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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 1^5/5/ Iff/ fif 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 in the Department of Biology and Botany. We accept t h i s thesis as conforming to the standard required from candidates for the degree of MASTER OF ARTS. Members of the Department of Biology and Botany THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1951. -1-Abstract. An Investigation into the Physical Basis of the Pirouette Mutation In the House Mouse (Mus musculuV). fhe object of this investigation was to study the abnormal behavior produced by the pirouette mutation i n the house mouse (Mus musculus) and to determine i t s physical basis. The pirouette mutation f i r s t appeared i n 1943 and was described by Wolley and Dickie (1945) who gave It the genetic symbol p i . This recessive mutation i s located on the t h i r d chromosome of the house mouse. Mice homozygous for t h i s mutation show mixed c i r c l i n g , head shaking and deafness. The abnormal movements commence fourteen days a f t e r b i r t h and these mice are evidently deaf throughout t h e i r entire l i v e s * Similar abnormal movements and deafness have been produced not only by other mutations in mice but also In other animals Including man. A survey of the l i t e r a t u r e revealed many possible sources of abnormalles which could produce the a t y p i c a l behavior of the pirouette mouse and these reported sources of abnormalities were Investigated with the following r e s u l t s : -2-1. Muscles of the head, neck and thorax were normal* 2. Liver gross and histological anatomy was normal. 3 . Brain * gross anatomy was normal. 4* Skull - gross anatomy was normal. 5 . Eighth nerve - gross and histological study revealed no tumors. 6. Bar - gross anatomy and "blood supply normal. .» histological studies showed degenerative changes within the spiral 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 of degenerative changes are different* In the pirouette mouse the extensive loss of nerve cells within the spiral ganglion later followed by degenerative changes In the s t r i a vas-cularis and in the organ of Corti suggests that the cause of these changes lies outside the cochlea. The fact that the degenerative changes of a l l structures are found f i r s t and most extensively In the basal whirl and later in the middle and upper whirls of the cochlea also supports the contention that the change is 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 in the pirouette mouse no tumors were present and since the nerve was intact, there remains only the possibility, of osseous compression or lesions within the central nervous system causing this abnormality. Acknowledgements. May I take this opportunity to express my sincere thanks to the following people, whose interest and help have made the preparation of this thesis possible. I am especially indebted to Mrs. R. Brink, Assistant Professor, by whom the problem was f i r s t suggested and under whose direction the work was carried on. Her valTsable advice and assistance when the problem seemed most d i f f i c u l t is 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 slides and for his aid in photography. To Dr. W. Gibson, lecturer, of the Department of Physcology, for his advice and assistance in 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 useful suggestions. I am indebted to the President's Committee on Research for a grant from the Board of Governors in 1948-1950, which made the preparation of this thesis possible. TABLE OF CONTENTS. Page I Introduction 1 1 : A. Origin of Pirouette 1 B. Statement of the Problem 1 II Description of the Mutation 2 A. Genetic Description of Pirouette 2 B. Effect of the Mutation 2 III Review of the Literature 3 A. Effect of Mutations on Behavior 3 B. Ghorie Mutations in 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* Jittery 14 8. Erelsler 15 9. v a r i t i n t waddler 16 10. Jerker 17 11. P a l l i d _ 18 12. Comparison of mutations 18 Page Ci Choric Mutations in Other Animals . 22 1. Rat '23 l - - i a. Waltzer 22 b. Wobbly 22 2. Peromyscus 23 Waltzer 23 3. Rabbit 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 in Man 27 IV Materials and Methods 34 A. Origin of the Mice 34 B. Care of Mice 34 1. Cages 34 2. Food 36 3. Records 35 G. Preparation of Material 36 1. K i l l i n g and fixing 36 2. Histological techniques 36 a. 5?he liver 36 b. The ear 36 Page v" Comparison of the Behavior of Normal and Pirouette Mioe >38 l A. Adult Mice 38 B. Development of Abnormalities 39 C. Comparison of Normal and Mutant Mice 40 VI Investigation of the Possible Causes for the Abnormal Behavior of the Pirouette Mouse. 48 A. Pathological Infection 48 Bi 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 Static Labyrinth 56 i i The Cochlea 60 Oi Post-natal development of the cochlea of a normal mouse. 67 2. Structure of the ear of the pirouette mouse 73 a. Position of the ear vessicle 73 b. The middle ear 74 o. The inner ear 74 S. Sku l l VII Discussion VIII Conclusion IX Summary X Biblography XI Plates XII Appendix Page 1 The s t a t i c Labyrinth 75 i i The cochlea 76 d. Post-natal development of the cochlea of a pirouette mouse 80 87 89 96 98 100 111 114 An Investigation into the Physical Basis of the Pirouette Mutation in the House Mouse ( Mus fftusculus). I Introduction A. Origin of Pirouette In 1943 a mutation appeared in a strain of mice at the Roscoe B. Jackson Memorial Laboratory, Bar Harbor.,Maine, which caused deafness, abnormal head shaking and ci 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 of this investigation is to study the abnormal behaviour produoed by the pirouette mutation in the-house mouse (Mus muaculus) and to determine its physical basis. oo 0 oo II Description of the Mutation. i A , Genetic description of pirouette,. The inheritance of pirouette (pi) was investigated by <J. W. Woolley and M.M. Dickie (1946), who established the faot that i t was a re*oessive mutation located on the third chromosome of the house mouse. Linkage tests showed that pirouette was located 7,2 map units 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 hr pi wv T: § 3B75 7,£ B. Effect of the mutation. Mice homozygous for this recessive mutation show mixed cir 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 Affecting Behaviour. Uhe triad of symptoms exhibited by the pirouette mice have been produced by other mutations In mice and also by mutations in many other animals including man. These chorio mutations appear to alter the developmental reactions oocuring within the animal in such a way as to produce changes in i t s struotnlre and to modify its physiology. Some of these mutant effects may be telly slight and thus d i f f i c u l t to detect or they may produoe changes that are easily recognized. J.P. Soott (1942) and C.E. Eeeler (1942) noticed that mutations affecting coat colour in mice also produoe slight changes in the behaviour pattern. Thus mice with grey coats are found to be wilder than those having black pelages. While studying autogenetio seizures in mice, C. Hall (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 dilute brown mice. It has been suggested by Keller (1947) that mutations affect-ing coat colour also produce slight behaviour changes by pleitroplc action* Henoe, a mutationnay produce its main effect on some definite 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 their main effect on the behaviour of the animal. - 4 -B. Choric Mutations in the House Mouse. Other mutations have occurred in the house mouse which produce choric "behaviour, similar to that of pirouette. The following mutations: Japanese waltzer (v), shaker-1 (sh-l), #haker~2 (sh-i2), shaker-short (st), figet ( f i ) , jerker (je), ji t t e r y ( j i ) , Erelsler (k) and p a l l i d (pa) a l l produce peculiar movements of the head and body, incessant activity 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 is the oldest and most commonly known of these behaviour mutations. According to Keeler (1931) the waltzing mutation appeared in China as early as 80 B.C. in Mus baotrianus and was introduced into Europe during the year 1890. 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 circles or whirling around on the same spot with incredible rapidity. These mice also show a characteristic head shaking movement and are deaf. Yerks (1907) found that the restless, jerky movements and general exoitability appear in these mice during the tenth to the fourteenth day after b i r t h . The ears of the waltzers open between the twelfth to the fourteenth day hut only in a few oases did the mice react to sound between the thirteenth and seventeenth days. Grmneberg (1943) suggests that ... there is some variation in the time of onset of deafness, so that, in most animals it is almost oomplete by the time an auditory response can f i r s t be evoked in the normal mouse" Yerks (1907) noted that some of the adult waltzers were mixed oirclers while others always appeared to circle in one direction only. He also reported that the waltzer Was able to run up and down inclines or vertical rods, cross narrow bridges and maintain it s balance; it s equilibrium, however, was not as good as that of the normal mouse. It was also noted that these mutant mice never jumped when frightened as did normal mice. Waltzing mice were unable to swim when placed in water and da not become dizzy when rotated. Gruneberg (1943) stated that t;v<- Alexander and Ereidl (1900) found that although waltzers did not become dizzy when rotated in 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 is looated on the tenth chromosome of the house mouse. When homozygous this recessive mutation produoes abnormalities whioh cause the mouse to run i n circles, whirl, show a restless, jerky, ve r t i c a l movement of the head and become deaf. Many theories have been developed to explain the physical basis of this mutation. Yerks (1907) reported that Saint Loup stated the abnormal movements of the waltzers were due to nervous defect while Landais believed this defect was a bil a t e r a l abnormality in the brain. Yerks (1907) stated that Hawitz (1899) reconstructed the ear of the waltzer in wax from microsoope slides and found that the anterior vertical canal was normal but the horizontal canals were crippled or fused together, the utriculus 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 in 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 structural defects within the cerebellum. Yerks (1907) reported that Baginsky in 1902 also found the Inner ear of the waltzer to be normal and that Alexander and Ereidal (1900) (quoted from Yerks, 1907) stated that: Structural peculiarities of the waltzer are the result of primary changes...The facts of behaviour are almost certainly due to pathological changes...in the nerves, ganglia and especially in 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 of the ganglion spirals. Gruneberg (1943) states that Kishi (1902) found a reduction in the size of the s t r i a vascularis cochlea and a slight diminuatlon of the hair cells in the waltzer's organ of Corti. Acoording to Gruneberg (1943), Tan Lennep's (1910) results indicated that: •••• shortly after birth, the s t r i a vascularis cochlea undergoes a process of degeneration; as this organ is reputed to secrete the endolymph necessary for the nutrition of Corti ?s organ (which lacks blood vessels of i t s own) the degeneration of that organ and the degeneration of the ganglion splrale in Rosinthal's canal, is regarded as secondary to. the changes in the s t r i a vascularis; no anomalies were found in the pars superior of the labyrinth. Gates (1926) stated that in 1911 Morgan supported the findings of Alexander and Zreidl (1900) by producing a r t i f i c i a l waltzers by injecting acetyl-atoxyl into normal mice* These mice waltzed like the mutant animals and remained abnormal for the remainder of their lives. The drug whieh caused these permanent changes in the mice is known to cause degeneration of certain nerve fibers in man. Thus, i f the drug affects mice in the same manner, i t would appear that the waltzing mutation can be explained by either a degener-ation or an arrest in development of 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 is known to affect the nerves and that Ealper (1913) found degeneration of the nerve fibers in the acoustic tracts of the waltzer*s brain. Euiper believed that this degeneration Is the primary factor and that the degeneration of the s t r i a vascularis cochlea is secondary. Prom the above discussion of the results obtained by the various research workers, i t is evident that the fundamental cause of this mutation is controversial. The above conflicting statements lead one to conclude that the waltzing mice are normal at birth and later a degeneration of the acoustic tracts of the brain and of the structures and nerves In the organ of Oprti are partly, If not wholly, responsible for the behaviour seen in adult Japanese waltzing mice. 2. Shaker - 1 Shaker-1 produces a similar nervous, rapid up and down head movement but cir c l i n g is not as common as in the waltzing mice. When ci r c l i n g is present in the shaker-1 the animals turn more frequently in a counter-clockwise than In a clockwise direction. Shaker-1 mice are able to hear u n t i l they are three to six months old. The deafness and ohorie head movement are the most noticeable part of the abnormal syndrome produced by this mutation. The shaker-1 mutation is 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 effect which results In deafness at the age of six months. from his study of the development of the shaker*! mice, Gruneberg (1943) found that the organ of Oorti is normal twelve days after birth. Degenerative changes within the organ of Corti set in after the vas spirale has dis-appeared. The vas spirale nourishes the organ of Corti during the embryonic and early post-natal l i f e of the mouse. After this vessel atrophies the organ of Corti depends for i t s nutrition entirely on the endolymph^ which is secreted by the s t r i a vascularis. The s t r i a vascularis is 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 in the organ of Corti pf these mutant mice are caused by malnutrition and the changes within the ganglion spirale are probably secondary. Zlmmermann (1935) found atrophy of the corpus striatoma in the brain of the shaker-1 mouse accompanied by a slight hydrocephalus internus. These findings resemble the pathalogical changes which oocur in Huntington's chorea in man. These changes explain the nervous head movements and lack of muscle co-ordination but their connection with those which occur in the inner ear s t i l l remains obscure* 3. Shaker-2 Shaker-2 is 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 is located on the seventh chromosome. Mice heterozygous for both these mutations show normal behaviour and hearing. The pathological changes produced by this mutation are as yet unknown. 