and organ of C o r t i suggests that the cause of these changes l i e s outside the cochlea. degenerative  The fact that the  changes of a l l structures are found f i r s t and  most extensively i n the lower whirl and later i n the middle and upper whirls, also supports the contention that the ohahge i s external to the cochlea and gradually Involves the whole organ.  Similar changes are known to be produced by  - 97 tumors, peseous compression, severing o f the eighth nerve or lesions in the central nervous system.  Since no tumors  were present and the eighth nerve was intact, there remains only the p o s s i b i l i t i e s of osseous compressi:on or lesions within the brain causing t h i s abnormality.  , 98 -  I X Summary. A r e v i e w o f the l i t e r a t u r e i n d i c a t e d s e v e r a l p o s s i b l e sources o f a b n o r m a l i t i e s which would account f o r the deafness and abnormal movements seen i n the p i r o u e t t e mouse. T.  The f o l l o w i n g s t r u c t u r e s were found t o be normal, i n  the p i r o u e t t e mouse.  II  A.  Muscles of the head, neck and t h o r a x .  B.  Liver.  C*  Absence o f tumors on the e i g h t h n e r v e .  D.  P o s i t i o n o f the ear v e s s i o l e s .  E.  Development o f the middle e a r .  ]?.  Osseous l a b y r i n t h o f the inner e a r .  G.  P o s i t i o n o f the s t r u c t u r e s w i t h i n the c o c h l e a .  Changes were found in the f o l l o w i n g s t r u c t u r e s w i t h i n  the cochlea o f t h e p i r o u e t t e mouse. A.  S p i r a l ganglion .1. Loss o f nerve c e l l s occurred in, ... the b a s a l w h i r l o f the g a n g l i o n at 20 days and became p r o g r e s s i v e l y more pronounced with I n c r e a s i n g age. The l o s s o f c e l l s commenced i n the b a s a l w h i r l and progressed t o the apex o f the cochlea*  B.  Stria vascularis S t r i a t l o n s not as pronounced as i n the normal mouse at 23 days. Cytoplasm becomes more granular and c e l l s are l e s s compact. These changes more pronounced In the b a s a l than the o p i c a l w h i r l s o f the c o c h l e a .  99 -  Organ o f C o r t i Outer h a i r c e l l s show degenerative changes i n the 55 day o l d mouse. 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Howell,  W.H.  1946  A Textbook of Physiology, Philadelphia, W.B.  Saunders.  - 106 H u e s t i s , R.H.  and Bart o,  1936  An  -  E.,  i n h e r i t e d tremor i n Peromyscus,  J o u r n a l o f H e r e d i t y , V o l . 27, p. 436-438* Hunt,  H.R. 1936  A L a b o r a t o r y Manual o f the Anatomy o f the Rat, New  York, The Macmillan  Go.  Jones, I . H. 1918  E q u i l i b r i u m and V e r t i g o , P h i l a d e l p h i a , Lippincott  Co.,  l a p p e r s ^ A.C.V., Huber, C.6. 1936  The  and Crosby,  Comparative Anatomy o f the Nervous  System o f V e r t e b r a t e s New Keeler,  E.C.  Including  York, The Macmillan  Cp.  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P i 1946  Conditional Reflexes, An Investigation of the Physiological a c t i v i t y of the Cerebral CorteXi London, Oxford University Press*  Peterson, E.W., Mac Goxm, H.W.,  MeCulloch, W.S. and  Lindsley, D.B. 1949  Production of posterior tremor, Journal of Neurophysiology, S p r i n g f i e l d , I l l i n o i s , C.C. Thomas, V o l . XII, No. (6) Nov. pp. 371-384.  Raseda, B. 1941  Jackson Memorial Laboratory, Biology of the Laboratory Mouse, Philadelphia^ Blakiston Company.  Schoemaker, Daniel M. and Byeiesbymer, Albert G. 1911  A Cross-Section Anatomy, New York, D'JLppleton and Go.  Scott, J.P. 1942  Genetic differences i n the s o c i a l behavior o f inbrad strains of mice; Journal of Heredity, V o l . 33 ( 1 ) .  - 109 -  S n e l l , G.D. 1941  Biology of the Labaratqry House, The Blakiston Op., Philadelphia.  1945  linkage of j i t t e r y and waltzing i n the mouse, Journal o f Heredity, V o l . 36 (9) pp. 279-280.  S n e l l , G.D. and Law, Lloyd W., 1939  A linkage between shaker-2 and wavy-2 in the house mouse, Journal of Heredity, 30, (10) Oct.  Stevens, S..S, and Davis, H. 1947  Hearing  i t s Psychology and Physiology,  New York* John Wiley and Sons, Inc. Streeter, S.L. The histogeneses  and growth of the  o t i c capsule and i t s contained  piriotic  tissue spaces i n the human embryo, Contributions to Embryology, No. 20, V o l . 7, pp. 6-24. Van Egmond, A.A.J., Groen, J . J . and Jongkees, L.B. 1949  The mechanics of the semicircular canal, The Journal of Physiology, London, Cambridge U n i v e r s i t y Press, V p l , No. 1, Dec. 15,  no,  - 110 Waddlngton, C.H*, 1940  Organisers and Genes, Cambridge, University Press,  Weiss, Paul 1939  Principals of Development, New York, Henry Holt and Co,  Yerkes, RJI. 1907  The Dancing Mouse, New York, The Macmillan Company.  Zimmermann, E., 1935  Srbliche Cehirnerkrankungen der Housmaus B e l l .z* dtsch, Aerglebl, Z  %  119-180.  - Ill  -  PLATES  -i  112 «  PLATE I L o n g i t u d i n a l s e c t i o n through the cochlea  o f a.  260 day o l d mouse showing b a s a l and c e n t r a l w h i r l s . Note densely s t a i n i n g s p i r a l g a n g l i o n . w i t h haemotoxylin and e o s i n .  Siction stained  Magnification of i l l /  norOM.1  -  114  PLATE  -  II.  S t r i a v a s c u l a r i s o f the "basal w h i r l o f cochlea o f a 260 day old mouse showing the blood v e s s e l s and the A  deeply s t a i n i n g s t r i a t e d cytoplasm. haemotoxylin and e o s i n .  S e c t i o n s t a i n e d with  Magnification of  48^.  -  115  -  - 116 PLATE  III.  S e c t i o n of the c o c h l e a of a normal 60 day mouse showing densely of  staining  s p i r a l ganglion  tthe a u d i t o r y branch o f the e i g h t h n e r v e .  s t a i n e d with M a l l o r y s f  and  old fibers  Section  t r i p l e stain.' Magnification of  4-«4  P L A T E III  118 PLATE 17. Area of the s p i r a l ganglion of the 60 day old normal mouse (see plate T) showing neuroglia nerve c e l l s and their stain.  fibers.  Section stained with M a l l o r y s t r i p l e  Magnification of  f  154-0.  PLATE IV  - 120 PLATE V.  S p i r a l ganglion of the "basal whirl of a normal 35 day old mouse showing the normal dove-tailed pattern foamed hy the nerve c e l l s and their f i b e r s . was stained with haemotoxylin and eosin. of 4(.(.  Section  Magnification  - 121 -  PLATE V  - 122 « PLATE  VI.  Ganglion c e l l s from the s p i r a l ganglion of the basal whirl of a normal 35 day old mouse (see plate V . ) Note the fibers leaving the deeply s t a i n i n g nerve c e l l s the l i g h t e r n u c l e i of the neuroglia c e l l s . haematoxylin and eosin.  Magnification o f  Stained with /540.  and  -  123  -  PLATE VI  12a • PLATE  HI.  Section through the cochlea of a 35 day old pirouette mouse showing a thinning of the c e l l s within the s p i r a l ganglion.  The degenerative  changes are more  pronounced i n the ganglion within the basal whirl of the cochlea than in the central w h i r l .  i85x.  - 125 -  PLATE VII  - 126 PLATE T i l l . Organ of COrti of the basal whirl of the cochlea of a 60 day old pirouette mouse.  Showing the  degenerative changes in the outer hair c e l l s . with Mallory's t r i p l e s t a i n .  Stained  Magnification of 14-81.  PLATE VIII  - 128  -  PLATE IX.  S p i r a l ganglion from the basal whirl of a 50 day old pirouette mouse. Note the small nuclei of the neuroglia c e l l s and the loss of nerve c e l l s * with haemotoxylin and eosin.  