The General Visceral Efferent Component of the Eighth Cranial ‘r2

MURIEL D. ROSS Department of Anatomy, University of Michigan, Ann Arbor, Michigan

ABSTRACT Study of the brain stem, stato-acoustic nerve, and inner of the mouse by a cholinesterase technique has disclosed that the acetylcholinesterase-positive centrifugal fibers to the are part of a general visceral efferent system which supplies fibers to the facial, vestibular and cochlear . This system largely corre- sponds in its central course to that of the bundle of fibers which has been called “olivo- cochlear,” but its cells of origin do not appear to lie in the . Instead, the preganglionic cells of origin are chiefly organized into four nuclei: one medial to the genu of the ; one lateral to the genu; the superior salivatory nucleus; and a small nucleus lying within the borders of the lateraI vestibular nucleus. The fibers to the inner ear , perhaps completely, on postganglionic cells located along their peripheral courses. Additionally, orthosympathetic acetylcholinesterase- positive fibers are present in the ; they arrive via the anterior inferior cerebellar artery. Postganglionic fibers supply not only vascular walls and secretory epithelium in the inner ear but also (1) contact the somata of some acetylcholinester- ase-positive, bipolar cochlear and (2) ramify among, or terminate upon, distal, acetylcholinesterase-positive processes of some sensory neurons at the foramina nervosa. These findings indicate that, on morphological grounds, autonomic nerve activity may influence auditory input at the periphery.

The original purpose of this investigation MATERIALS AND METHODS was to determine whether or not observable Thirty-three normal mice were prepared for changes occurred in the olivo-cochlear bun- microscopic examination. The mice were of Pero- dle prior to, concurrent with, or following myscus municulatus bairdi stock obtained from the Laboratory of Mammalian Genetics at The the loss of the and spiral University of Michigan. Twenty-six normal ani- cells in a species of mice which mals between 17 and 26 days postnatal age were becomes progressively deaf with age (Ross, beheaded; the fur, snout, and lower jaw were ’65a,b). It was hoped that the study would removed; the remaining portions of the skull with brain in situ were frozen on dry ice without prior yield information concerning a possible decalcification, mounted, and sectioned at 14 p in neurotrophic influence of the efferent sys- a cryostat at -18°C. Sections were mounted on tem upon the receptor organ. A cholinester- coverslips, kept frozen until all sectioning was completed, and then prepared by Holmstedt’s (‘57) ase technique was used to selectively stain modification of Koelle’s technique for cholinester- in their entirety the neurons whose ases except that development with ammonium comprise the bundle. Sections of skulls sulfide was carried out. The sections were washed briefly three times in distilled water, held in a with brains and inner in situ were pre- 10% ammonium sulfide solution for 30 seconds, pared so that the origin, course and termi- washed again briefly in distilled water, and then nation of the bundle could be determined placed on slides with glycerin jelly as the mount- in serial sections. Normal and abnormal ing medium. Different incubation periods and pH values were used on individual series, with one mice were used so that valid comparisons hour and fifteen minutes incubation in solutions could be made. It became apparent as the at pH 6.4 proving optimal for most tissues pre- study progressed that, in the mouse, the pared. Four complete series were counterstained with acetylcholinesterase-positive fiber bundle cresyl violet following treatment with ammonium which supplies the inner ear does not arise sulfide. The sections were washed briefly in dis- chiefly from the superior olivary complex. tilled water, fixed in buffered 10% formalin for With this finding, attention was turned to- * Supported by Deafness Research Foundation; Uni- of versity of Michigan H. H. Rackham Fac. Res. Proj. ward a complete investigation the cen- 787; and U.S.P.H.S. NB-07306-01. trifugal bundle to the inner ear as it occurs 2 Presented in part at the annual meeting of the American Association of Anatomists, New Orleans, in the normal mouse. La., April, 1968.

J. COMP. NEUR.,135: 453-478. 453 454 MURIEL D. ROSS

30 minutes, rinsed again, then immersed for three tent of the nucleus. The closely grouped minutes in a 0.25% solution of cresyl violet ad- and highly acetylcholinesterase-positive justed to pH 3.5 and heated to 56°C. Sections were differentiated in three changes of 95% alcohol, cells along the caudoventral border of the for a total of 15 minutes, then dehydrated and superior olivary nucleus are of medium- mounted on slides with permount. size, plump, multiangular, and have eccen- In three series, pretreatment of some sections tric nuclei. Prominent dorsal processes ex- with eserine in concentrations ranging between tend from these cells into the rostra1 part and 10-8 molar was carried out. In one se- ries, some sections were pretreated with mo- of the facial nucleus (fig. 2). This small lar concentration of DFP. clump of neurons merges anteriorly with The brains, only, of four additional normal and is replaced by a group of scattered, mice over three months of age were treated iden- moderately acetylcholinesterase-positive, tically in order to determine whether or not differ- ences in staining quality due to age exist. No such fusiform neurons with eccentric nuclei. differences were noted between these animals and These latter cells have long dorsal, medial those 19 to 21 days old. Animals of 17 days post- and lateral processes, but it could not be natal age generally yielded better stained tissues when longer incubation times were used (one and determined which was the . The dor- one-half to two hours). sal process occasionally extends through Recently, pretreatment with EDTA of skulls the superior olive into the tegmentum; the with brains and inner ears in situ of animals over medial, into the region of the medial pre- three months of age followed by the histochemical procedure outlined above has been fairly success- olivary nucleus; and the lateral, into the ful in yielding typical patterns of distribution of . These fusiform cells are acetyl- and butyrylcholinesterase. The enzymes loosely continuous at the lateral border are diminished quantitatively, however, as the re- of the superior olive with more closely actions are never so intense as those in the fresh material. These results, obtained in three speci- grouped multipolar neurons of less enzy- mens, are included in this report. The method fol- matic reactivity which possess a cen- lowed was essentially that given by Balogh and tral nucleus. Rostrally, this group of cells Nomura (’64), except that the pH of the incu- merges with the ventral nucleus of the lat- bating medium used in the histochemical pro- cedure was lowered to pH 6.4. The authors recom- eral lemniscus. mend a pH of 7.4 which, in the case of the mouse, There are exceedingly few neurons in results in considerable diffusion artifact of the en- the medial preolivary nucleus (nucleus zymes. This artifact frequently masks the cells of trapezoidalis ventralis). Those present are origin of the acetylcholinesterase-positive fibers centrally and leads to staining of all the vestibular similar morphologically and histochemi- and cochlear neurons peripherally. Use of a lower cally to the acetylcholinesterase-positive, pH eliminates some of this artifact. fusiform neurons of the lateral preolivary nucleus. DESCRIPTION OF FINDINGS Although positively stained fibers of fine Sziperior olivary complex. None of the diameter course vertically through the typical fusiform cells of the superior olive, field, dorsal to the superior olivary com- accessory superior olive, or nucleus trape- plex, none of them can be traced to an ori- zoidalis medialis stain positively for acetyl- gin in any portion of the latter complex in cholinesterase with the incubation periods the present material. or pH used (figs. 1, 2). A few multipolar Visceral efferent pathway. The cells of cells possessing cytoplasmic acetylcholin- origin of the acetylcholinesterase-positive esterase are sometimes scattered in the re- pathway referred to as the “olivo-cochlear gions of infolding of the superior olivary bundle” are distributed along the central nucleus or along its dorsolateral border. course of their centrifugal bundle and These cells correspond to the marginal and are loosely organized into three nuclear retro-olivary neurons described by Cajal groups: (1) a nucleus medial to the genu (’09), but not to the neurons described by of the facial nerve; (2) a nucleus closely Poliak (’26) or Rasmussen (’46) as retro- related to the ventrolateral border of the olivary. genu of the facial nerve; and (3) the SU- There are acetylcholinesterase-positive perior salivatory nucleus. Additionally, a neurons in the lateral preolivary nucleus fourth small nucleus of autonomic cells (nucleus trapezoidalis lateralis), differing located ventrally in the lateral vestibular morphologically and in strength of enzy- nucleus is linked by fibers to the “olivo- matic reaction through the caudorostra1 ex- cochlear bundle.” VISCERAL EFFERENTS OF EIGHTH NERVE 455