4. Hertwig's Shaker Hertwig (1942) produced shaker mice by x-radlatlon. This abnormal change produces its effect on the homozygotes when they are ten days old, causing them to r o l l over on their backs, shake their heads, drag their hind legs and sometimes c i r c l e . These abnormal movements are almost completely lost during the second month. Hertwig 1s mice were also found to be deaf, unable to swim and did not become dizzy when rotated. - 11 5. Shaker Short Another ehorio mutation, shaker-short (st) produces deafness, c i r c l i n g , ataxia of the head and muscular inco-ordination similar to that found among waltzing mice, This recessive mutation also results in a shortening of the t a i l j and at birth the mice show one or two hernias near the middle of the parietooccipital suture. The abnormal behaviour in these mice has been observed as early as five days after birth. Similar to the other mutations shaker-short produces deafness in its homozygotes. Bpnnevie (1936) made an embryonic study of the shaker-short mioe and reported that at eight days ah abnormal compression of the brain is evident. After the medullary tube closes a coalescence between the epidermis and the dorsal median line of the myelencephalon ocourts. The roof of the latter is narrow due to a compression of the brain. The choroidal pleicus remains rudimentary and the foramen magendie is absent. During the last days of embryonic develop-ment the roof of the myienoephalon and cerebellum is 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 . If this disturbance is 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, laterally compressed vesicle without any endolymphatic appendage, semicircular canals or division into separate chambers. These abnormalities are caused by changes in the posterior part of the head and brain, surrounding the ear vesicles. Bonnevie (1936) found that in normal embryos the myelencephalon is retarded and con-sequently the surrounding tissues including the ear vesleles become abortive. Thus, she concluded that the primary effect of the shaker-short mutation is to change the developmental rate of parts of the brain in suoh a manner as to also affect the inner ear. Gruneberg (1947) reported that the primary effect of the shaker-short mutation is a disturbance of the eotoderm which forms a transitory connection between the skin and the dorsal olosure region of the brain. This Interferes with the formation of the plexus choroideus of the fourth ventricle, while the plexus of the lateral ventricles also remain rudimentary, no foramen of Magendie is formed. As the rudi-mentary plexuses f a i l to produce a sufflcienoy of cerebro-spinal f l u i d , the whole ventricular system is considerably narrowed down. This narrowness of the brain case results in an abnormally high pressure within the brain cavity and a - 13 -reduced pressure on the ear vesicles which as a consequence develop abnormally. Thus, the shaker-i-short mutation acts direct ly on the extoderm in 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 atypical behaviour of these mice. The effect of the short t a i l has not yet been investigated. 6. Elget Figet mutation oocured in 1941. Mice homozygous for this recessive faotor show side to side head movements, c i rc l ing in one direction and deafness. These mice are hyper-sensitive 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 to four months o l d . Like the waltzers the figets are unable to swim and Immediately lose a l l sense of direction when placed in water. The figet mutation also affects the eyes of the homozygote. After the eyes are open a discharge from the oonjuotima occurs and the eyelids tend to st ick together and permanently close the eyes. - 14 -The morphological effect of this mutation has not been determined. The phenotypic effect of the mutation differs from the other members of this group in that the head is shaken from side to side rather than in a dorso-ventral direction. The physical basis for this mutation is unknown. 7. Jittery Jittery is a reoessive juvenile lethal mutation which is characterized by muscular incoordination and tetany. These mice when twelve days of 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 readily right them-selves. A few days latter Snell (1945) observed tetany involving the fore legs. These mice died when thirty-two days old. De Ome (1945) found that when a ji t t e r y mouse was parabiotieally joined to a normal sib i t could live for about fifty-one days. In j i t t e r y mice the anterior pituitary shows increased c e l l u l a r i t y and engorged blood vessels. Implanta-tion of normal pituitaries was found to be without effect. In later stages the thyroid gland of the ji t t e r y mouse became hypoactive and involution of the thymus ooours in the more advanced stages. It is also recorded that the blood oalclum is normal but a vaouolar degeneration is seen in the motor cells of the lumber region of the spinal cord. Jittery differs from - 15 previously mentioned mutations in that i t is a juvenile lethal. The lethal action of the mutation appBars to he through an upset of the hormones secreted by the pituitary, thyroid and thymus glands. In the other mutations affecting the musculature and sense of balance, the main abnormalities appear to be in the development of the ear and the brain. 8. Ereisler The mice homozygous for the Ereisler mutation are affected at the age of ten days. At this time they are seen to crawl in circles, t i r e rapidily and when fatigued f a l l into a r i g i d position on their backs or sides. The adults show deafness, dance and occasionally toss their heads. The Ereisler mice shake their heads less frequently but circle more often than do the shaker-1 mice. Hertwig (1942) found that in nine day old Ereisler embryos the ear vesicles are displaced laterally 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 vesicles. In older embryos ductus and sacculus endolymphatics are absent and the semi-circular canals are incomplete. The saoculus and utriculus are incompletely separated, the cochlea is abnormal and its ganglia are absent. The absence of the endolymphatic duct results in increased pressure of the endolymph whieh causes a oyst to evaginate into the subarachnoid space. After birth these cysts continue to grow and interfere with neighbouring parts of the brain. The abnormal configuration of the labyrinth leads to changes within the os petrous . The fossa acuata which normally harbours the flocculus cerebelli is absent and consequently this structure of the brain is not formed. Thus the main effect of the Ereisler mutation is produced by changes within the ear of the mouse whioh In turn produoe abnormalities within the brain of the mutant animal. 9. Varltint Wadaier. T&e dominant varitint waddler mutation (va) pro-duces a variously tinted coat and causes the mice to have a duck like walk. Mice having this mutation are nervous, restless and deaf. These mice also show choric head move-ments and mixed cir c l i n g fourteen days after birth. The choreic tendency is more pronounced in homozygous varltint waddler mice. At fourteen days old these animals are ex-tremely excitable and deaf. If suddenly disturbed they r o l l over and over then leap into the air several times and fi n a l l y end with a convulsive stiffening of the body. The heterozygote is not as excitable as the homozygote but is in constant motion unless asleep. Mice which are homozygous for this - 17 -mutation are unable to swim while the mice which are heterozygous for varitint waddler are able to swim in oircles. Both types of mice show head-shaking similar to that seen in shaker-1 mice. They waddle when walking, sometimes c i r c l e , and do not become dizzy when rotated. Since this mutation affects both the integument and nervous system Qloudman (1945) believed that the disturbances of hearing, equilibrium and pigment formation might con-ceivably be related in their early embryonic origin* He suggests that since pigment arises from the neural crest a single predisposing factor might operate quite early in embryonio development to affect both the integument and nervous system. However, nothing is known of the physical basis of this mutation. 10. Jerker Jerker is 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 is thus linked with ruby eye and s i l v e r . No investigation has been oonducted on the physical basis of this 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 in some of these mutant mice and further investigation revealed that the affected mice showed continual nystagmus• The head weaving is presumably a compensation for the nystagmus. The physical cause of this mutation is unknown. 12. Comparison of the Mutations. The above mutations are similar In that they a l l produce abnormal behaviour in the house mouse. They cause nervous, excitable movements which keep the mice in oonstant motion when they are awake* These mutations, with perhaps the exception of ji t t e r y , cause deafness in the adult mice* In some cases (shaker-short and Ereisler) the ear vesicles have been founa to be abnormal, while in others (shaker-1 and waltzer) only certain structures within the cochlea are affeotea. Some of these mutations (waltzer, shaker-1, shaker-short ana Krelsler) proauee aegeneratlon or de-struction of the nerve traets in the brain. These changes within the central nervous system are apparently responsible for the abnormal movements of the b o a y . A comparison of the symptoms produeea by these mutations with their physical changes is given in table 1. * 19 -SABLE I MUTATION SYMPTOMS PHYSICAL CHANGES WALTZER (v) Chromosome 10 C i r c l i n g , head shaking, deafness, restlessness, e x c i t a b i l i t y . Head shaking at 17 days deaf at 14 days, c i r c l e i n either d i r e c t i o n . Do not become dizzy-when rot a t e d . Cannot swim. Abnormalities i n sensory nerve supply to the ear. Pathological change in the peripheral nerve endings of the ear. Deafness due to defect-ive cochlea and lack o f dizziness due to diminuation of nerve fib e r s i n the canal organs. Degeneration of the ganglion s p i r a l e . Degeneration of nerve fibers i n acoustic t r a c t o f the b r a i n . SHAKER-1 (sh) (Chromosome-l) Head shaking in a rapid 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 dizzy when rotated. Cannot swim. In the organ of C o r t i the abnormal s t r i a vascularis f a i l s to secrete enough endol* ymph whioh r e s u l t s i n degeneration of the former structure and also leads to the de-generation of the ganglion s p i r a l e . Muscular incoordina-t i o n i s a r e s u l t of an atrophy of the corpus striatum in the b r a i n . SHAKER- 2 (sh-2) (CHROMOSOME 7) Same as i n Shaker-1 unknown. SHAKER SHORT (st) (Chromosome?) Deafness, c i r c l i n g , headshaking (up and down), and muscular incoordination, shortening of the t a i l and 2 cerebral hernias on the parieto-o c c i p i t a l suture. Eight day embryos show an abnormal compression of the brain. Choroid plexus i s rudimentary, foremen Magendle i s absent. Thus, drainage of the brain is hampered and pressure develops, so * MUTATION SYMPTOMS SHaker-Short continued PHYSICAL CHANGES which causes the brain roof to break and hernias are formed. The inner ear remains an oval com-pressed vessicle lacking semicircular canals. These abnormalities are due to the retarded growth of the myelencephalon, PI GET (fir;.) 