Stained  Magnification of 4-1  - 129 -  PLATE IX  - 130 PLATE  X .  Area of the s p i r a l ganglion o f a 30 day old pirouette mouse • (see plate I X )  Note the small  neuroglia c e l l s , blood vessels and loss of nerve c e l l s * Stained with haemotoxylin and eosin.  Magnification of i5<vo.  - 131 -  PLATE X  -i 132  -  PLATE 21.  Area of s p i r a l ganglion o f the basal whirl of a 60 day old pirouette mouse.  Note the s c a r c i t y of nerve  c e l l s and the open network formed by the remaining f i b e r s . Stained with haemotoxylin and eosin.  Magnification  PLATE XI  -134-  PLATE XII  L o n g i t u d i n a l s e c t i o n through the ductus c o c h l e a r i s o f a 260  day  o l d p i r o u e t t e mouse s t a i n e d w i t h M a l l a r y ' s  triple stain, s p i r a l ganglion of Zo6-  ^ote and  the degenerative changes w i t h i n the  organ o f C o r t i .  the  Magnification  PLATE  XII  -136v  PLATS XIII  The s p i r a l ganglion cochlea XII.  o f the b a s a l w h i r l o f the  o f the S60 day o l d p i r o u e t t e mouse shown i n p l a t e  Note the l o s s o f merve c e l l s and t h e i r  Magnification  o f \5ZL>  fibers.  -137-  PLATE  XIII  -138-  PLATS XIV  Area of the day  s p i r a l ganglion  o l d mouse s t a i n e d w i t h C a j a l ' s s i l v e r  Note the nerve c e l l s and of  of a normal  i5Z(*-  their  fibers.  60  stain.  Magnification  PLATE  XIV  -140-  PLATS XV  Area o f the s p i r a l ganglion  of a 30 day o l d  p i r o u e t t e mouse s t a i n e d w i t h C a j a l ' s s i l v e r  stain.  Note the spaces between the c e l l s and the r e d u c t i o n im  the number o f nerve c e l l s .  Magnification  o f .52-1*.  -141-  PL/ATE  XV  -142-  PLATB XVI  Area of the s p i r a l ganglion o f a 60 day o l d p i r o u e t t e mouse s t a i n e d w i t h C a j a l ' s s i l v e r Note the r e d u c t i o n their  fibers.  stain.  i n the number o f nerve c e l l s and  Magnification  o f I5ZU-  PLATE  XVI  APPENDIX  C H A R T  A  COMPARISON OF THE INNER EARS OF NORMAL AND PIROUETTE M i d . TYPE OF MOUSE  ACE IN DAYS  Normal  H & E  Pirouette  H & E Mallory  Pirouette  2.  Semicircular canals,saeculus and u t r i c u l u s are a l l r e p r e s e n t e d . The maculae and c i t ^ T S B ? have n o t e n t i r e l y attained t h e i r a d u l t form. T h e i r sensory c e l l s a r e column a r , few nerve f i b e r s , pass i n t o Scapa's Ganglion.  Broken end organs appear normal  H & E  Broken  H & 1  Kormal  ORGAN OF CORTI  . SPIRAL GANGLIA  2-3 rows ©f e e l l s . Deeply staining nuclei l i e a t the p e r i f e r a l edge o f the organ and their irregular cytoplasm extends inwards. Basal nuclei l i e ©n the i n n e r edge ©f the organ. Between these layers i s a central layer and many b l o o d vessels.  undifferenti a t e d colmninar e e l l s . Tect o r i a l membrane r e s t s on the s u r f a c e s ©f the f u t u r e hair cells.  C o n s i s t s ©f l a r g e round nuclei surrounded by granular cytoplasm. C e l l s have a dove-tailed appearance.  As above,  As above  As above  Cytoplasm more s t r i a t e d than a t 1 day.  As above  As above  STRXA VASCULARIS  VESTIBULAR  STAIN  -  Broken. End ©rgans b e t t e r developed.  Striated cytdplasm d e e p l y staining nuclei. Many c a p i l l a r i s and b l o o d spaces present. 3 l a y e r s o f c e l l s can be d i s t i n g u i s h e d .  Arches o f C o r t i and h a i r e e l l s can be d i s tinguished . Hair c e l l s were t o r n .  N u c l e i smaller and cytoplasm more f i b r o u s than a t 2 days.  As  As Above.  As  .  Pirouette  5.  H & E  As  Normal  12.  H & E  Labyrinth normal. End organs w e l l developed. More f i b e r s seen passing t o Seapa*s g a n g l i a .  