Medial to the genu of the facial nerve smaller, but prominent, fascicle loops dor- two types of neurons can be distinguished solateralward toward the small nucleus of both morphologically and histochemically autonomic cells in the lateral vestibular (fig. 3). Only the most dorsomedial neu- nucleus; the later course of this fascicle is rons appear to be typical preganglionic also considered below. The remaining two cells and to contribute to the visceral sys- fascicles distribute with nervus interme- tem described here. The more ventral ap- dius; one of them leaves the ventral aspect pear to be reticular cells. They have two of the superior salivatory nucleus to join long processes, one of which extends ven- nervus intermedius at the ventrolateral tralward toward the superior olivary com- border of the brain stem. The other follows plex and the other rostomedialward toward a lateral course from the superior saliva- the midline (fig. 3). tory nucleus, cutting through the spinal The nucleus ventrolateral to the genu of nucleus and tract of the the facial nerve consists of small, multi- to reach the medial border of the vestibular polar and acetylcholinesterase-positive neu- root. This fascicle may join the centrifugal rons. Distribution of the enzyme is heavier fibers to the inner ear in their course here, in the cell body than in the axon (figs. 1, or may remain independent as it travels 3). Some of these cells send their axons ventralward to join nervus intermedius. ventralward with the distal root of the There are two centrifugal bundles to facial nerve, while either collaterals of the inner ear structures at the lateral border same cells or axons of others are sent into of the medulla in the mouse: a large and the laterally coursing efferent bundle. a small bundle. The large bundle is the The superior salivatory nucleus is a continuation of the visceral efferent system small cluster of multipolar, acetylcholines- lateral to the superior salivatory nucleus, terase-positive cells situated dorsolaterally whereas the small bundle arises particu- in the medulla, directly in the path of the larly from the cells related to the lateral laterally coursing efferent bundle (fig. 1). vestibular nucleus. These two fascicles dif- This nucleus is located inferior to the lat- fer not only in size and in origin, but also eral vestibular nucleus and medial to the in position in the vestibular root, in num- nucleus of fasciculus solitarius. The su- ber of collaterals and in termination. perior salivatory nucleus is connected with The small bundle enters the central the larger inferior salivatory nucleus by a region of the vestibular root and remains slender column of cells and fibers. somewhat independent in its course, giving These three nuclei are linked together off few or no collaterals to either the prim- by the laterally coursing efferent fibers to ary acoustic nuclei or to the large efferent which their cells give rise, and to the con- bundle. It terminates in whole or in part tralateral nuclei by a decussation of some in a small plexus of postganglionic neurons of the fibers at the most rostra1 limits of and fibers at the ventrolateral edge of the the facial genu (fig. 4). Whether or not all medulla in the superior part of the vesti- of the nuclei contribute fibers to the contra- bular ganglion (fig. 5). It receives a few lateral efferent bundle cannot be deter- collaterals from the main efferent bundle mined in the available material. Only the near its termination (fig. 5). Postganglion- medially situated group of neurons can be ics from the neurons of this plexus are ex- shown with certainty to send some axons tremely difficult to trace, but appear to across the midline. supply the ampullae of the superior and The bundle thus formed of ipsilateral the lateral semicircular canals and the and contralateral fibers has been called macula utriculi. This distribution corre- “olivo-cochlear.”It splits into smaller f asci- sponds to that of the superior ramus of the cles and then recombines in its course in the human. across the dorsolateral tegmentum to the The large efferent bundle to the inner superior salivatory nucleus. In this region ear courses lateralward from the superior four main fascicles diverge from the bun- salivatory nucleus over the dorsal border dle, only one of which ultimately supplies of the spinal tract of the trigeminal nerve the . This branch, the largest, will and enters the medial aspect of the vesti- be considered in detail below. A second bular root. This bundle projects collaterals 456 MURIEL D. ROSS into an elongated nucleus (Martin’s nu- nerve has the same general pattern of cleus) which extends from the base of the distribution as described for the olivo-coch- restiform body along the medial border lear bundle (Rasmussen, ’53). The main of the primary acoustic nuclei (fig. 6). course of the fascicle is along the cochlear The collaterals ramify greatly in this nu- nerve to midmodiolar regions, then upward cleus and end in knobs, intensely acetyl- through the to the intra- cholinesterase-positive, around the somata ganglionic spiral bundle where it bifur- of the neurons. Some of the latter cells are cates; one branch turns apicalward and slightly acetylcholinesterase-positive, while the other, basalward in the intraganglionic the majority are not (fig. 7). spiral bundle. However, individual fibers or The large efferent bundle continues small bundles of them leave the large across the vestibular root at the ventral fascicle along its course; these follow two limit of the ventral , gives different routes through the cochlea. Some off a few collaterals to the small efferent of these fibers wind around the inferior bundle already described, and penetrates border of the spiral ganglion to reach the the . Some fibers are root of the osseous spiral lamina where given off from this bundle in its course they join the intraganglionic spiral bundle. through the ganglion but the majority of Others cut through the spiral ganglion to them run together to the inferior part of enter the intraganglionic spiral bundle di- the ganglion. The bundle then forks, giving rectly. off one branch which curves around the Postganglionic neurons can be demon- cochlear nerve and is the classical “olvio- strated histochemically in the intragan- cochlear bundle.” while the other branch glionic spiral bundle of the mouse, which turns sharply posteriorly toward the thus corresponds to an autonomic plexus. and posterior ampulla. As they emerge These postganglionic neurons differ from from the main trunk of fibers. the Schwann one another in size, shape, and histochem- and myelin sheaths of these preganglionic ical reaction (figs. 9-12). Some are small fibers begin to stain for butyrylcholines- (5-7 K), have eccentric nuclei, rounded terase as well as for acetylcholinesterase. contours, and are slightly reactive for The preganglionic fibers to the saccule acetylcholinesterase (fig. 12). Others are terminate among small clusters of post- of medium size (around 10 u), appear ganglionic neurons in the tunica propria rounded or sometimes bipolar or multipolar of the saccular macula or in similar cells and are moderately reactive for the enzyme located more proximally along the efferent (figs. 10, 11); still others are large (12- bundle within the vestibular ganglion. The 14 K), multipolar, and react moderately to postganglionjc neurons related to the sac- heavily for acetylcholinesterase (fig. 9). cule are minimally or moderately acetyl- After counterstaining with cresyl violet an cholinesterase-positive and are butyryl- occasional large, multipolar which cholinesterase-negative. The distribution of is not reactive to any extent for acetyl- their axons corresponds to that of the in- cholinesterase, but which receives acetyl- ferior branch of the vestibular nerve. cholinesterase-positive terminals and gives The preganglionic fibers to the posterior rise to an axon highly reactive for the ampulla end on postganglionic neurons enzyme js evident (fig. 10). which occur singly or in small ganglia Distribution of vestibular postganglionic along their course. Many of these postgan- fibers. Not all of the terminals of the glionic neurons are minimally acetylcholin- fibers to the vestibular organs have been esterase-positive, but an occasional cell is identified with certainty. Some end in rela- highly reactive for the enzyme (fig. 8). No tion to blood vessels, but the latter are not butyrylcholinesterase activity was detected shown well in this type of preparation. in these postganglionic neurons. Axons of However, within each ampulla, some of the these postganglionic cells distribute with fibers turn toward the cells at the base of the posterior branch of the vestibular the crista where they terminate in a net- nerve. work (fig. 13). These cells differ from The fascicle which forks off from the those of the upper parts of the crista in main efferent bundle and joins the cochlear his tochemical re action, staining positively VISCERAL EFFERENTS OF EIGHTH NERVE 457 for both acetylcholinesterase and butyryl- thereupon gave rise to three tiny collaterals cholinesterase, whereas the upper cells which terminated in greatly enlarged, remain minimally stained or unstained in acetylcholinesterase-positive knobs upon either substrate. In the macular areas, the distal process of a (fig. postganglionic terminals are most conspic- 19). Whether the stem fiber of the term- uous basally in the epithelium (fig. 14). inals was ortho- or parasympathetic could Distribution of cochlear postganglionic not be conclusively shown with the present fibers. Some of the cochlear postgan- technique. glionic fibers appear to ramify in a vascular Spiral ganglion. In the species of plexus at the root of the osseous spiral mouse reported upon here there are some lamina. Other postganglionic fibers run acetylcholinesterase-positive bipolar neu- along the tympanic face of the osseous rons in the spiral ganglion (fig. 20). These spiral lamina to its distal border (fig. 15) neurons are more numerous basally and in where some of them bifurcate and may mid-portions of the cochlea than apically. form loops or a plexus. Still other fibers They are located most often at the edge of ramify in relation to the axons of bipolar the spiral ganglion, near the intragan- neurons near the foramina nervosa. The glionic spiral bundle. The cells sometimes peripherally coursing fibers are fine, in- possess an eccentric nucleus which is tensely acetylcholinesterase-positive and drawn toward the proximal process. Acetyl- unmyelinated. cholinesterase is concentrated at the cell Orthosympathetic fibers. Acetylcholin- membrane, particularly of the , al- esterase-positive fibers turn off from the though the proximal portions of both proc- anterior inferior cerebellar artery to pene- esses of the bipolar neurons are reactive trate the cochlear nerve (fig. 16). Such fi- along the axolemma when the pH of the bers are presumed to be orthosympathetic incubation solutions is high (7.2). The on the basis of their course along an artery. stain occurs cytoplasmically after pro- The fibers run independently of the para- longed incubation times. Neither the cells sympathetic bundles and of one