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 hyper-sensitive to sounds then gradually become aeaf when 5-4 months old. Eyelias also tend to fuse. unknown JITTERY (It) (Chromosome -10) A juvenile lethal mutation producing muscular inco-oraination 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 aiffieulty in righting them-selves. Tentany in the fore-limbs. They ale when 32 aays ola* Mice have abnormal an-terior 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 EREISLER 10 day old mioe ( k r l ? ) crawl in oirlees, (Chromosome 5) t i r e rapidly and f a l l into a r i g i d position on either their hacks or sides. Adults are deaf, circle and ocoasion-ally show head shaking. 9 day embryos show an ear vessiele which is displaced laterally and is separated from the neural tube by mesenchyme. Abnormal-ities of the labrinth develop, Abnormal pressure of the endol-ymph produces cysts in the subarachnoid space whieh grow and interfere with the surrounding brain tissue. The folcculus cerebelli is absent. VARITINT WADD1ER ( V a T ; ) (Chromosome ?) Duck-like walk* some cir c l i n g and head shaking, nervous-ness and restless. Mice are also deaf. The pigment of the skin is effected and produces a variously tinted coat • Disturbance in the very early development of the neural crest material. PIROUETTE ( p i l l ) (Chromosome 3) mixed cir c l i n g , head shaking, deaf, do not be-come dizzy.cannot swirn^ , Young have a crab-like walk.. unknown JEREER (le) (Chromosome 4) mixed ci r c l i n g , head shaking, deafness, rest-lessness, and excitability unknown PALLID (pa) (Chromosome (5) pink eye, head-weaving and nystagmus• headweaving due to nystagmus, cause of nystagmus unknown. * 22 •? C # Chorie Mutations in Other Animals, 1. Rat A. Waltzer Waltzing resembling that seen in the mouse has been observed in the ra t , (Rattusnorvegicus). the waltzing mutation in the rat causes the animal to circle and shake it s head but its hearing is unimpaired. This mutation acts as a recessive gene which is influenced by modifying factors which may favour or inhibit its expression. In the rat waltzing is linked with albino, red-eyed yellow and pink* eyed yellow, B. Wobbly Castle, Zing and Daniels (1941) have described another mutation in the rat whieh they have named wobbly. Animals affected by this simple reeessive mutation show a peculiar, locomotion which is f i r s t notioeable when the eyes / open at about fourteen days* The movements of these animals are jerky and the young animals walk a step at a time with a jerk of the body as each step is taken. These movements suggested an infection of the inner^ear but Castle, King and Daniels (1941) state that histological sections did not reveal any pathological condition within this structure. - 23 i 2. Peromyscus. Waltz lng. Waltzing has occur eel in the Peromyscus similar to that which occurs in the mouse and the rat, Gruneberg (1947) stated that waltzing Peromyscus tend to run in circles, often ao rapidly t h a t they seem to be rotating on their legs. The same mouse may rotate either right or l e f t . Affected mice may waltz with or without external stimuli. Being placed in a strange cage, the Jangling of keys, etc. will, stimulate a waltzer to oircle, This recessive mutation varies in the degree of its expression according to the species, Thus, the head tremor is most pronounced in Peromyscus ermatus (catus mouse) but these individuals may not waltz. The exprer ssion of this mutation also varies within a subspecies. Thus, Peromyscus maniculatus artisinae and P*m. bairdii differ in that the former show deafness except when very young, but the latter respond to sound at a l l ages. When a cross was made between waltzing mice of these sub-species the hybrids obtained appeared normal, indicating that the waltzing mutations are caused by different hereditary factors. The cause of this abnormal behaviour in Peromyscus has not been investigated . 84 -3. Rabbit, A, Waltzer Waltzing rabbits have also been recorded by Gruneberg (1947), The behavior of these animals closely resembles the condition seen in the house mouse. In the waltzing rabbit the vestibular and cochlea apparatus is apparently normal, B, Shaking Paralysis. Another mutation produces shaking paralysis in the rabbit* Gruneberg (1947) states that the affected animals when 1G days old develop a fine tremor which only ceases when they are at rest, f l a c c i d paralysis gradually develops in the hind limbs. Gruneberg (1947) states that pathological study showed a "degeneration of nerve oells in the basal ganglia of the brain; the neo striatum is poor in big cells and there is a great scarcity of ganglion eells In the pallidum." These changes are apparently responsible for the abnormalities produoed by this mutation. C, Ataxia. Another mutation resulting in a degenerative disorder Involving the brain stem of the rabbit is ataxia. This recessive mutation produces its abnormalities when the rabbit is 58-86 days old* It f i r s t expresses i t s e l f as a - 2 5 clumsy gait, caused by the Inability of the legs to support the body. Later a coarse tremor of the head and neck develop, accompanied by a lateral nystagmus. Gruneberg (1947) reports the occurrence of degenerat-ive 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 their nuol&4 and to some extent in the vestibular v nerve. Within the brain degenerative ohanges occurred in the trapezoid body, the lateral lemniscus and the vestibulo-cerebellar fasiculus. Gruneberg (1947) states that t h e B e changes in the brain and nerves account for the abnormal behavior produced by this mutation since "changes in the vestibular system and the medial lemniscus probably leads to loss of normal sensation from the feet and causes postoral d i f f i o u l t i e s i " Involvement of the oerebellum accounts for the tremor of head and neck, combined cerebellar and vestibular lesions result in nystagmus• - 26 4, Guinea-pig A. Waltzing. Waltzing in the guineafpig is produced hy a simple recessive factor. The movements seen in these animals resemble those of the waltzing house mouse hut with a re-duction of the circus movements. G i r d i n g is predominantly in one direction, hut the animals can reverse the movements. Head shaking is common and the animals are deaf. When these animals are rotated they do not show nystagmus or compensatory head movements. If the waltzer can see when i t is dropped It w i l l land on its feet hut when blindfolded i t w i l l land on Its baek. Gruneberg (1947) states that the structure of the cochlea is these animals is normal u n t i l they are ten days old* After this time degenerative changes ocour within the stfta vascularis cochlea, the organ of Corti and the ganglion spirals. The vestibular part of the ear is apparently normal. The c i r c l i n g and head shaking movements probably originate in the central nervous system. B. Congenital Palsy. Congenital palsy in the guinea-pig resembles j i t t e r y in the house mouse and shaking paralysis in the rabbit. It is caused by a recessive juvenile lethal mutation whieh produces a jerky paralytic 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 of the parathroid glands* - 2 7 -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 con-ditions: 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 • T o r t i c o l l i s is produced by mechanical misalignment and changes in the cervical region of the spinal cord. Labyrinthine t o r t i c o l l i s , as i t s name suggests, is due to i r r i t a t i o n of the semicircular canals on one side of the head while ceatral t o r t i c o l l i s is the result of organic changes of the basal ganglia. Thus, the same symptoms can be produced by three distinct pathological changes. Abnormal movements of the trunk and limbs occur in L i t t l e f s disease* Preldreiok's disease, Huntington's chorea, Hereditary chorea, Wilson's disease, Multiple sclerosis* St. Vitus' dance, Parkinson's disease and dystonia musculorum. The congenital stiffness of the limbs and spastic adduction of the thighs which results in the cross legged walk characteristic of L i t t l e ' s disease has been found to be caused by atrophy or sclerosis of the lateral columns of the spinal cord. Hereditary lesion of the posterior columns and pyramidal tracts of the spinal cord and degeneration of the cerebellar tracts and cerebellum result 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 result of dis-- 29 -Integration of the motor oells within the basal ganglia. The pathological changes produced by hereditary chorea which only affects the muscles of the face and a r m s is as yet unknown. The inherited bilateral athetoid movements of hands, a r m B and legs which increase on voluntary movements are seen in Wilson's disease. The pathological changes produced by this disease are a hobnail cirrhosis of the liv e r and changes within the lenticular nuclei. Degeneration of multiple sclerosis results in a spastic condition of the affected muscles and often produces a tremor of the head and upper extremities. In St. Vitus 1 dance tempory lesions In the subcortical regions produce coarse tremors which a r e lost during sleep. Parkinson's disease results in a rhythmical tremor of the resting limb which is lost during voluntary motion. Dystonia muscularum causes abnormal movements which closely resemble those seen in the pirouette mouse • Patients having this disease show irregular, involuntaryj clonio contortions of the trunk and proximal muscles of the extremities. These symptoms a r e lost when resting but re-occur with voluntary motion. The abnormal movements are most pronounced when the patient i s walking and as he proceeds the body is bent and twisted forward and sideways. These abnormalities are caused by a progressive degeneration - 3 0 -of the lenticular nucleus. Thus, In the above diseases the main pathological changes are found in the lenticular nuclei of the brain, The following table contains a summary of the nervous diseases in man whieh produce abnormal movements similar to those seen In the pirouette mouse* - 31 -TABLE II DISEASES OF THE NERVOUS SYSTEM OP MAN PRODUOINS ABNORMALITIES SIMILAR TO THOSE PRODUCED BY THE PIROUETTE MUTATION DISEASE L i t t l e ' s disease Friedreich's disease Huntington's disease Hereditary chorea Wilson's disease (progressive lenticular degeneration) SYNDROME Congenital stiffness of the limbs, cross-legged walk due to spastic adduction of the thighs. Arks less involved than the legs. Hereditary ataxia nystagmus, head tremor and."nodding, asynergy of "both arms • Hereditary, jerky, irregular movements of the entire body. Disturbed gait; head, shoulders and arms twisted from side to side as patient walks. Dementia dev e lops gr adually • Only affects the muscles of the face and arms. Inherited bi 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 spastic. PATHOLOGY Atrophy or sclerosis of the lateral columns of the spinal cord. Lesion of the posterior columns and pyramidal tracts of the spinal cord and degeneration of the cerebellar tracts and cerebellum. Degenerative changes in motor cells of caudate nuoleus and the putman of the corpus striatum and of some pyrimidal c e l l s . undetermined Hobnail cirrhosis of the l i v e r and changes in the lenticular nuolei. - 38 -DISEASE SYNDROME P^ATHOLOGY Multiple sclerosis Symptoms varied, spastic conditions predominate. Ataxic tremor of the head and upper extremit-ies and nystagmus occasionally occur. Degeneration of mul-tiple areas of the brain mainly involving the motor centers. St. Vitus 1 Infectious disease dance producing coarse (acute chorea)muscular twitchings, incoordination of muscles, ataxia which are lost during sleep. Temporary lesion in subcortical region, perhaps the caudate nucleus • Parkinson's disease (Paralysis agitans) Rhythmical tremor of the resting limb. Organic affection of the central nervous system. Gerebello-pont ine angle tumor Tinnitus and vertigo followed by gradual deafness. Tumor on cerebellopon-tine angle. T o r t i c o l l i s ; A tonic-olonic spasm of the neck muscles. Occurs in Pott's disease produced by cervical changes and mechanical misalignment. Labyrinthine T o r t i c o l l i s ; As above Due to i r r i t a t i o n of the semioircular canals on one side of the head. Central T o r t i c o l l i s As above Follows organic changes in the region of the basal ganglion* 33 D I S E A S E SYNDROME PATHOLOGY Dystonia museularum (Tortlpelvis) Abnormal constant shifting tonus of the muscles character-ized by irregular, Involuntary, clonic contortions of the trunk and proximal muscles of the ex-tremities. Symptoms are lost when rest-ing but reoccur with voluntary movement. Abnormality most pronounced in walking when the body is bent and twisted forward and sideways. Produoed by progressive degeneration in the lenticular nucleus* Pyramidal tracts are normal. Reflexes sen-sation 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 tinnitus and increasing d i f f i c u l t y in hearing. A sen-sation of rotation of the body accom-panies the attacks. Gross dilation of the endolymph system and particularly the saccule, which occupies nearly the entire vestibule, The dilated soala media of the cochlea dis-places the Re is snap's membrane to the wall of the soala vestibule and thus obliterates the perilymph space. This increase in pressure causes the inner ear to be ex-tremely sensitive and slight .changes in pressure results in disturbances in the function of the labyrinthine end organs. - 34 -TV Materials and Methods A. Origin 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 built up from these original mice has been used in this Investigation into the physical