Striated cytoplasm d e e p l y staining nuclei, Numerous b l o o d spaces and capillaries. Has g e n e r a l s t r i a t e d appearance o f a d u l t organ.  B r ©ken. Remaining h a i r c e l l s normal. Rods o f G o r t i als© i d e n t i f i e d .  Large and small n u c l e i distinguished i n ganglia.  Pirouette  12.  H&I  As  As  As  As  above.  Normal  15.  Mellary  As above.  Cytoplasm more striated.  Broken, r e maining h a i r e e l l s and r o d s ©f C o r t i w e l l developed.  As  above.  Pirouette  15.  I kW Marchi  As above. None o f the m y e l i n sheaths were blackened w i t h the osmie acid.  Deeply s t a i n i n g nuclei, striated darkly staining cytoplasm,  lr©ken. Remaining h a i r c e l l s w e l l developed•  G a n g l i a composed o f many l a r g e nucleated e e l l s and s m a l l n u e l e i F i b e r s between eells.  Pirouette  20.  Marchi  Mall©ry s t a i n does n o t r e v e a l any change. Marchi b l a c k e n s many m y e l i n sheaths o f vestibular n e r v e s . A few o f these a p p a r e n t l y are degenerating.  Nuclei closely paeked. C y t o plasm n o t as s t r i a t e d as a t 15 u a y s .  Poorly stained but a p p a r e n t l y normal.  Mallory*s stain shoved a r e d u c t i o n i n number o f large n u c l e i i n the b a s a l w h i r l . M a r c h i blackened 3th nerve and some o f these f i b e r s shewed degenerative changes.  Broken.  Cells closely packed. Nerve and n e u r o g l i a c e l l s and fibers well developed.  Mallery  Normal  21.  March! Mallory  Pirouette  23.  Marchi & Mallory  above.  above.  above.  above.  L a b y r i n t h broken. Cytoplasm s t r i End organs w e l l a t e d . Many developed. blood vessels visible.  above.  As a t 20 days. Broken Cytoplasm g r a n u l a r * Blood v e s s e l s visible.  Nerves n o t blackened w i t h Marchi s t a i n and are apparently normal.  above.  As i n 20 day p i p i . However greater l o s s ©f c e l l s i n basal whirl.  Pirouette  25.  H & E  Broken. Remaining e r i s t a apparent* l y normal.  Granular c y t o plasm few s t r i a t i o n s . Few eentral nuelei surrounded by clear cytoplasm. Few v a s c u l a r spaces and c a p i l l a r i e s  P i l l a r s normal. Hair c e l l s torn.  Normal  28.  Mallory  As i n 21 day o l d normal mouse •  As i n 21 day o l d normal mouse  As i n 21 day o l d normal mouse.  Labyrinth normal, end ©rgans w e l l developed. Scapa's g a n g l i o n normal.  Deeply s t a i n i n g . Cytoplasm mere g r a n u l a r than striated. L i g h t areas around some nuclei.  Organ ©f C o r t i tern. Pillars^ normal, l e - * maining h a i r cells apparently normal.  Broken. End organs apparentl y normal.  Broken. S t r i a t i o n s not marked and c y t o - P i l l a r s and remaining h a i r plasm g r a n u l a r . e e l l s appear I n lower w h i r l t o be normal. striations lost. Amount ©f c y t o plasm reduced. Many b l o o d v e s s e l s .  Few nerve c e l l s . Many f i b e r s and neuroglia c e l l s . Blood v e s s e l s also present.  Pirouette  30.  H &1 Mallary Cajal.  Pirouette  35.  H & E Mallory  I n upper w h i r l ganglia c e l l s slightly less dense than normal. Lower whirls large n u c l e i sparse and many f i b e r s and n e u r o g l i a cells.  As i n 21 day ©Id normal mouse.  Basal ganglia degenerating. Few f i b e r s seen l e a v i n g organ o f C o r t i and passing t o nerve c e l l s . Nuclei lost i n central ganglia.  Normal  37  H & E  Removed b e f o r e sectioning.  W e l l developed. Cytoplasm s t r i a t e d and deeply stained.  H a i r c e l l s and p i l l a r s of Corti are d i s t i n c t and w e l l developed.  