another nor their sheaths stain positively for bu- through the cochlear nerve. It is impossible tyrylcholinesterase, the neurons resembling to trace them to their termination in the in this respect the rest of the bipolar neu- present material as the staining quality of rons of the spiral ganglion. Peripheral the parasympathetic and of the orthosym- processes of some of these neurons enter pathetic fibers is very similar. However, the intraganglionic spiral bundle. some of the reactive fibers which course Efferent fibers frequently appear to form individually in the cochlear nerve continue a network around the somata of the qcetyl- through the spiral ganglion to the intra- cholinesterase-positive bipolar neurons, to ganglionic bundle. Therefore, the possibil- coil upon them, or to touch them in pass- ity exists that some of the orthosympa- ing by (fig. 20). So far, this contact be- thetic fibers enter this bundle. tween efferent fibers and bipolar neurons Autonomic fibers related to sensory has been observed only in the cochlea, axms. The distal processes of certain but might also exist in the vestibular areas. bipolar neurons are acetylcholinesterase- Future research must determine whether positive in the mouse (figs. 15, 17-19), or not such contact relationships exist be- from approximately the first node distally tween parasympathetic fibers and bipolar into the organ of Corti. Some butyrylcholin- neurons, or between acetylcholinesterase- esterase activity occurs in the glia-myelin positive orthosympathetic fibers and bi- sheath of the first internode and around polar neurons. these processes up to the foramen nervo- sum, but this enzyme is not present distal DISCUSSION to the foramen. Some of the positively-re- Centrifugal fibers in the eighth nerve acting distal sensory processes have fine, have been described in the literature since unmyelinated fibers winding around or the latter part of the nineteenth century running close to them (figs. 17, 18). In one (for comprehensive review, see Addens case such a fine fiber was traced to the '34; Rasmussen, '46; Rossi and Cortesina, region of the foramen nervosum where it '65a). Many workers thought such fibers 458 MURIEL D. ROSS to be preganglionic salivatory components Gacek and Balogh ('65), Gacek, Nomura of nervus intermedius. Since the reports and Balogh ('65), and Rossi and Cortesina of Papez ('30) and Rasmussen ('46), how- ( '63, '65b ) . ever, the origin of the centrifugal fibers Churchill, Schuknecht and Doran ('56) has been considered to be the superior described acetylcholinesterase activity in olivary complex. Both workers made ex- the centrifugal fiber bundle in the osseous perimental lesions in the region of the spiral lamina, and in the region of the superior olivary complex which resulted inner hair cells. Wersall, Hilding and in degeneration of a bundle which traveled Lundquist ('61) and Hilding and Wersall dorsalward, then contralateralward ventral ('62) found acetylcholinesterase activity to the facial genu, and across the dorso- in the large vesiculated terminals. Vesi- lateral part of the field to enter the contra- culated terminals ending on hair cells of lateral vestibular nerve. Rasmussen was the organ of Corti have been described by successful in tracing the bundle into the Spoendlin ('57), Engstrom ('58, '60), cochlea and appropriately named it "olivo- Smith ('61), Smith and Sjostrand ('61a, cochlear." Papez thought that the cells of b), Iurato ('62), Hilding and Wersal ('62), origin for this peduncle were located either and others. in the accessory superior dive, the medial The findings reported here support Ras- preolivary nucleus, or the nucleus of the mussen's thesis that the bundle he de- . Rasmussen postulated that scribed is parasympathetic, although the the cells of origin were in the retro-olivary cells of origin do not appear to be related nucleus and in the medial preolivary nu- to the superior olivary complex in the cleus. mouse nor are its fibers centrifugal to the Rasmussen ascribed a parasympathetic cochlea alone. The cells of origin as deter- function to the bundle, basing this hypo- mined histochemically are spread over a thesis on the morphology of the predicated broad area at the level of the genu of the cells of origin and on the small size and facial nerve from medially near the raphe thin myelinization of the fibers. Later, after to the ventral border of the lataral vesti- the work of Fernandez ('51), Portmann bular nucleus laterally. The cells are sub- and Portmann ('51), and Portmann ('52), grouped into three nucei: one medial to who indicated that the efferent fibers of the genu; one ventrolateral to the genu; this bundle reached the inner hair cells of and one at the ventral border of the lateral the organ of Corti, Rasmussen ('53) post- vestibular nucleus. An additional nucleus ulated that the bundle might be inhibitory of morphologically and histochemically to the hair cells. He found in 1953 that similar cells is located within the borders not all of the peripheral fibers of the bundle of the lateral vestibular nucleus. The neu- degenerated when he cut the efferent bun- rons are typically small to medium-size, dle centrally. He thought the persisting multipolar and acetylcholinesterase-posi- fibers to be axons of bipolar spiral gan- tive. The enzyme is cytoplasmically local- glion cells misplaced along the efferent ized, as Gerebtzoff ('59) found to be the fascicles. Later ('60), he described a homo- case in rabbit preganglionic cells. lateral origin for some of the fibers of the Most of the axons of these cells form a bundle. Rasmussen worked on the rat, common bundle which is partly crossed, opossum, dog, cat, and human and found (through the decussation ventral to the the bundle remarkably similar in all these facial genu) , traverses the dorsolateral forms in respect to size, origin, and distri- part of the field and splits into groups of bution. Among other workers who have in- fibers which accompany the vestibular, vestigated the bundle are Borghesan ('59), cochlear, or facial nerves distally. The rest Smith and Rasmussen ('63, '65), Gacek, of the axons, few in number, join the facial Nomura and Balogh ('65), and Rossi and root in the brain stem. Although the cells Cortesina ('63, '65b). Among the authors of origin of the particular groups of fibers who have discussed the efferent innerva- may differ in location, this could not be tion to the vestibular end organs are Petroff determined in the present, normal ma- ('55), Rasmussen and Gacek ('58), Gacek terial. It appears that the visceral efferents ('60), Hilding and Wersdl ('62), Nomura, to the lacrimal and salivary glands and to VISCERAL EFFERENTS OF EIGHTH NERVE 459 the vestibular and cochlear areas arise vestibular root intermingle in the dog and from similar regions, share a common human, but not in the cat. decussation, and largely pursue the same Literature pertaining to the origin of the course through the brain stem. olivo-cochlear bundle is scarce and findings Results of much previous work supports are not always in agreement. Some support the anatomical and histochemical evidence exists for Rasmussen's predication that a presented here that the system described portion of the retro-olivary neurons give is broadly distributed and visceral efferent. rise to the crossed olivo-cochlear fascicle Van Gehuchten ('27) indicated that the (Rossi and Cortesina, '63, '65b; Shute and centrifugal fibers to the vestibular root re- Lewis, '65). According to the last-quoted semble autonomics and arise bilaterally authors medial preolivary neurons also con- from cells situated medial to the genu of tribute to the crossed bundle. Aside from the facial nerve near the midline. Both these points of agreement, wide disagree- Kohnstamm ('02a,b) and Kaida ('29) con- ment prevails. Rasmussen's ('46) own cluded, on the basis of experimental work, retrograde degeneration studies do not sup- that the preganglionic neurons associated port his thesis that the medial preolivary with salivary glands are spread over the neurons contribute to the crossed olivo- wide area defined above. That the cells are cochlear bundle, for he found only an chiefly laterally situated, in the superior occasional atypical cell in this nucleus fol- salivatory nucleus, was indicated by these lowing section of the decussating fascicle. authors and many others (Yagita and Ha- The direct component to the efferent bun- yama, '09; Yagita, '09; Windle, '33; Addens, dle out of the superior olivary nucleus de- '34; and others). The cells clustered around scribed by Rasmussen ('60) lacks confirm- the genu of the facial nerve were described ation. Rossi and Cortesina ('63, '65b) have on the basis of histochemical results as described four nuclei of origin €or the un- salivatory by Lewis and Shute ('59) and crossed centrifugal bundle, which are not Shute and Lewis ('65). Cells dorsal to the in agreement with Rasmussen's results. facial nucleus (Yagita, '14) or lateral to These nuclei are located as follows: (1) the genu (Chouard, '62) have been de- ventralward between the medial and the scribed as lacrimal. The decussation of lateral preolivary nuclei in a region made some of the fibers ventral to the genu has up of cells morphologically similar to retro- been described by virtually all students of olivary neurons; (2) in a reticular nucleus nervus intermedius except for Yagita and along the dorsal part of the raph6; and (3) Hayama ('09) and Yagita ('09). The cen- and (4) in the lateral part of the dorsal tral course of visceral efferent fibers field, with one nucleus lying ventral to and through the dorsolateral part of the field the other within the lateral vestibular and into the vestibular root has been de- nucleus. scribed by Kohnstamm ('02a,b), Van Ge- Attempts to discover the origin of vesti- huchten (W), Kaida ('29), Lorente de N6 bular efferents experimentally have been ('33) and Addens ('34). similarly fraught with difficulty. Gacek The fact that the central route and de- ('60), without anatomical proof, thought cussation of the centrifugal fibers to the the lateral vestibular nucleus to be the vestibular root is, for the most part, the source of ipsilateral, centrifugal fibers to same as that described for a portion of the vestibular nerve. In contrast, Carpen- nervus intermedius has been ignored by ter, Bard and Alling ('59) and Carpenter most students of the olivo-cochlear bundle. ('60) thought that all of the vestibular The acetylcholinesterase-positive centrifu- nuclei except the lateral gave origin to gal fibers to the inner ear should be con- ipsilateral fibers, while the fastigial nu- sidered a part of nervus intermedius. This cleus contributed crossed efferent fibers to concept has not been suggested in the the peripheral vestibular regions. previous literature, although Windle ('33) The present results do not resolve these actually described the component from the discrepancies in the literature. The cells facial root into the vestibular ganglion in the superior olivary, in the accessory without naming it; and Van Buskirk ('45) olivary and in the trapezoid body nuclei, noted that nervus intermedius and the and in Poliak's ('26) and Rasmussen's 460 MURIEL D. ROSS