basis of the pir.ouette mutation; B. Care of Mice: 1. Cages Plywood cages measuring 24"~£l2lt divided by a central partition into two compartments were used to house the mice. A semicircular hole out in the base of the partition enabled the mice to pass from one side of the box to the other. The only opening through which light and air could enter the cage was by a window 3^ X4"' covered with wire mesh. Two wooden lids fitted over the top of the box allowed easy aooess for feeding and cleaning. Twice weekly the mice were put into clean oages, soiled boxes were emptied, washed and thoroughly 4Med before being used again. Water dishes were r e f i l l e d daily, while food containers were washed when the mice were moved into clean cages. The floor of the oage was covered with dry peat moss. A Jar of water and a petri dish of food were placed below the window and a small amount of cotton batting was put in the other side of the box. Bight to ten mice were kept in the cages * 35 that were used for maintaining the stock. Only two mice were placed in the breeding cages and the female was often isolated while she was raising her young. This method of housing kept the mice warm and protected them from drafts. The room in which the cages were kept was heated during the winter months to prevent the mice from beooming chilled. This method of care and feeding proved to be satisfactory u n t i l the room was re-quired for other purposes. The mice were then removed to the animal husbandry laboratory where they continued to receive similar care. 3. Pood The mioe were given a balanced diet by feeding them fox cheokers or dog meal pellets. In addition they were frequently fed; grass, clover, groundsel and dandelions. A plentiful 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 Identification. The system of numbering suggested by G.B. Snell was used In order to keep adequate records of the mice* By this method the mice can be numbered from one to ninety-nine. A record of each mouse was kept on a 4Z5* library card. Its number, sex, mutations, date of birth and death were recorded on the card* Observations, on the behavior of the mouse were also noted as well as matlngs and the number of l i t t e r s produced. - 36 C* Preparation of Material. 1* K i l l i n g and Fixing* The mioe used for gross and histological studies were k i l l e d with ether. The parts required were immediately dissected out and fixed in 10% saline formaldehyde E. Histological techniques. 4.The l i v e r . Pieoes of l i v e r , fixed in formaldehyde, were embedded in paraffin. Seotions 10^ thick were stained with either alum haematoxylin and eosin or Feulgen's nuclea* stain and light green. b* The ear After fixation in formaldehyde the inner ears of the mice were decaljoifled with either a 5$ solution of n i t r i c acid in 70$ alcohol or a 5$ aqueous solution of formic acid. The latter method was found to be preferable since i t did not adversely effect the stains which were employed* The ears were stained either before or after embedding in paraffin. After the tissues were dehydrated i t was found advisable to clear them with n-amyl acetate before embedding in paraffin 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 in paraffin the ears were cut into sections 151* thick and mounted on slides with egg albumen. - 37 -Several stains were employed to show the various structures within the ear. Alum haematoxylin and eosin were employed as a general s t a i n . Mallary's t r i p l e s t a i n was employed for a study of the blood vessels and connective t i s s u e . March's osmic acid s t a i n was used to study changes within the mylen sheaths of the nerve f i b e r s . Cajal*s s i l v e r s t a i n was used on the normal nervous tissue within the cochlea of the mouse. ~ 38 • ¥ Comparison of the Behavior of  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 their effect on the be-havior of the mutant mice. Dickie and Woolley (1945-1946) have described the general effect of this imitation and have determined i t s position on the third chromosome of the house mouse• A. Adult Mice. When awake the pirouette mouse Is in constant motion; nodding its head, moving restlessly around its 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 active. The head is tossed back over the shoulders and is often drawn slightly to one side so that the mouse appears to be constantly sniffing the a i r . If the mouse is held in the hands one can feel the trembling r muscles of the neck. This tremor gradually increases u n t i l the head is suddenly pulled back. The neck muscles then relax before the tremor begins again. Thus the head nodding is apparently due to involuntary tonio-olonic spasms of the neck muscles. Pirouette mice are able to move along straight lines as do normal mice; however, the mutant mouse frequently whirls or runs i n a c i r c l e . The circlin g movement occurs more often among the younger mice, and is usually preceded by violent head shaking. The mouse takes a few steps backwards and then either whirls in the same spot or describes a larger c i r c l e . The cir c l i n g lasts for varying lengths of time and may be either clockwise or anticlockwise. Thus, the mixed circ 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 subjected to the above stimuli the normal mice reacted in several ways. They usually respond to whistling by pricking up their ears and remaining motion-less. When tested with louder noises they respond either by starting violently and darting away or by remaining perfectly quiet and often trembling. The pirouette mouse did not rer 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 re-port of 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* The mutant mouse is indistinguishable from the normal animal u n t i l i t is * 40 -ten days old. At this age, a normal mouse tends to walk in a straight line and seldom shows the cir c l i n g movement; while a pirouette mouse of the same age moves more frequently in circles and is less steady on its feet than the normal mouse. The equilibrium of the mutant mouse is poor and i t frequently loses its balance and r o l l s over on its back; When twelve days old the pirouette mouse takes longer to right 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 latter then responds to sound while the former remains deaf. Atthis age the mutant mouse begins to show head shaking which increases in intensity u n t i l the mouse is approximately eighteen days old. During this period of growth the ci r c l i n g movements occur very frequently. This triard of symptoms gradually increases in intensity u n t i l maturity is reached j after which time the circling movements occur less frequently. The development of the pirouette mouse is compared in greater detail with that of the normal mouse in 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 is more active than the normal. It is constantly in motion and shows incessant head shaking which increases when - 41 -i t becomes excited. The pirouette mouse does not respond to sounds but,,like the normal mouse, shows response to a i r currents, changes i n the intensity of l i g h t and v i b r a t i o n s . It was noted that when a shadow was suddenly cast over the pirouette and the normal mouse both responded by leaping into the a i r and darting away. In t h i s respeot both the_ pirouette and the normal mouse d i f f e r from the waltzing mouse which never jumps. The constant a c t i v i t y of the pirouette mouse can be observed when i t i s placed on a table. The animal moves around and appears to be exploring i t s new environment. It runs aimlessly back and forth on the table and w i l l often o i r c l e . I f i t comes in contact with a wall i t w i l l attempt to climb i t . The normal mouse under the same conditions i s less active and moves cautiously about, often stopping to s n i f f the a i r or to l i s t e n . The locomotor a b i l i t i e s of the pirouette and normal mice were tested to determine the effect of the mutation on the general behavior of the mouse. It has already been r e -ported that mice homozygous for thi 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 Dickie and Woolley (1946). Further observation reveals that normal adult mice when placed i n water are able to swim d i r e c t l y to the edge o f the container and climb out of the water. I f a normal mouse i s submerged - 4 2 -i t r i s e s r a p i d l y to the surface and commences swimming. However, when an adult pirouette mouse i s placed in water i t reacts in either of two ways. It may lose a l l sense of d i r e c t i o n and position and twist and turn under the water, in which case i t w i l l quickly drown, or i t may manage to swim to the side o f the container. |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 of d i r e c t i o n and was unable to regain the surface. Additional tests indicated that i f the adult pirouette mouse was c a r e f u l l y placed i n the water so that i t s head remained dry i t was usually able to swim, but i f i t once l o s t i t s balance i t was unable to regain i t s sense of d i r e c t i o n . It was also observed that the pirouette mouse became fatigued r a p i d l y and i t s movements were not well coordinated. The difference in swimming movements was noted as early as the eigth day. At thi s age the pirouette holds i t s hind legs more s t i f f l y than does the normal mouse. A further comparison of the swimming movements of the normal and pirouetting mice i s contained i n the table i n the following section. The r i g h t i n g reflexes of these mice were i n -vestigated. It was found that when the normal mouse was dropped i t always landed on i t s feet while the pirouette mouse frequently landed on i t s back or side. Thus the equilibrium of these mice i s also effected by the pirouette mutation* The mice were further tested by placing them on - 43 -the wooden handle of the dissecting needle which was held horizontally. The wild type mice were able to balance on the stick, and when i t was slowly revolved they were able to maintain their upright position. The pirouette mice were able to balance on the stick bat eould not maintain an upright position when the stick was revolved. The mutant mouse clung tightly to the stick at f i r s t but tired rapidly and fin a l l y i t let go with its 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 this mutation are of the same intensity in both the males and females. The pirouette males are more aggressive than the wild type males. This tendenoy to fight results in 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 this mutation and the time of their occurrence. Deafness, ci r c l i n g , and head shaking are the main distinguishing characteristics of the pirouette mutation. The iKitant mice do not become dizzy when rotated, are unable to swim as easily and become fatigued more rapidly than do normal mice. Thus the changes produced by- this mutation have a manifold effect 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 rapidly i t is able to move in circles and show the normal righting reflexes. The circling appears to be due to inco-ordination of the hind legs. When placed in water the mouse makes swimming movements with Its fore legs but Is un-able to keep i t s head above the water. The behavior and re-actions of the mutant mouse are indistinguish-able from that of the normal animal. 2. The mouse is able to hold i t s head up as i t travels in circles Swimming Is the same as at 1 day. Indistinguishable from the normal mouse. 3. The mouse usually moves In circles, however, i t can travel along a straight l i n e . The legs are more eo* ordinated when the mouse is in water than at 2 days. Indistinguishable from the normal mouse. 4. The mouse is more active and is able to travel either in circles or along a straight l i n e . A l -though i t s t i l l swims In circles, the hind limbs are used more often. The mouse is able to hold its head above water The movements are the same as seen in the normal animal. In the majority of the pirouette mice the distal end of the pinna of the ear is s t i l l bent down. - 45 -AGE IH DAYS. ij ieRMAl MOUSE rPIROUETTE MOUSE. 4. while swimming. The d i s t a l pinna of the ear which was bent down over the external auditory meatus is now free. Movements as above. Mouse is more active. Movements as in normal mouse. The d i s t a l end of the pinna has be-come free. 6. 7. As at 5 days. Circling movements s t i l l oecur and the coordination of the fore-limbs is s t i l l better than that of the hind limbs. Same as normal mouse. Same as normal mouse. 8. Mouse usually moves in circles but when disturbed It w i l l progress straight forward. The mouse swims with the head held high above the water. At this 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 its 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 stiffness and inco-ordination of the hind legs and one fore leg is used more frequently - 4 6 -AGE IN DAYS NORMAL MOUSE (PIROUETTE MOUSE. 9.'. 'than the other. The mutant mouse swims in circles with its nose under water. When dropped i t does not always land on its feet. 10. The mouse circles or walks in a straight line and is able to ewim. Teats are visible on the females. Mouse moves in circles; and is unsteady on its feet. It swims with its head under water and i t s body tends to lean to one side. The mutant mouse becomes fatigued more rapidly than the normal animal. 