W e l l developed c l o s e l y packed Nerve and neuroglia c e l l s . F i b e r s seen between c e l l s .  Pirouette  50.  Cajal  Broken. End organs apparentl y normal. Seapa* s g a n g l i a normal.  S l l v e s t a i n does net s t a i n c e l l s ©f s t r i a vascularis. Amount o f c y t o plasm appears t o be redueed.  In lower w h i r l hair cells degenerated and Hods o f C o r t i not d i s t i n c t . In central w h i r l outer hair eells indistinct.  Number o f nerve c e l l s reduced. Many remaining nerve c e l l s are undergoing chromomatolysis.  Pirouette  55*  Mallory  Broken. Seapa*s g a n g l i o n , c r i s t a e and maculae normal.  C e l l s n o t as compact spaces between c e l l s , Cytoplasm g r a n u l a r . Blood v e s s e l s normal.  Rods p r e s e n t . Cytoplasm i n outer h a i r c e l l s redueed. H a i r cells flattened,  Lower w h i r l s ©f ganglia contain few s m a l l n u e l e i f i b e r s and b l o o d vessels. Ganglia i n apex appears almost normal.  Normal  60.  H & E Mallory Marchi Cajal  Few nerve f i b r e s blackened evenly with Marchi s t a i n . End organs and membranes as above •  As i n 35 day As i n 35 day o l d normal, mouse. o l d normal mouse, Blood v e s s e l s w e l l developed.  As i n 35 day o l d normal mouse. Some f i b e r s blackened w i t h Marchi s t a i n . These f i b e r s are 1; normal.  Pirouette  60.  Wk E Mallory Marchi Cajal  End organs normal• S c a p a s g a n g l i o n normal.  Less cytoplasm, v a e u a l i s around a few o f the n u c l e i , spaces between c e l l s . Cytoplasm o n l y s t r i a t e d a t apex.  Rods normal. Inner h a i r c e l l s normal. Outer hair cells l y i n g down and not d i s t i n c t .  Ganglia i n 55 day p i p i mouse,  Mallory  Broken. Scapa's g a n g l i o n normal.  L e s s Cytoplasm, granular appearance• Number ©f n u e l e i reduced. Few bl©od vessels.  Broken. Cytoplasm ©f hair cells did not s t a i n . Nuclei stained blue, instead ©f r e d .  Most nerve c e l l s have been l o s t i n basal whirl. Many dropped out ©f c e n t r a l whirl.  Marchi Mallory  Broken. End organs w e l l developed.  i n the 60 day o l d normal mouse.  Broken.  As i n 60 day o l d normal mouse •  As i n t h e 60 Broken. day ©Id p i r o u e t t e mouse •  Pirouette  85.  Normal  90,  1  Pirouette  100.  Mallory  Appears normal.  Normal  10 *.  Mallory  As i n 60 day o l d As i n 60 day o l d Broken. normal mouse. normal mouse.  1  Number o f n u c l e i redueed i n b a s a l whirl. Apical w h i r l apparently normal.  As i n 60 day o l d normal mouse.  B a s a l w h i r l organ of C o r t i r e duced t o l a y e r of u n d i f f e r e n tiated tissue. Broken i n central whirl.  Firouette  260  Mallory  Broken. End organs appear t© be normal.  As i n t h e 60 day ©Id mouse.  Normal  262  H & E Marchi  Broken.  Organ o f C o r t i Deeply s t a i n w e l l developed, ing, striated as i n 35 day o l d normal mouse.  N rmal  390  1 1 1  Removed before sectioning,  As i n 262 day normal mouse.  1 & E  Broken.  In b a s a l w h i r l G r e a t l y reduced the organ o f i n width. L e s s Corti Is recytoplasm, number o f n u c l e i duced t o an u n differentiated reduced. knob o f t i s s u e . V a s c u l a r spaces and b l o o d v e s s e l s I n c e n t r a l w h i r l the p i l a r s are l o s t . No s t r i a t i o n s present present but outer h a i r c e l l s have degenerated.  0  Pirouette  Broken.  Few nerve c e l l s present i n b a s a l w h i r l . Loss of nerve c e l l s i n central whirl.  As i n 35 day ©Id normal mouse.  As i n 262 day normal mouse.  Nerve and ganglia consist o f few f i b e r s , small n u c l e i and b l o o d vessels.  

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