('46) retro-olivary nucleus are not acetyl- Connections from the efferent bundle cholinesterase-positive. This agrees essen- into the were de- tially with Koelle's ('54) and Gerebtzoff's scribed by Rasmussen ('60) for the cat. ('59) findings. The latter author ascribed Comparable relays exist in the mouse, al- moderate staining to cells of the rat trape- though their distribution is more restricted, zoid body but the present research indicates the collaterals terminating around the that, based on their location and morphol- somata of the small cells of Martin's nu- ogy, these cells lie in the lateral preolivary cleus. Most of these cells are not acetyl- nucleus. This accords well with prior de- cholinesterase-positive, so their axons can- tailed descriptions of this nucleus in the not be followed to a termination with the mouse (Cajal, '09; Poliak, '2E), the rat histochemical technique employed. Neither (Harrison and Warr, '62), and the cat Martin (1894) nor Fuse ('13), who have (Poliak, '26; Taber, '61). The few acetyl- described this nucleus previously, were cholinesterase-positive neurons, located successful in learning the distribution of more medially and embedded in trapezoid its axons. These cells may be important in fibers, are morphologically and histochem- regulating acoustic input at the primary ically similar to the fusiform neurons of nuclei, or in feedback for regulation of the lateral preolivary nucleus and may be parasympathetic outflow to acoustic stim- displaced neurons of this nucleus. Cajal uli. ('09) described the medial preolivary nu- The peripheral course of the cochlear cleus as small and superficial in the mouse, fascicle to the level of the intraganglionic Harrison and Warr ('62) failed to locate spiral bundle in the mouse is in close agree- it in the rat, and Taber ('61) found it ment with that originally given by Ras- "ambiguous" in the cat. The paucity of mussen ('46, '53) for the olivo-cochlear acetylcholinesterase-positive cells in the re- bundle in the cat, dog, man, and other gion of the medial preolivary nucleus pre- forms. New findings are that the parent cludes this nucleus as a primary source of bundle provides vestibular as well as coch- acetylcholinesterase-positive efferent fibers lear efferents and that both sets of efferent to the inner ear. fibers terminate, perhaps completely, upon Two of the nuclei described by Rossi postganglionic neurons scattered along and Cortesina ('63, '65b) as the origin of their courses. Within the cochlea, the nu- vestibular efferents probably correspond to Ions are all embedded within the fibers two similarly located nuclei in the mouse: of the intraganglionic spiral bundle. their "interposed" nucleus is comparable Differences in histochemical reaction in position to the superior salivatory nu- which are pronounced between the various cleus; the other nucleus lies within the postganglionic neurons in the inner ear of borders of the lateral vestibular nucleus. the mouse may possibly be related to A third nucleus which they describe as a differences in function. That autonomic source of direct reticular fibers to the inner cells differ from one another histochem- ear is present in the mouse, but no con- ically has been reported previously (Gia- clusion could be reached concerning its cobini, '56; Holmstedt, '57; Learning and projection. However, by definition, retic- Cauna, '61; Cauna et al., '61). ular cells are confined to the central ner- Vestibular postganglionic fibers termi- vous system (Rossi and Zanchetti, '57). nate predominantly among epithelial cells Thus, results of the present study do in the maculae and ampullary crests. In not concur that the centrifugal, acetyl- the latter case the innervated cells are lo- cholinesterase-positive fibers to the inner cated at the base of the crista. These cells ear arise in the superior olivary complex. would appear to be secretory, based upon It is possible that the olivo-cochlear bundle their histochemical reaction for cholines- as described by Rasmussen is not acetyl- terase and their nerve supply, as predicated cholinesterase-positive, or that the contri- by Dohlman ('64, '65). bution to this system from the superior In the cochlea, many postganglionic fi- olivary complex is small in the mouse and bers do not terminate in relation to blood was overlooked. vessels or secretory epithelium. Some of VISCERAL EFFERENTS OF EIGHTH NERVE 461 these fibers cannot be traced to their termi- minals in the organ of Corti with the axons nation, but are lost at the distal border of of cells lying in the medulla when, in fact, the osseous spiral lamina. Others, which they do not. In order to prove this, the en- have the same histochemical properties as tire neuron from its origin to its terminals postganglionic autonomic fibers, form spi- must be demonstrated; no one has done rals or networks around the somata of the this. Such studies have shown unequiv- acetylcholinesterase-positive bipolar neu- ocally, however, the importance of an in- rons in the spiral ganglion of the cochlea, tact centrifugal bundle to the well-being some wrap around the distal myelinated of the vesiculated terminals, for if the processes of bipolar neurons, and some end former is largely or completely destroyed, in knob-like terminals on the unmyelinated many or all of the latter will degenerate. segment of the latter neurons proximal to The exact reason for the degeneration of the foraminanervosa (fig. 19). Wolff ('60) the terminals upon removal of the para- has mentioned observing fibers stopping on sympathetic innervation to the inner ear some vestibular and cochlear bipolar gan- must be found in future research. glion cells. The fibers described here do not Although the present findings indicate terminate in regions classically considered that the mouse intraganglionic spiral bun- to be the distribution of the visceral effer- dle is largely a parasympathetic plexus, at ent system. These results might suggest least two other types of fibers may travel that there is a morphological basis for an with the bundle. Orthosympathetic fibers autonomic modification of sensory input, appear to enter the bundle, and axons as some experimental evidence indicates of the acetylcholinesterase-positive bipolar (Seymour and Tappin, '53; Beickert et al., neurons run with it. The latter finding sup- '56; Loewenstein and Altamirano-Orrego, ports results obtained by Lorente de N6 '56; Eldred et al., '60; Hunt, '60; Kimura, ('37) and Fernandez ('51). '61; Chernetski, '64). They may also pro- The acetylcholinesterase-positive bipolar vide a morphological basis for the finding neurons are unusual in that they share that stimulation of the centrifugal bundle some of the morphological and histochem- to the inner ear in anesthetized animals ical characteristics of both the sensory results in suppression of auditory nerve ac- bipolar neurons and of the autonomic post- tivity (Galambos, '56) and increased coch- ganglionic cells. They are structurally bi- lear microphonics (Fex, '59, '62; Desmedt polar neurons and have no butyrylcholines- and Monaco, '61). terase activity, thus resembling the other A possible neurotrophic influence of the sensory neurons of the spiral ganglion. parasympathetic system on sensory ter- However, unlike the sensory neurons, they minals should be considered. This may be are acetylcholinesterase-positive (Ross, '66), direct or indirect, through regulation of often possess an eccentric nucleus, are vascular supply or fluid balance. Much ex- contacted by autonomic fibers, and send cellent research has shown that when the their distal processes into the intragan- centrifugal fibers to the inner ear are cut glionic spiral bundle. In these ways, the centrally (Engstrom and Fernandez, '61; neurons more closely resemble autonomic Iurato, '62; Kimura and Wersiill, '62; cells. Smith and Rasmussen, '63; Spoendlin and The presence of acetylcholinesterase in Gacek, '63; Spoendlin, '66) or peripherally the axons of some bipolar neurons prox- (Spoendlin and Gacek, '63) the large vesic- imal to the foramina nervosa in the mouse ulated terminals on the hair cells degen- may be a species variation. Nevertheless, erate. Additionally, some investigators have it demonstrates that acetylcholinesterase- reported that the acetylcholinesterase at positive terminals within the organ of Corti the base of the hair cells largely disappears may arise from sensory neurons and are when the centrifugal fibers are cut cen- not necessarily efferent. Acetylcholinester- trally (Schuknecht, Churchill and Doran, ase has been found to occur in sensory ter- '59; Smith and Rasmussen, '63). These re- minals elsewhere (Cauna, '61 ; El Rakhawy sults have been interpreted to prove the and Bourne, '61). Its function in receptors physical continuity of the vesiculated ter- is completely unknown. 462 MURIEL D. ROSS