11. Locomotion as above. When dropped i t lands on its feet. Mouse is unsteady on its feet and moves in circ l e s . When dropped i t lands more often on its back or side than on i t s feet. 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 in circles and is able to dart around rapidly and swims easily. Its eyes are partly open and i t responds to sounds. The mouse is less steady bn i t s feet than the normal animal. It is very active and circles either to the right or l e f t . A slight head tremor is visible when the mouse is walking. It loses i t s orienta-tion when placed in water and r o l l s and twists below the sur-faoe. Although the mouse does not respond to sound the ears are open. The eyes are partly open. 15. Byes open, active, darts around the cage. Responds to sounds. Eyes open, very active, circle rapidly. It is deaf. Head shaking more pronounced. 16. 17. As above As above As above. More active than the normal mouse, constant-l y in motion, frequent ci 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 trying to feed i t s e l f . 19. Mouse Is active, responds to sounds, swim£ etc. It acts like the adult normal mouse. Mouse is able to care for i t s e l f but is s t i l l more active than adult pirouette mouse. Some animals show swimming movements but when they lose their balance they drown• - 48 -Vf "Invest igat ion of the Possible Pauses for the  Abnormal Behavior of the Pirouette Mouse* A review of the literature revealed many different physical causes for deafness and choreic behavior in 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 in the pirouette mouse. A. Pathological Infection. It is known that patholdgieal Infection of the inner ear w i l l produce deafness and. disturbances of equilibrium in man(Jones, 1918), The abnormalities of these mioe are evidently not due to infection since mioe heterozy-gous for this mutation, although raised among homozygous pirouette slbs, 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 be-havior seen in the mice is produced by some unknown inherited factor. Thus pathological infection as a cause of the abnormal behavior is eliminated. - 49 _ B. Muscular Abnormalities; The possibility 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 significant differences between the normal and the pirouette mice* In a l l cases the muscles of the mutant mice were found to be well de-veloped* C. The Liver. The movements produced by the pirouette mutation are similar 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 this disease are pathological changes within the li v e r , causing i t to become lumpy and discoloured. This abnormal condition is referred to as "hobnail" cirrhosis of the liver (Cecil, 1944). The pirouette mice show abnormal twisting which increases with voluntary movement. Since this condition resembles that of Wilson's disease in man, the livers of < - 5 0 * normal and pirouette mice were compared. Gross examination revealed no difference between the livers of the normal and the pirouette mice. Pieces of liver tissue from both pirouette mice and normal mice were prepared for histolog-i c a l examination as described on page 36. Uo variation be-tween the livers of the normal and the mutant mice was observed. From these results i t is apparent that the pirouette mutation, unlike Wilson's disease, has no visible 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 brain. A gross comparison of brains of normal and pirouette mice was undertaken to determine whether there was an abnormal com-pression of the brain as reported in shaker-short; (Bonnevie 1936). cysts within the subarachnoid space and absence of the flocculus cerebelli as found in Ereisler: (Her twig, 1942 ).y or hydrocephalus interims as in shaker-1 (Gruenberg,1943). A study of the brain of the pirouette mouse did not reveal any of the above abnormalities. The gross anatomy of the pirouette brain appears to be identical to that of the normal animal. However, i t is possible that a histological study ustfing 'special staining techniques might reveal histological abnormalities. - 51 -B. Tumors * Abnormal movements and deafness similar to those produced by the pirouette mutation are known to 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 in the pirouette mouse since a similar syndrome is found to be produced by von Recklinghausen^ disease (neurofibromatosis) in man. This disease is pro-duced by a dominant mutation which results in 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 cranial nerve. The pressure exerted by these tumors causes degeneration of both the auditory and vestibular nerves resulting in deafness and abnormal movements. The possibility of the formation of tumors upon the eighth cranial nerve of the pirouette mouse was in-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 If tumors were inter-fering with any of the nerves in such a way as to result in abnormal movements. The results of this 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 in both the old normal and the old pirouette mice. These results indicated that the abnormalities produced by the pirouette mutation apparentlyv«re not produced by tumors. P. The Bar. The pirouette mutation produces deafness, head shaking and circling similar to that caused by the Ereisler, shaker-short, waltzer, and shaker-1 mutations in mice, It is known that the Ereisler and shaker-short mutation's cause developmental changes which result in lateral displacement of the ear vessieles while the waltzer and shaker-1 mutations produoe changes within the organ of Corti of the inner ear. Since i t seemfl l i k e l y that the pirouette mutation might also produce changes in the ear the position and structure of the ear of the wild-type mouse was studied and compared with that of 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 literature was available on this subject. - 53 -1. The Structure and Function of the Normal Bar. The general structure of the ear of the normal mouse is 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 skull where they come in contact with the tympanic membrane. The tympanic membrane separates the external auditory meatus from the middle ear. a. The Middle Bar. The middle ear is located in a hollow cavity within the temporal bone. The sound waves set the tympariio membrane in motion and its vibrations are transmitted through the middle ear by three small ossicles, the malleus, incus and stapes which form an irregular chain across the cavity of the middle ear. The handle of the malleus is pressed against the ear drum while its head is attached to the incus. The inner surface of the incus is 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 fluid of the inner ear* (see figure 1.) - 5 4 -THE MIDDLE EAR OF THE HOUSE MOUSE. X I 5 FIGURE | EUSTACHIAN TUBE TENSOR TYM PA Nf CAVITY OF AUDITORY BULLA TYMPANIC MEMBRANE MALLEUS INCUS STAPES COCHLEA INTERNAL CAROTID ARTERY FACIAL NERVE (VII) This method of transmission reduces the strength of the vibrations picked up by the ear drum to about one-third of their original strength (Beatty, 1932). 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 hits the ear drum the tensor typani and.the stapedius muscles, which are attached to the malleus and stapes, contract by a reflex movement which lessens the strength of the vibrations striking the inner ear. The vibrations produced by the sound waves may also be conducted through the air found in the hollow oavity of the auditory bulla, to the round window and hence to the perilymph of the inner ear; or they may pass directly through the temporal bone to the inner ear (Stevens and Davis, 1947). The air 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 - 56-b. The Inner Ear* The general structure of the inner ear of the normal mouse is similar to that of man. It is embedded in the petrous region of the temporal bone and is composed of three parts, namely, the vestibule, semicircular canals and cochlea, each of 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 lies 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 of the osseous vestibule is found the fenestra ovalis. 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 is connected to the membranous cochlea by the canal reunions. Into the posterior wall of the vestibule open the five orifices of the three semicircular canals, (see figures 2&3). d. Static Labyrinth The membranous labyrinth of the inner ear is sus-pended in a f l u i d , the perilymph, and contains another liquid known as endolymph. The membranous vestibule is composed of two small sac-like structures; the upper one is - 57 -F I G U R E S 28r3 (GRAY, 1949) -58 -the utriculus, and the lower larger one is the saeculus. The latter communicates with the ductus coehlearis of the cochlea by the canal reunions and with the utriclus by a narrow passage, the ductus utriculosaccular i s . The vestigial saeculus endolymphaticus arises from a small opening at the base of the saeculus. The five orifices of the semicircular canals open into the utriolus. The semi-circular canals are named acoording to their position in the head; anterior v e r t i c a l , horizontal and posterior vertical.(see figure 4). The static organs are located within the saeculus and utriculus and also in swellings, the ampullae, at the base of each of the semicircular oanals. The sense organs of the saeculus and utriculus are the macula. These structures are composed of clumps of hair cells whose short hairs project Into a gelatinous membrane, the otolithic membrane within which are embedded the otoliths. The maculae u t r i c u l i lies on the lateral wall of the utriclus while the maculae sacouli occupies the medial wall of the saeculus so that the surface of both maculae are per-pendicular to each other. The pull of gravity upon the otoliths act as the stimulus which is transmitted by the nerve fibers to the brain and informs the animal of the position of its head. The static organ of each semioircular - 5 9 -F I G U R E 4 60 -canal, the crista acoustioa, is composed of groups of .hair cells 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 rise to three branches. The superior branch supplies nerve fibers to the cr i s t a within the ampulla of the anterior and horizontal canals and the maculae of the utriculus; the inferior branch sends fibers to the maculus of the saeculus, while the posterior branch serves the crist a of the posterior semicircular canal, (see figures 5 and 6). a The Cochlea The osseous cochlea appears as a tube which diminishes in diameter as i t eoils around a central axis, the modiolus. A bony shelf, the osseous spiral lamina, projects from the modiolus into the tube and imperfectly divides i t into two passages. (Refer figure 7). The division is completed by the basilar membrane which stretches from the free edge of the osseous spiral lamina to the s p i r a l cochlear ligament which is attached to the outer wall. A third passage is formed by the membrane of Reissner which extends from the crista spiralis to the spiral ligament. These three passages thus formed within - 61 -3 4 S r, v v ., II i„ |f F l O . 9-5.—Ripht human membranous labyrinth, remove*! from its bony enclosure anil viewe.1 front the antcro-laleral aspect. ((!. Ketzius i F l O . 92G.—The same from the po.stero-me.lial aspect. 1. Latera l semicircular eaual; 1'. Us ampul la: 2. I'osle-rior canal; 2', its ampulla. 3. Superior canal; .'I'. its ampulla. 4. ( W j o i i i e i l luiib of *upcn..r an.I posterior canals (sinus utriculi tuprrior). 5. I'triele. V liecessus ulrieiil i . U". Sinus utrieuli posterior. Ii Ductus f l n l i * lymphaticus. 7. Canal i s utriculosaccular!*. S. Nerve to ampul la of suix-rior canal 9. Xervv to ampulla »rf lateral canal". 10. Nerve to recessus utrieuli ( in Fiir. 925. the three branches a M s a r euiijoineil) ID'. Kii i l iui: o l nerve in recessus utriculi. 11. Kaeial nerve. 12. I.atfena cochlea-. I.I Nerve of cochlea within spiral lamina II. Ila.iil.ir membrane. 15. Nerve fillers to macula of saccule. Hi Nerve to ampulla of posterior canal. 17 Saccule. i s Secondary membrane of t.vinpaiiuiii. 19. Caual is reuniens 20. Vestibular end ol ductus lib-iris. 2.1. Section of the facial ami acoustic nerves within internal acoustic meatus (tlie wparatuKi bet we., i t fo in i s n i l apparent in tha section), Retzius J (GRAY, 1948) F I G U R E S 5 8 6 .62-APEX GALLERY Fir,. !>. -Sectional view of the bony cochlea (I'oirier's Anatomy) (GRAY, 1948) F I G U R E 7 .63 -the cochlea are the soala vestibuli, the ductus cochlear is and the soala tympani (See figure 8). The latter is 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 vestibuli is 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 is the central passage, i t s three walls are formed by the basilar membrane, which separates i t from the scala typani; by the membrane of Relssner, which separates i t from the soala vestibuli; and by the sp i r a l ligament which is attached to the walls of the cochlea. This passage follows the spiral windings of the coohlea and ends blindly at the apex, while at its base i t communicates by the canalis reunions with the sacculus. The periferal end organs for hearing are located in the organ of Corti which rests on the inner edge of the basilar membrane within the ductus cochlearis. The organ of Corti consists of the rods of Corti, hair cells and supporting c e l l s . It is 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 of Corti, which rest upon the basilar membrane. The inner rods of Corti 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 of attachment of the basilar mem-brane to the sp i r a l lamina. On the inner side of the inner rods and on the outer side of the outer rods are three or four rows of smaller external hair cells and several rows of supporting c e l l s . The terminal fibers of the acoustic nerve end In contact with these hair cells, which, according to Stevens and Davis (1947) are: "the ultimate sensory cells of the organ of hearing. Above the hair cells of the organ of Corti lies the tectorial membrane which arises from the crista spirales, looated on the inner edge of the osseous spiral lamina. Prom the posterior part of the crista s p i r a l i s , just behind the origin of the tectorial membrane, arises the membrane of Reissner. The periosteum forming the outer wall of the ductus oochlearis becomes thickened to form the spiral ligament whioh is attached to the outer edge of the basilar membrane. The upper portion of the spiral ligament, the s t r i a vascularis, contains small blood vessels and capillary loops. This organ secretes the endolymph which nourishes the organ of Corti. This spiral ligament decreases In size from the vestibule to the hellcatrema, whereas the tectorial membrane increases progressively in size from the basal to the oplcal end of the cochlear canal. (See figure 9). THE ORGAN OF CCRTI FROM A NORMAL MOUSE (227 DAYS OLD) BLOOD VESSEL STRIA VASCULOSA C R I S T A SPIRALIS TECTORIAL MEMBRANE HENSON'S C E L L S BORDER CELLS SPIRAL LIGAMENT PHALANGEAL CELLS - 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 Corti are located within the spiral canal of the modiolus of the osseous cochlea. In the normal adult animal, each bipolar nerve c e l l of the spiral ganglion sends one fiber along the edge of the osseous spiral lamina to the hair eells of the organ of Corti, while the other branch passes along the aeotuatio nerve to the brain. According to Stevens and Davis (1947) the nerve cells within the spiral ganglion are not evenly distributed along the basilar membrane but are more densely congregated in the upper portion of the basilar whirl of the cochlea. The auditory branch of the nerve leaves the spiral canal of the modiolus and joins the vestibular branch at the base of the internal acoustie meatus to form the eighth nerve. This cranial nerve leaves the internal acoustic meatus and passes medially to enter the brain at the base of the inferior cerebral peduncle. The fibers of this nerve ter-minate in the dorsal and ventral cochlear nuclei 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 birth but a l l of them are represented. In the day old mouse - 68 -the s t r i a vascularis is composed of three layers of c e l l s . In the superficial 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 cells the nuclei l i e along the inner edge of the s t r i a vascularis and the cytoplasm projects outward to the center of the organ. The central layer consists of irregular cells with a network of blood vessels and spaces between them* At birth the organ of Corti appears as a layer of high columnar cells whioh latter differentiate into the hair c e l l s , rods of Corti and their supporting c e l l s . The upper surface of these cells come in contact with the tectorial membrane while their bases are separated from the basilar membrane by supporting c e l l s . The spiral ganglion, within the modiolus is composed of closely packed oells 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 10). Five days after birth the cytoplasm of the super-f i c i a l cells of the s t r i a vascularis is beginning to show the striations which produced the characteristic striated appearance of the adult organ. The c e l l membranes of the polyhedral epithelial c e l l s , which are arranged haphazardly - 70 between the superficial and basal cells, are not as distinct as in the day old mouse. At this age the organ of Corti has begun to differentiate and the hair cells and rods of Corti can be distinguished* The nuclfe* of the nerve cells of the spiral ganglia of the 5 day old mouse do not appear to be quite as large as in the day old mouse. In some areas thin f i b r i l s can be seen passing between the cells and out towards the organ of Corti. m the twelve day old mouse the outlines of the cells forming the s t r i a vascularis have become less and their cytoplasm has become more striated than at 5 days of age. Many blood capillaries as well as numerous blood spaces can be seen passing between the cells of this organ* The organ of Corti was broken during sectioning but the remaining rods and hair cells appear to have reached their adult form. The cells of the spiral ganglia were closely packed and two types of cells can be distinguished. The large palyhedral nerve cells have large round nuel£fc surrounded by granular cytoplasm. The nuolet of the small neuroglia cells are dispersed between the nerve cells and their outlines are indistinct. Fibers can also be identified as they pass from the organ of Corti and between the cells of the spiral ganglion. - 71 -No variation 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 characteristic striated appearance. The three separate c e l l layers of the organ can he distinguished and numerous blood spaces and capi l laries can be seen between the c e l l s . (See figure 11 and plate I I ) . The organ of Corti is well developed and the rods and hair cel ls are dis t inct , and deeply staining. The sp ira l ganglia is composed of many closely packed nerve cel ls which have large nuclei;. Their dendrites can be seen passing to the organ of Corti while their axons can be followed into the auditory nerve within the modiolus. The supporting ce l l s , dispersed among the nerve ce l l s , have small nuel&c. Thus these two types of cel ls within the sp ira l ganglion can be readily distinguished by the size of their nuclfep. Several blood vessels which A nourish this organ can be seen passing between the cells of the s p i r a l ganglia. (See plate I I I , IV, V t and V I ) . - 73 -2* The Structure of the Bar  of the Pirouette Mouse After having obtained a knowledge of the structure of the ear of a normal mouse the sources of possible ab-normalities within the ear of the pirouette mouse were ex-amined. Since many choric mutations such as E r e i s l e r , shaker-short, waltzer and shaker-1, are known to produce abnormal-i t i e s within the ear a car e f u l investigation of the pirouette ear was undertaken. In order to determine when the abnormalities f i r s t occured and whether there was pro-gressive change and degeneration the ears of pirouette mice Of the following ages: 1, 2, 5, 12, 15, 20, 23, 25, 30, 35, 55, 60, 85, 100, 260 and 434 days were studied and com-parison made with the ears of normal mioe of approximately the same ages. a. P o s i t i o n of the Bar Yesaiole. A l a t e r a l displacement of the ear ve s s l c l e s and mal-development of the inner ear has been found to occur i n both the E r e i s l e r (Hertwig, 1944) and shaker-short (Bonnevie, 1936) mutations. Hence, the po s i t i o n of the auditory capsule of the pirouette mouse was Investigated. Q T O S B dissections of the ears of both newly born and adult normal and pirouette mioe were made and the position and development of 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 Static Labyrinth Abnormalities in the development of the vestibular system have been recorded in the waltzer,(Yerks 1902), shaker-short (Bonnevie 1936) and E r e i s l e r (Hertwig, 1 9 4 4 ) _ mutations. It is also known that, in man, the occurrence of pathological changes within the vestibular system eauses disturbances of equilibrium (Jones, 1918)* Normally when the static labyrinth is stimulated i t causes nystagmus as well as reaction movements of the body. If 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 rotation 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 in the position or develop-ment of the semicircular canals. Microscopic study of this system indicated that unlike the waltzer, shaker-short and Ereisler 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 12 , 15, 20 , 2 3 , 30 , 3 5 , 50, 55, 60 and 100 day old pirouette mice were examined, m a l l cases the end organs of the vestibular system were well developed. The nerves leaving these structures were traced to Soapa1s ganglion. When Mallory's stain was employed no variation was observed in the branches of the vestibular nerve in normal and pirouette mice. However, Marchi's osmic acid stain revealed some signs of degeneration within the myelin sheaths of the vestibular nerve of a twenty day old mutant mouse. In this case the osmic acid had blackened the majority of the myelin sheaths but many of these appeared unbroken and evenly stained and did not show the broken nature typical of the degenerating sheaths. The few de-generating myelin sheaths were traced to the cristas and maculae. These end organs, however, appeared completely normal. 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 in any of the ears examined. Cabal's silver stain was employed to study the nerve cells of Seapa's ganglion in a 60 day old pirouette mouse. This method of staining failed 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 this 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, ab-normalities 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 ab-normalities 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 of Corti the degeneration is most pronounoed in the "basal whirl of the cochlea. In this region the rods of Corti are apparently normal hut the outer hair oells and supporting cells have become less distinct and are not upright as in the normal animal. (See plate VIII). The degenerative changes are most pronounced within the s p i r a l ganglion. In the basal whirl of the spiral ganglion the majority of the nerve cells have dropped out and the structure appears to be composed mostly of thin fibers, small scattered nuel& of the neuroglia cells and;a-few blood vessels. (See figure 14 and plate IX, X^  and XI.) d. Post-natal -Development of the Cochlea of  the Pirouette Mouse. Since a histological study of the cochlea of an adult pirouette mouse revealed abnormalities within the spiral ganglion, stifia vascularis and organ of Corti a de-velopmental investigation was undertaken to determine the age at which these changes occur. A summary of the results of this 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 birth the structures within the eochlea of the pirouette mouse are" identical to those found in the normal animal. Changes it) " SPIRAL GANGLION' OF A PIROUETTE MOUSE (35 DAYS) CRISTA SPIRALIS SPIRAL GANGLION NERVE CELL NEUROGLIAL CELL BLOOD VESSEL FIGURE 14 1 CD -82 -within the spiral ganglion were f i r s t observed in the basal whirl of the cochlea of a 20 day old mutant mouse. In this region the cells of the ganglion were not as closely packed and the number of nerve oells had been reduced. These de-generative changes within the spi r a l ganglion become more pronounced as the mouse becomes older. The loss of the nerve cells within this ganglion commenced in the basal whirl of the cochlea and gradually progressed to the apex. (See table V.) The number of nuclei within a given area of spiral ganglia of normal and mutant mice was oounted to de-termine the extent of the nuclear loss. A Howard mold count disc was used in making these counts and the number of nuclei within a single square was recorded. In a l l specimens nuclei of comparable regions were counted and the numbers averaged. These counts indicated that the greatest loss occurs in the basal whirl of the cochlea and the apical whirl is least affected (See table V). Counts were also made of the large and small nuclei within the basal whirl of the cochlea of normal and pirouette mice to determine which type of nuclei was being lost. (Refer table VI.) The results of these counts indicate that there is a progressive loss of the large nuclei while the small nuclei remain f a i r l y constant. Cajal's silver stain indicated that the large nuclei were those oi the nerve cells while the small nuclei belonged to the neuroglia cells.(plate X) - 83 -T A B L E V . Number of nuclei In the basal, central and apical whirls of the s p i r a l ganglion in 1 square of an occular g r i d counter. A. Normal Mouse. BASAL WHIRL. • CENTRAL WHIRL. * APICAL WHIRL.. 1 50 48 47 5 45 46 48 I E 40 40 45 1 5 49 42 40 S I 58 43 3 7 3 5 56 41 36 60 49 40 4 4 1 0 4 5 4 42 43 S6S 59 45 50 3 9 0 53 48 47 B. Pirouette Mouse* A1AY S W B A S A L WHIRL. CENTRAL WHIRL. APICAL WHIRL. 