ACKNOWLEDGMENTS Dohlman, G. F. 1964 Secretion and absorption of endolymph. Trans. Amer. otol. SOC.,52: 96 Animals used in this study were ob- 113. tained from the Laboratory of Mammalian 1965 The mechanism of secretion and Genetics through the generosity of Dr. absorption of endolymph in the vestibular ap- paratus. Acta Oto-laryng. (Stockh.), 59: 275- Elizabeth Barto, to whom the author is 288. deeply indebted. Eldred, E., H. N. Schnitzlein and J. Buchwald I thank Dr. Rita Ping Liu for her consci- 1960 Response of muscle spindles to stimula- tion of the sympathetic trunk. Exp. Neurol., entious technique work and for her assis- 2: 187-195. tance with photography. El-Rakhawy, M. T., and G. H. Bourne 1961 Cholinesterases in the human tongue. Histo- LITERATURE CITED chemistry of Cholinesterase, Symposium Basel Addens, J. L. 1934 A critical review of the 1960. Bibl. anat. 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SOC.,49: 58-60. Heilk., 168: 495-507. Fernandez, C. 1951 The innervation of the Borghesan, E. 1959 On the significance of the cochlea (Guinea Pig). Laryngoscope, 61: 1152- intraganglionic spiral bundle. Acta Oto-laryng. 1172. (Stockh.), 50: 154-162. Fex, J. 1959 Augmentation of the cochlear mi- Cajal, S. Ram& y 1909 Histologie du systhme crophonics by stimulation of efferent fibers to nerveux de l'homme et des vertkbrks. Maloine, cochlea. Acta Oto-laryng. (Stockh.), 50: 54C- Paris. 541. Carpenter, M. B. 1960 Experimental anatomi- 1962 Auditory activity in centrifugal cal-physiological studies of the vestibular nerve and centripetal cochlear fibers in cat: A study and cerebellar connections. In: Neural Mecha- of feedback system. Acta physiol. scand., 55, nisms of the Auditory and Vestibular Systems, (Suppl. 189). edited bv G. L. Rasmussen and W. F. Windle. Fuse, G. 1913 Das Ganglion ventrale und das Charles C Thomas, Springfield, Illinois. Chap. Tuberculum acusticum bei einigen Sauglingen XXII, 297-323. und beim Menschen. Arb. Hirn-Anat. Inst. Carpenter, M. B., D. S. Bard and F. A. Alling Zurich, 7: 1-210. 1659 Anatomical connections between the fas- Gacek, R. R. 1960 Efferent component of the tigial nuclei, the labyrinth and the vestibular vestibular nerve. In: Neural Mechanisms of the nuclei in the cat. J. Comp. Neur., 111: 1-26. Auditory and Vestibular Systems, edited by G. Cauna, N. 1961 Cholinesterase activity in cu- L. Rasmussen and W. F. Windle. Charles C taneous receptors of man and some quadrupeds. Thomas, Springfield, Illinois. Chap. XX, 276- Histochemistry of Cholinesterase, Symposium 284. Basel 1960. Bibl. anat. (Basel), Fasc., 2: 128- Gacek, R. R., Y. Nomura and K. Balogh 1965 138. Acetylcholinesterase activity in the efferent Cauna, N., N. T. Naik, D. B. Learning and P. fibers of the stato-acoustic nerve. Acta Oto- Alberti 1961 The distribution of cholinester- laryng. (Stockh.), 59: 541-553. ases in the autonomic ganglia of man and of Galambos, R. 1956 Suppression of auditory some mammals. Histochemistry of Cholinester- nerve activity by stimulation of efferent fibers ase, Symposium Basel 1960. Bibl. anat. (Basel), to the cochlea. J. Neurophysiol., 19: 424437. Fasc., 2: 90-96. Gerebtzoff, M. A. 1959 Cholinesterase (A Chernetski, K. E. 1964 Sympathetic enhance- Histochemical Contribution to the Solution of ment of peripheral sensory input in the frog. Some Functional Problems). Pergamon Press, J. Neurophysiol., 27: 493-515. New York. Chouard, C. H. 1962 Recherches sur l'organisa- Giacobini, E. 1956 Histochemical demonstra- tion intra-axiale des formations motrices et tion of acetylcholinesterase activity in isolated parasympathiques de nerf facial. Private publi- nerve cells. Acta physiol. scand. (Suppl. 123- cation. 124), 36: 276-290. Churchill, J. A., H. F. Schuknecht and R. Doran Harrison, J. M., and B. Warr 1962 A study of 1956 Acetylcholinesterase activity in the coch- the cochlear nuclei and ascending auditory lea. Laryngoscope, 66: 1-15. pathways of the medulla. J. Comp. Neur., 119: Desmedt, J. E., and P. Monaco 1961 Mode of 341-380. action of the efferent olivo-cochlear bundle on Hilding, D., and J. Wersall 1962 Cholinesterase the inner ear. Nature, 192: 1263-1265. and its relation to the nerve endings in the VISCERAL EFFERENTS OF EIGHTH NERVE 463