1 49 46 48 5 40 4 4 41 12 42 4 5 43 1 5 48 43 3 9 SO 4 4 47 48 S3 47 3 4 42 85 4 5 3 7 46 3 0 36 3 4 3 5 3 5 37 3 4 36 55 1 7 31 37 60 3 4 36 38 1 0 0 37 3 3 41 8 6 0 39 3 3 3 4 4 3 4 30 41 39 - 84 -TABLE VI Number of small land large nuclei i n the basal whirl of the s p i r a l ganglia i n 1 square of an occular g r i d counter. A. Normal Mouse. AGE IN DAYS • LAP.GS NUCLEI. . SMALT, NUCLEI. . TOTAL 12 13 33 46 15 19 30 49 21 33 25 58 35 34 ' 32 66 60 25 27 52 104 21 33 54 262 33 37 70 390 34 28 62 B. Pirouette Mouse. ^DAYS3 LARGE NUCLEI. SMALL NUCLEI. TOTAL 45 50 46 47 45 38 29 17 42 29 27 29 20 12 13 32 15 16 34 20 14 32 23 27 2Q 25 12 33 30 9 29 35 7 22 55 1 16 60 8 34 85 11 18 100 i i 16 260 5 24 434 2 18 - 85 -Changes within the s t r i a vascularis were f i r s t observed in the basal whirl of the cochlea of the 23 day old mutant mouse. The cytoplasm of the s t r i a vascularis gradually lost its normal striated appearance and became granular. As the mouse became older (35 days) there was a slight reduction in the amount.of oytoplasm but the number of nuclei remained approximately the same as in the normal animal. This results in the cells 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 in the 434 day old mutant mouse in which the entire s t r i a vascularis was reduced to a thin layer of undifferentiated tissue. It was not possible to trace the early progress of the degenerative changes within the organ of Corti of the mutant mouse since this structure was frequently broken during the preparation of the microscope slides. However changes within the organ of Corti were observed in the 55 day old pirouette mouse. In the basal whirl of the cochlea the outer hair, cells and supporting cells did not stand as upright as in the normal mouse. These changes within the organ of Corti were more pronounced In the 60 day old mutant mouse. In the 85 day old mutant mouse the rods of Corti were normal but the hair cells are less distinct a&d - 86 -t h e i r nuclei, do not s t a i n as. deeply as in the 60 day old animal. In the oldest pirouette mouse examined (434)days) the organ of C o r t i within the "basal whirl of the cochlea was reduced to a mass of undifferentiated t i s s u e . In the central whirl only the rods of Co r t i could be r e a d i l y distinguishea while at the apex the hair e e l l s s t i l l remained although they showea signs of aegeneration. Thus the changes within the cochlea became progressively more pronounced as the age of the pirouette mouse increased. (See appendix). -87 -G . The Skull. Mellanhy (1938 & 1843) observed that vitamen A deficiency in dogs produced deafness and choric movements similar to those found in pirouette mice. He found that vitamen A deficiency produced bone overgrowth which re-sulted in increased intercranial pressure and a stretching and compression of the cranial nerves. This osseous pressure upon the eighth nerve produced degeneration of the spiral ganglion similar to that found in the pirouette mouse. Hence, the heads of normal and pirouette mice were sectioned longitudinally through the sagittal suture of the skull so that the relation of the cranial bones to the brain could be observed. Measurements were also made of the thickness of some of the bones (see tables VI & VII) This investigation of the bones of the skull and their relationship to the brain did not reveal any significant differences between the normal and pirouette mice. However, i t is possible that special techniques may reveal regions of bone overgrowth in the foramena of the cranial nerves. - 88 -TABLE.VII Measurements of Bones of the S k u l l . Adult Normal Mouse. LEEISTH OF DEPTH OF DEPTH OP PARIETAL FRONTAL PARIETAL PARIETAL BONE . . PARIETAL . OCCIPITAL . BONE SUTURE . SUTURE -89.7 8 3 7 7 4 85.7 7.7 7.5 4 90 8 7 5 86.5 . 9 9 5 87.5 8 10.5 5 85.f 8 4 83 6 6 3£ 77 . H 7 4 80 9 7 4* 82 8| 7 4 Aver-age. 81 8 7 4 TABLE VIII. Adult Pirouette Mouse. LENGTH 6P DEPTH OP DEPTH OP :  PARIETAL . FRONTAL PARIETAL PARIETAL BONE _ PARIETAL OCCIPITAL BONE . SUTURE - SUTURE -94.5 8 9.5 87.7 7 9.5 95.5 7.7 8.7 95.5 7.7 7 90 8 7 77 9 6 82 7 7 80 7 8.5 85 8 7.5 Aver- -age.86 4.5 4.3 4.5 4 4.5 4 4 4.5 4.5 8 - 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) in the cochlea of the shaker-1 mouse. In both these mutant animals the structures within the cochlea were apparently normal twelve days after birth. 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 old whereas the pirouette mouse was deaf throughout i t s entire l i f e . In the cochlea of the shaker-1 mice abnormalities were f i r s t observed in the s t r i a vascularis then In the organ of Corti and f i n a l l y within the spiral ganglion. The degenerative changes within the s t r i a vascularis resulted in a lose of cytoplasm with a relative increase of the nuclei and vascular spaees. In the organ of Corti the de-generative changes commenced in the outer hair cells and progressed Inwards toward the rods of Corti, u n t i l the entire organ was reduced to a small and compact hillock of undifferentiated tissue. Changes in the density of the ganglionic tissue were observed within the spiral ganglion. This thinning of the ganglion cells 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 in starvation of the cells forming Corti*s organ and that this, in turn, would result in degeneration of the nerve fibers and ganglion serving this organ. In the pirouette mouse changes were observed with-in the spiral ganglion 20 days after birth 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 old. 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 in the same order. Degeneration of the spiral ganglion, organ of Corti and s t r i a vascularis also occurs in the Japanese waltzing mouse. According to Gruneberg (1943) this mu-tation results in a reduction in the size of the s t r i a vascularis, slight degeneration of the hair cells within the organ of Corti and degeneration of the spiral ganglion. He states that van Lennep suggests that a degeneration of the s t r i a vascularis would result in changes within the organ of Corti and secondary degeneration within the spiral ganglion On the other hand Gruneberg reports that Yasuhara (1935) found no degeneration in the organ of Corti and regards the degeneration of the spiral ganglion as primary. Thus the exact mature, extent and sequence of the physical changes / - 91 -in the waltzing mouse are s t i l l contraversial. However the changes occuring in the pirouette mouse are similar in sequence to those reported hy Yasuhara. Inherited ataxia in the rabbit produces similar changes within the inner ear. Gruneberg (1947) states that this mutation results in 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 in the guinea-pig produces degenerative changes within the s t r i a vascularis, organ of Gorti and later in the spiral ganglion. This mutation does not produce changes within the vestibular nerve. Gruneberg (1947) suggests that these ehanges are due to lesions in the central nervous system. Since in pirouette mice the degenerative changes occur f i r s t in the spiral ganglion i t suggests that the primary change may likewise be due to lesions in the central nervous system. The waltzing mutation in Peromyscus also produces degenerative changes within the spiral ganglion of the cochlea. In this case the degeneration of the cochlear nerve was fou#d to be caused by bilateral tumors on the eighth cranial nerve. In man, similar degenerative changes within the spiral ganglion are produced by the presence of tumors on the eighth cranial nerve. Since no tumors - 92 -were present on the eighth nerve in the pirouette mouse, the primary degenerative changes must have another cause* Arteriosclerosis of the labyrinth in man is known to produce degeneration of the s t r i a vascularis, organ of Gorti and spiral ganglion. When the "blood supply passing through the internal auditory artery is partially or wholly cut off atrophy occurs within the s t r i a vascularis, organ of Corti, spiral ganglion and nerve endings within the vestibular system. These structures are replaced hy de-posits of connective tissue. The "blood supply to the organs of the inner ear was imvestigated in the pirouette mouse and was found to he similar to that of the normal animal. Even after degener-ation had occurred the affected structures had a normal blood supply. Hence, i t is unlikely that degenerative changes are produced by alteration of the blood supply to the inner ear. In man, otosclerosis, which is produced by a dominant mutation, results in 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 result in degeneration of the eighth cranial nerve. Similar degenerative changes were reported by Mellanby (1938, 1943) who observed that^in dogs, a vitamin A deficiency produced bone overgrowth which r e s u l t e d in a stretching and compression of the eighth nerve. The abnormal pressure caused the nerve fibers of the cochlear division of the eighth nerve and the spiral ganglion to degenerate. In severe cases a l l the nerve cells within the spiral ganglion were lost and a newly formed bone was ob-served within the mftdiolus. Many of the nerve fibers of the vestibular portion of the eighth nerve were destroyed but Scapa's ganglion unlike the spiral ganglion, was more resistant and in most oases remained unchanged. Examination of the cochlea of the pirouette mouse revealed similar de-generative changes within the spiral ganglion but no abnormal deposits of spongy bone were observed within the modiolus. However, the possibility of an overgrowth of bone occurring within the foramen of the eighth nerve has not been in* vestigated. Experimental studies have shown that severance of the eighth nerve w i l l cause degeneration of the spiral ganglion although the peripherial 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 in 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 -cells of Corti's organ were well preserved and the vestibular system showed slight changes although l i t t l e remained of the nerve fibers and cells within the spiral ganglion. The central canal of the modiolus and the canal of Rosenthal were occupied chiefly 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 spiral ganglion of the mutant mouse showed degenerative changes before any abnormalities were observed in the s t r i a vascularis or in the organ of Corti, i t seems possible that the primary change originates in -either the eighth cranial nerve or within the central nervous system. - 95 -VIII COBTCLTJSIOH. Mioe homozygous for the pirouette mutation show characteristic c i r c l i n g and head shaking movements when they are fourteen days old. ffiese mioe are apparently deaf throughout their entire lives. A survey of the literature revealed many possible sources of abnormalities which could produce the atypical behavior of the pirouette mouse. These reported sources of abnormalities were investigated in the pirouette mouse with the following results: 1. Muscles of the head, neck and fore-limbs -- normal. £. l i v e r - gross and histological structure normal. 3. Brain >- gross anatomy - normal. 4. Skull - gross anatomy - normal. 5. Eighth nerve •* gross and histo-logical studies revealed, no tumors. 6. Ear - gross anatomy of middle and inner ear, and blood supply -normal. 7. Oochlea n histological studies showed abnormalities in the following structures: - 96 -a. Spiral ganglion - -Loss of nerve cells observed in basal whirl of ganglion at 20 days after birth. Loss of cells progresses from base to apex and becomes progressively more pro-nounoed with Increasing age ( refer chart A) b. Stria vascularis -Striations not as pronounced as In normal mouse at 23 days. Cytoplasm becomes more granular, cells less compact, (refer ehart A). c. Organ of Corti -Outer hair cells less upright in 55 day old pirouette, loss of dlstlnet appearance. De-differentiates f i r s t in basal whirl. At 260 days organ of Corti in basal whirl a mass of undifferentiated tissue (refer chart A). These changes are similar to those reported in waltzing and shaker-1 mice, but the time of onset and se-quence of the degenerative changes are different. The extensive loss of nerve cells within the spiral ganglion later followed by degenerative changes in the s t r i a vascularis and organ of Corti suggests that the cause of these changes lies outside the cochlea. The fact that the degenerative changes of a l l structures are found f i r s t and most extensively in the lower whirl and later in the middle and upper whirls, also supports the contention that the ohahge is external to the cochlea and gradually Involves the whole organ. Similar changes are known to be produced by - 97 -tumors, peseous compression, severing of 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 possibilities of osseous compressi:on or lesions within the brain causing this abnormality. , 98 -I X Summary. A review of the l i t e r a t u r e indicated several possible sources of abnormalities which would account for the deafness and abnormal movements seen i n the pirouette mouse. T. The following structures were found to be normal, i n the pirouette mouse. A. Muscles of the head, neck and thorax. B. L i v e r . C* Absence of tumors on the eighth nerve. D. Position of the ear v e s s i o l e s . E. Development of the middle ear. ]?. Osseous labyrinth of the inner ear. G. Position of the structures within the cochlea. II Changes were found in the following structures within the cochlea of the pirouette mouse. A. S p i r a l ganglion - .1. Loss of nerve c e l l s occurred in, ... the basal whirl of the ganglion at 20 days and became progressive-l y more pronounced with Increasing age. The loss of c e l l s commenced in the basal whirl and progressed to the apex of the cochlea* B. S t r i a vascularis -Stri a t l o n s not as pronounced as in the normal mouse at 23 days. Cyto-plasm becomes more granular and c e l l s are less compact. These changes more pronounced In the basal than the opical whirls of the cochlea. 