inner ear. Acta Oto-laryng. (Stockh.), 55: 205- Poliak, S. 1926 Untersuchungen am Oktavus- 217. system der Saugetiere und an den mit diesem Holmstedt, B. 1957 A modification of the thio- koordinierten motorischen Apparaten des Hirn- choline method for the determination of cho- stammes. Eine morphologisch-biologische Stud- linesterase. 11. Histochemical application. Acta ie. J. Psychol. Neurol. (Lpz.), 32: 170-231. physiol. scand., 40: 331-337. Portmann, M. 1952 Les fibres nerveuses ef- Hunt, C. C. 1960 The effect of sympathetic fbrentes cochl6aires. Thesis, Imprimerie-Librai- stimulation on mammalian muscle spindles. J. rie Delmas, 6, place Saint-Christoly, Bordeaux. Physiol. (Lond.), 151: 33Z341. Portmann, M., and C. Portmannn 1951 Les Iurato, S. 1962 Efferent fibers to the sensory fibres spirales de la cochl6e. Rev. Laryng., 72: cells of Corti’s organ. Exp. Cell Res., 27: 162- 737-750. 164. Rasmussen, G. L. 1946 The olivary peduncle Kaida, Y. 1929 Wber den Ursprung und den and other fiber projections of the superior peripheren Verlauf der sog. zentrifugalen Vesti- olivary complex. J. Comp. Neur., 84: 141-219. bularisnerven nach Leidler (Fasciculus vesti- 1953 Further observations of the ef- bularis medialis nach Kaplan). Arch. 0hr.-, ferent cochlear bundle. J. Comp. Neur., 99: Nas.-, u. Keh1k.-Heilk., 123: 62-69. 61-74. Kimura, K. 1961 Factors affecting the response 1960 Efferent fibers of the cochlear of taste receptors of rat. Kumamoto med. J., nerve and cochlear nucleus. In: Neural Mech- 14: 95-99. anisms of the Auditory and Vestibular Systems, Kimura, R. and J. WersU 1962 Termination edited bv G. L. Rasmussen and W. F. Windle. of the olivo-cochlear bundle in relation to the Charles C Thomas, Springfield, Illinois. Chap. outer hair cells of the organ of Corti in guinea VIII, 105-115. pig. Acta Oto-laryng. (Stockh.), 55: 11-32. Rasmussen, G. L., and R. R. Gacek 1958 Con- Koelle, G. B. 1954 The histochemical localiza- cerning the question of an efferent fiber com- tion of cholinesterase in the central nervous ponent of the vestibular nerve of the cat. Anat. system of the rat. J. Comp. Neur., 100: 211- Rec., 130: 361-368. 235. Ross, M. D. 1965a A comparison of the normal Kohnstamm, 0. 1902a Vom Centrum der cochlea with the abnormal of the waltzing Speichelsekretion, dem Nervus intermedius und mouse. (Abstract). Anat. Rec., 151: 408. der gekreuzten Facialiswurzel. Verh. dtsch. 1965b The auditory pathway of the Kongr. inn. Med., 20: 361-373. epileptic waltzing mouse. 11. Partially deaf 1902b Der Nucleus salivatorius chordae mice. J. Comp. Neur., 125: 141-164. tympani (Nervi intermidii). Anat. Anz., 21: 1966 Cholinesterase activity in the 362-363. auditory pathway in normal and abnormal Learning, D. B., and N. Cauna 1961 A qualita- mice. (Abstract). Anat. Rec., 154: 414. tive and quantitative study of the myenteric Rossi, G., and G. Cortesina 1963 Research on plexus of the small intestine of the cat. J. Anat. the efferent innervation of the inner ear. J. (Lond.), 95: 160-169. Laryng., 77: 202-233. Lewis, P. R., and C. C. D. Shute 1959 Selective 1965a The efferent innervation of the staining of visceral efferents in the rat brain inner ear. A historical-bibliographical survey. stem by a modified Koelle technique. Nature, Laryngoscope, 75: 212-235. 183: 1743-1744. 196513 The “efferent cochlear and vesti- Lorente de N6, R. 1933 Anatomy of the eighth bular system” in Lepus cuniculus L. Acta anat. nerve. Laryngoscope, 43: 1-38. (Basel), 60: 362-381. 1937 Symposium: The neural mech- Rossi, G. F., and A. Zanchetti 1957 The brain anisms of : I. Anatomy and physiology stem reticular formation: Anatomy and physiol- (b). The sensory endings in the cochlea. Laryn- ogy. Arch. ital. Biol., 95: 199-435. goscope, 47: 373-377. Schuknecht, H. F., J. A. Churchill and R. Doran Loewenstein, W. R., and R. Altamirano-Orrego 1959 The localization of acetylcholinesterase 1956 Enhancement of activity in a Pacinian in the cochlea. Arch. Otolaryng., 69: 549-559. corpuscle by sympathomimetic agents. Nature Seymour, 3. C., and J. W. Tappin 1953 Some (Lond.), 178: 1291-1293. aspects of the sympathetic nervous system in Martin, P. 1894 Zur Endigung des Nervus relation to the inner ear. Acta Oto-laryng. acusticus im Gehirn der Katze. Anat. Anz., 9: (Stockh.), 43: 618-635. 181-184. Shute, C. C. D., and P. R. Lewis 1965 Cholin- Nomura, Y., R. R. Gacek and K. Balogh 1965 esterasecontaining pathways of the hindbrain: Efferent innervation of vestibular labyrinth: Afferent cerebellar and centrifugal cochlear Histochemical demonstration of acetylcholin- fibers. Nature, 205: 242-246. esterase activity in the guinea pig inner ear. Smith, C. A. 1961 Innervation pattern of the Arch. Otolaryng., 81: 335-339. cochlea - the internal . Trans. Amer. Papez, J. W. 1930 Superior oIivary nucleus-its OtoI. SOC.,49: 35-60. fiber connections. Arch. Neurol. Psychiat., 24: Smith, C. A., and G. L. Rasmussen 1963 Re- 1-20. cent observations on the olivo-cochlear bundle. Petroff, A. E. 1955 An experimental investiga- Ann. Otol. (St. Louis), 72: 489-506. tion of the origin of efferent fiber projections 1965 Degeneration in the efferent to the vestibular neuroepithelium. Anat. Rec., nerve endings in the cochlea after axonal sec- 121: 352-353. tion. J. Cell Biol., 26: 63-76. 464 MURIEL D. ROSS

Smith, C. A., and F. S. SjSstrand 1961a A bulaire et sur les voies vestibulaires centrales. synaptic structure in the hair cells of the Rev. Oto-neuro-oculistique, 5: 777-791. guinea pig cochlea. J. Ultrastruct. Res., 5: Wersu, J., D. Hilding and P. G. Lundquist 1961 184-192. Ultrastruktur und Innervation der Cochlearen - 1961b Structure of the nerve endings Haarzellen. Arch. 0hr.-, Nas.-, u. Keh1k.-Heilk., on the external hair cells of the guinea pig 178: 106-126. cochlea as studied by serial sections. J. Ultra- Windle, W. F. Neurofibrillar development struct. Res., 5: 1933 523-556. in the of cat embryos Spoendlin, H. H. 1957 Elektronenmikroskop- ische Untersuchungen am Cortischen Organ between 8 and 12 mm long. J. Comp. Neur., des Meerschweinchens. Pract. oto-rhino-laryng. 58: 643-723. (Basel), 19: 192-234. Wolff, D. 1960 Discussion of neural mech- - 1966 The Organization of the Cochlear anisms in the . In: Neural Receptor, edited by Prof. Dr. L. Ruedi, Basel Mechanisms of the Auditory and Vestibular (Switzerland), S. Karger/New York. Systems, edited by G. L. Rasmussen and W. F. Spoendlin, H. H., and R. R. Gacek 1963 Elec- Windle. Charles C Thomas, Springfield, Illinois. tron microscopic study of the efferent ankl af- Chap. XXIV: 359-361. ferent innervation of the organ of Corti in the Yagita, K. 1909 Weitere Untersuchungen iiber cat. Ann. Otol. (St. Louis), 72: 660-686. das Speichelzentrum. Anat. Anz., 35: 70-75. Taber, E. 1961 The cytoarchitecture of the 1914 Einige Experimente an dem Ner- brain stem of the cat. I. Brain stem nuclei of vus petrosus superficialis major zur Bestim- cat. J. Comp. Neur., 116: 27-69. mung des Ursprungsgebietes des Nerven. Folia Van Buskirk, C. 1945 The seventh nerve com- neuro-biol. (Lpz.), Bd. VIII: 361-382. plex. J. Comp. Neur., 82: 303-335. Yagita, K., and S. Hayama 1909 Uber das Van Gehuchten, P. 1927 Recherches exp6ri- Speichelsekretionscentrum. Neurol. Centralbl., mentales pur les terminaisons du nerf vesti- Bd., 28: 738-753.