99 -Organ of Co r t i -Outer hair c e l l s show degenerative changes i n the 55 day old mouse. The organ of Corti f i r s t de-di f f e r e n t i a t e s in the basal whirl of the cochlea. - 100 -BIBLIOGRAPHY 1 ; Anderson, W.A.D. 1946 Synopsis of Pathology, St. Louis^ G. V. Mosley Co. Amadon, A.M. 1917 Atlas of Physiology and Anatomy of the Human Body, Boston, L i t t l e , Brown and Company. Barr, B.£», 1947 The Structure of the Brain and Spinal Cord in Man, -Department of Anatomy, Faculty of Medicine, University of Western Ontario. Beatty, R.L., 1932 Hearing in Man and Animals, London, Gr. B e l l and Sons L t d . Beers, C.V. and Cheever, E.A., 1945 Hereditary ataxia, The Journal of Heredity, Vol.36, no.11, Nov. pp 335-344. Bing, R. 1940 Compendium of Regional Diagnosis In Lesions of the Brain and Spinal Cord, St. Louis, C.V. 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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 University Press, Vpl, no, No. 1, Dec. 15, - 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 Bell .z* dtsch, Aerglebl, Z% 119-180. - I l l -P L A T E S -i 112 « PLATE I Longitudinal section through the cochlea of a. norOM.1 260 day old mouse showing basal and central w h i r l s . Note densely staining s p i r a l ganglion. S i c t i o n stained with haemotoxylin and eosin. Magnification of i l l / - 1 1 4 -P L A T E I I . S t r i a vascularis of the "basal whirl of cochlea of a 260 day old Amouse showing the blood vessels and the deeply staining s t r i a t e d cytoplasm. Section stained with haemotoxylin and eosin. Magnification of 48^. - 1 1 5 -- 116 PLATE III. Section of the cochlea of a normal 60 day old mouse showing densely staining s p i r a l ganglion and f i b e r s of tthe auditory branch of the eighth nerve. Section stained with Mallory fs t r i p l e s t a i n . ' Magnification of 4-«4 P L A T E III 118 -PLATE 17. Area of the spiral ganglion of the 60 day old normal mouse (see plate T) showing neuroglia nerve cells and their fibers. Section stained with Mallory fs triple stain. Magnification of 154-0. PLATE IV - 120 -PLATE V. Spiral ganglion of the "basal whirl of a normal 35 day old mouse showing the normal dove-tailed pattern foamed hy the nerve cells and their fibers. Section was stained with haemotoxylin and eosin. Magnification of 4(.(. - 121 -PLATE V - 122 « PLATE V I . Ganglion cells from the spiral ganglion of the basal whirl of a normal 35 day old mouse (see plate V.) Note the fibers leaving the deeply staining nerve cells and the lighter nuclei of the neuroglia c e l l s . Stained with haematoxylin and eosin. Magnification of / 5 4 0 . - 1 2 3 -PLATE VI 12a • PLATE HI. Section through the cochlea of a 35 day old pirouette mouse showing a thinning of the cells within the spiral ganglion. The degenerative changes are more pronounced in the ganglion within the basal whirl of the cochlea than in the central whirl. 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 . Stained with Mallory's tr i p l e stain. Magnification of 14-81. PLATE VIII - 128 -PLATE IX. Spiral ganglion from the basal whirl of a 50 day old pirouette mouse. Note the small nuclei of the neuroglia cells and the loss of nerve cells* Stained with haemotoxylin and eosin. Magnification of 4-1 - 129 -PLATE IX - 130 -P L A T E X . Area of the spiral ganglion of a 30 day old pirouette mouse • (see plate I X ) Note the small neuroglia cells, blood vessels and loss of nerve cells* Stained with haemotoxylin and eosin. Magnification of i5<vo. - 131 -PLATE X - i 132 -PLATE 21. Area of sp ira l ganglion of the basal whirl of a 60 day old pirouette mouse. Note the scarcity of nerve cells and the open network formed by the remaining f ibers . Stained with haemotoxylin and eosin. Magnification PLATE XI -134-PLATE XII Longitudinal section through the ductus cochlearis of a 260 day old pirouette mouse stained with Mallary's t r i p l e s t a i n , ^ote the degenerative changes within the s p i r a l ganglion and the organ of C o r t i . Magnification of Zo6-P L A T E X I I -136-v PLATS XIII The s p i r a l ganglion of the basal whirl of the cochlea of the S60 day old pirouette mouse shown in plate XII. Note the loss of merve c e l l s and t h e i r f i b e r s . Magnification of \5ZL> - 1 3 7 -P L A T E XIII -138-PLATS XIV Area of the s p i r a l ganglion of a normal 60 day old mouse stained with Cajal's s i l v e r s t a i n . Note the nerve c e l l s and t h e i r f i b e r s . Magnification of i5Z(*-P L A T E XIV -140-PLATS XV Area of the s p i r a l ganglion of a 30 day old pirouette mouse stained with Cajal's s i l v e r s t a i n . Note the spaces between the c e l l s and the reduction im the number of nerve c e l l s . Magnification of .52-1*. -141-PL/ATE XV -142-PLATB XVI Area of the s p i r a l ganglion of a 60 day old pirouette mouse stained with Cajal's s i l v e r s t a i n . Note the reduction in the number of nerve c e l l s and thei r f i b e r s . Magnification of I5ZU-P L A T E 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 STAIN VESTIBULAR STRXA VASCULARIS ORGAN OF CORTI . SPIRAL GANGLIA . Normal H & E Semicircular canals,saeculus and u t r i c u l u s are a l l represen-ted. The maculae and cit^TSB? have not e n t i r e l y attained t h e i r adult form. Their sensory c e l l s are colum-nar, few nerve f i b e r s , pass i n -to Scapa's Ganglion. 2-3 rows ©f e e l l s . Deeply staining n u c l e i l i e at the peri-f e r a l edge of the organ and t h e i r i r r e g u l a r cytoplasm ex-tends inwards. Basal n u c l e i l i e ©n the inner edge ©f the organ. Between these layers i s a central layer and many blood v e s s e l s . undifferent-iated colmninar e e l l s . Tec-t o r i a l mem-brane r e s t s on the surfaces ©f the future h a i r c e l l s . Consists ©f large round n u c l e i surrounded by granular cytoplasm. C e l l s have a dove-tailed appearance. Pirouette H & E Mallory Broken end organs appear normal As above, As above As above Pirouette 2. H & E Broken Cytoplasm more st r i a t e d than As above As above at 1 day. Kormal H & 1 Broken. End - ©rgans better developed. Stri a t e d cytd-plasm deeply staining n u c l e i . Many c a p i l l a r i s and blood spaces present. 3 l a y -ers of c e l l s can be distinguished. Arches of 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 torn. Nuclei smaller and cytoplasm more fibrous than at 2 days. Pirouette 5. H & E As above. As above. As Above. As above. Normal 12. H & E Labyrinth normal. End organs well developed. More f i b e r s seen passing to Seapa*s ganglia. Striated cyto-plasm deeply staining nuclei, Numerous blood spaces and c a p i l l a r i e s . Has general s t r i a t e d appear-ance of adult organ. Br ©ken. Remaining h a i r c e l l s normal. Rods of Go 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 above. As above. As above. As above. Normal 15. Mellary As above. Cytoplasm more s t r i a t e d . Broken, r e -maining h a i r e e l l s and rods ©f C o r t i w ell developed. As above. Pirouette 15. I kW As above. Marchi None of the myelin sheaths were blackened with the osmie ac i d . Deeply staining lr©ken. n u c l e i , s t r i a t e d Remaining h a i r darkly staining c e l l s w e l l de-cytoplasm, veloped• Ganglia composed of many large nucleated e e l l s and small nuelei Fibers between e e l l s . Pirouette 20. Marchi Mallery Mall©ry s t a i n does not reveal any change. Marchi blackens many myelin sheaths of vestibular nerves. A few of these apparently are degenerating. Nuclei c l o s e l y paeked. Cyto-plasm not as st r i a t e d as at 15 uays. Poorly stained but apparently normal. Mallory*s s t a i n shoved a reduction i n number of large n u c l e i i n the basal w h i r l . Marchi blackened 3th nerve and some of these f i b e r s shewed degenerative changes. Normal 21. March! Labyrinth broken. Cytoplasm s t r i -End organs well ated. Many Mallory developed. blood vessels v i s i b l e . Broken. C e l l s c l o s e l y packed. Nerve and neuroglia c e l l s and f i b e r s w ell developed. Pirouette 23. Marchi & Mallory Nerves not blackened with Marchi stai n and are apparently normal. As at 20 days. Broken Cytoplasm granular* Blood vessels v i s i b l e . As i n 20 day p i p i . However greater l o s s ©f c e l l s i n basal w h i r l . Pirouette 25. H & E Broken. Remaining e r i s t a apparent* l y normal. Granular cyto-plasm few s t r i a t i o n s . Few eentral n u e l e i surrounded by clear cyto-plasm. Few vascular 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. In upper w h i r l ganglia c e l l s s l i g h t l y l e s s dense than normal. Lower whirls large n u c l e i sparse and many f i b e r s and neuroglia c e l l s . Normal 28. Mallory As i n 21 day old normal mouse • As i n 21 day normal mouse old As i n 21 day old normal mouse. As i n 21 day ©Id normal mouse. Pirouette 3 0 . H & 1 Labyrinth Deeply st a i n i n g . Organ ©f C o r t i Basal ganglia normal, end Cytoplasm mere tern. P i l l a r s ^ degenerating. Mallary ©rgans well granular than normal, l e - * Few f i b e r s seen developed. s t r i a t e d . maining h a i r leaving organ C a j a l . Scapa's ganglion Light areas c e l l s of C o r t i and normal. around some apparently passing t o n u c l e i . normal. nerve c e l l s . Nuclei l o s t i n cen t r a l ganglia. Pirouette 35. H & E Mallory Broken. End organs apparent-l y normal. S t r i a t i o n s not marked and cyto-plasm granular. In lower w h i r l s t r i a t i o n s l o s t . Amount ©f cyto-plasm reduced. Many blood vessels. Broken. P i l l a r s and remaining h a i r e e l l s appear to be normal. Few nerve c e l l s . Many f i b e r s and neuroglia c e l l s . Blood vessels also present. Normal 37 H & E Removed before sectioning. Well developed. Cytoplasm s t r i a t e d and deeply stained. Hair c e l l s and p i l l a r s of C o r t i are d i s t i n c t and well developed. Well developed c l o s e l y packed Nerve and neuroglia c e l l s . Fibers seen between c e l l s . Pirouette 50. C a j a l Broken. End organs apparent-l y normal. Seapa* s ganglia 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 v a s c u l a r i s . Amount of cyto-plasm appears to be redueed. In lower w h i r l h a i r c e l l s degenerated and Hods of C o r t i not d i s t i n c t . In central w h i r l outer h a i r e e l l s i n d i s t i n c t . Number of nerve c e l l s reduced. Many remaining nerve c e l l s are undergoing chromomatolysis. Pirouette 55* Mallory Broken. C e l l s not as Seapa*s ganglion, compact spaces c r i s t a e and between c e l l s , maculae normal. Cytoplasm granular. Blood vessels normal. Rods present. Lower whirls ©f Cytoplasm i n ganglia contain outer h a i r c e l l s few small n u e l e i redueed. Hair f i b e r s and blood c e l l s f l a t t e n e d , vessels. Ganglia i n apex appears almost normal. Normal 60. H & E Mallory Marchi C a j a l 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 old normal, mouse. Blood vessels w e l l developed. As i n 35 day As i n 35 day old normal mouse, old normal mouse. Some f i b e r s blackened with Marchi s t a i n . These f i b e r s are 1; normal. Pirouette 60. Wk E Mallory Marchi C a j a l End organs normal• Scapa 1s ganglion normal. Less cytoplasm, vaeualis around a few of the n u c l e i , spaces between c e l l s . Cytoplasm only s t r i a t e d at apex. Rods normal. Ganglia i n Inner h a i r c e l l s 55 day p i p i normal. Outer mouse, hai r c e l l s l y i n g down and not d i s t i n c t . Pirouette 85. Mallory Broken. Scapa's ganglion normal. Less Cytoplasm, granular appearance• Number ©f nue l e i reduced. Few bl©od vessels. Broken. Cytoplasm ©f hai r c e l l s did not s t a i n . Nuclei stained blue, instead ©f red. Most nerve c e l l s have been l o s t i n basal w h i r l . Many dropped out ©f c e n t r a l w h i r l . Normal 90, Marchi Mallory Broken. End organs w e l l developed. i n the 60 day old normal mouse. Broken. As i n 60 day old normal mouse • Pirouette 100. Mallory Appears normal. As i n the 60 Broken. day ©Id pirouette mouse • Number of n u c l e i redueed i n basal w h i r l . A p i c a l w h i r l apparently normal. Normal 101*. Mallory As i n 60 day old As i n 60 day old normal mouse. normal mouse. Broken. As i n 60 day old normal mouse. Firouette 260 Mallory Broken. End organs appear t© be normal. As i n the 60 day ©Id mouse. Basal w h i r l organ Few nerve c e l l s of C o r t i r e -duced to layer of undifferen-t i a t e d t i s s u e . Broken i n central w h i r l . present i n basal w h i r l . Loss of nerve c e l l s i n cen t r a l w h i r l . Normal 262 H & E Marchi Broken. Deeply s t a i n -ing, s t r i a t e d as i n 35 day old normal mouse. Organ of C o r t i well developed, As i n 35 day ©Id normal mouse. N 0rmal 390 1 1 1 Removed be-fore sectioning, As i n 262 day normal mouse. Broken. As i n 262 day normal mouse. Pirouette 1 & E Broken. Greatly reduced i n width. Less cytoplasm, number of n u c l e i reduced. Vascular spaces In basal w h i r l the organ of C o r t i Is r e -duced to an un-d i f f e r e n t i a t e d knob of t i s s u e . and blood vessels In ce n t r a l w h i r l l o s t . No the p i l a r s are Nerve and ganglia consist of few f i b e r s , small n u c l e i and blood vessels. 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. 

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