Abbreviations AIC, Anterior inferior cerebellar artery MB, Main visceral efferent bundle B, Cells at the base of the crista ampullaris MS, Macula sacculi BP, Bipolar neuron distal process PG, Preganglionic cells CA, Crista ampullaris PGC, Postganglionic cells CN, Cochlear nerve RC, Reticular cells DR, Distal portion of facial root S, Superior salivatory nucleus EF, Efferent visceral fascicle SG, Spiral ganglion F, Facial nucleus SL, Spiral limbus FN, Foramen nervosum SO, Superior olivary nucleus G, Genu of facial nerve VCN, Ventral cochlear nucleus IG, Intraganglionic spiral bundle VE, Visceral efferent (“olivo-cochlear” ) bundle LB, Looping visceral efferent bundle VG, Vestibular ganglion

PLATE 1

EXPLANATION OF FIGURES

1 The laterally coursing visceral efferent bundle (“olivo-cochlear”) is shown here (VE). There is no discrete, stained fascicle out of the superior olivary complex. DR, distal portion of the root of the facial nerve; G, genu of the facial nerve; LB, looping visceral efferent bun- dle into the region of the lateral vestibular nucleus; S, superior salivatory nucleus; SO, superior olivary nucleus. pH 6.8. x 45. 2 The most caudal neurons of the lateral preolivary nucleus (heavy arrow) send their dorsal processes into the facial nucleus (F). SO, superior olivary nucleus. pH 6.0. Counterstained with cresyl violet. X 275. VISCERAL EFFERENTS OF EIGHTH NERVE PLATE 1 Muriel D. Ross

465 PLATE 2

EXPLANATION OF FIGURES

3 There is a small nucleus lateral to the genu (G) of the facial nerve which gives origin to a part of the efferent fascicle to the inner ear. PG, preganglionic neurons; RC, reticular cells; VE, visceral efferent bundle. The rounded black structures are air-bubble artifacts. pH 6.8. X 125. 4 Some of the fibers crossing the midline turn into the genu (G) or distal portion of the root (DR) of the facial nerve (slender arrows); others hook under the genu (heavy arrow) to join the laterally di- rected visceral efferent bundle. pH 6.4. X 150.

466 VISCERAL EFFERENTS OF EIGHTH NERVE PLATE 2 Muriel D. Ross

467 PLATE 3

EXPLANATION OF FIGURES

5 A small nucleus of postganglionic cells (PGC) is located at the tip of the ventral cochlear nucleus (VCN) in the vestibular ganglion (VG). Slender collaterals (arrow) from the main visceral efferent bundle (MB) to the inner ear project into this region. pH 6.0. Counter- stained with cresyl violet. x 200. 6 Slender collaterals (fine arrows) of the main efferent bundle (MB) to the inner ear enter the basal cells of the ventral cochlear nucleus (VCN). The pale staining fascicle indicated by the heavy arrow is part of nervus intermedius. pH 6.4. x 125. 7 The basal cells of the ventral cochlear nucleus are, for the most part, acetylcholinesterase-negative (heavy arrows). The fine arrow indicates a collateral from the visceral efferent system which ramifies in the region. pH 6.4. X 1500. 8 An acetylcholinesterase-positive postganglionic neuron (arrow) is shown along the course of the efferent fascicle (EF) to the posterior ampulla. pH 6.4. X 2000.

468 VISCERAL EFFERENTS OF EIGHTH NERVE PLATE 3 Muriel D. Ross

469 PLATE 4

EXPLANATION OF FIGURES

9 Two acetylcholinesterase-positive postganglionic cells in the intra- ganglionic spiral bundle are indicated by heavy arrows. They have numerous, positively reacting terminals on their somata; two of these terminals are indicated by slender arrows. pH 6.4. x 1600. 10 Three types of postganglionic cells lie side by side in the intragan- glionic bundle in this section. The arrow (1) indicates a highly reactive cell; the arrow (2) indicates the axon hillock of a neuron mildly reactive for acetylcholinesterase which has a heavily stained axon; the double arrows indicate a cell of intermediate staining qual- ity which has a markedly eccentric nucleus. pH 6.0. Counterstained with cresyl violet. x 1500. 11 An acetylcholinesterase-positive postganglionic neuron of intermediate size is shown in the intraganglionic spiral bundle. This cell has a few prominent acetylcholinesterase-positive , one of which is indicated by an arrow. pH 6.4. Whole head pretreated with EDTA. X 1700. 12 Two small postganglionic neurons with little or no enzymatic reaction located in the intraganglionic spiral bundle are shown here. pH 6.4. Whole head pretreated with EDTA. x 2000. 13 Cells at the base (B) of the crista ampullaris (CA) are cholinesterase- positive, They are innervated basally by a network of autonomic fibers (arows). pH 6.4. X 375. VISCERAL EFFERENTS OF EIGHTH NERVE PLATE 4 Muriel D. Ross

471 PLATE 5

EXPLANATION OF FIGURES

14 The macula sacculi (MS) is innervated basally by efferent fibers, indicated by arrows. pH 6.4. X 150. 15 The fibers coursing out of the intraganglionic spiral bundle (IG) run along the tympanic border of the osseous spiral lamina (arrow 1). The terminal portions of some of the bipolar neurons are acetyl- cholinesterase-positive (arrows 2 and 3). FN, foramen nervosum; SL, spiral limbus. pH 6.4. x 500.

472 VISCERAL EFFERENTS OF EIGHTH NERVE PLATE 5 Muriel D. Ross

473 PLATE 6

EXPLANATION OF FIGURES

16 Orthosympathetic fibers, two of which are indicated by arrows, join the cochlear nerve (CN) from the anterior inferior cerebellar artery (AIC). Many more such fibers are shown in the field. pH 5.5. Counter- stained with cresyl violet. X 500. 17 A Schwann cell nucleus is indicated by a heavy arrow. An unmy- elinated fiber (slender arrow) runs toward the distal process of a bipolar neuron (BP). FN, foramen nervosum. pH 6.4. X 200. 18 A fine fiber, indicated by a heavy arrow, runs toward the distal my- elinated segments of bipolar neurons (BP) where it appears to end. pH 6.4. x 2000.

4 74 VISCERAL EFFERENTS OF EIGHTH NERVE PLATE 6 Muriel D. Ross

475 PLATE 7

EXPLANATION OF FIGURES

19 A fine, acetylcholinesterase-positive fiber (arrow 1) runs toward the foramen nervosum (FN). Near the foramen it branches (arrow 2) into three terminals, two of which are shown here. An end knob of one of the terminals on the axon of a bipolar neuron at the foramen nervosum is indicated by arrow 3. pH 5.5. Counterstained with cresyl violet. x 200. 20 Acetylcholinesterase-positive fibers coil about some positively reacting bipolar neurons (arrows). The somata of other bipolar neurons can be seen in the background. IG, intraganglionic spiral bundle; SG, spiral ganglion, pH 5.5. Counterstained with cresyl violet. x 1100.

476 VISCERAL EFFERENTS OF EIGHTH NERVE PLATE 7 Muriel D. Ross

477