Development of the brain stem in the rat. III. Thymidine ...Bayer JCN 194 3.pdf · THE JOURNAL OF...

THE JOURNAL OF COMPARATIVE NEUROLOGY 1942377-904 (1980)

Development of the Brain Stem in the Rat. Ill. Thymidine-Radiographic Study of the Time of Origin of

Neurons of the Vestibular and Auditory Nuclei of the Upper Medulla

JOSEPH ALTMAN AND SHIRLEY A. BAYER Laboratory of Deuelopmental Neurobiology, Department of Biological Sciences, Purdue Uniuersity, West Lafayette, Indiana 47907.

ABSTRACT Groups of pregnant rats were injected with two successive daily doses of 3H-thymidine from gestational days 12 and 13 (E12+13) until the day before parturition (E21+22). In adult progeny of the injected rats the proportion of neurons generated on specific embryonic days was determined quantitatively in the vestibular and auditory nuclei of the upper medulla. In the vestibular nuclei, neurons are generated between days E l l and El5 in an overlapping sequential order, yielding a lateral-to-medial and a rostral-to-caudal internuclear gradient. In the lateral vestibular nucleus peak production time is day E12; in the superior nucleus, E13; in the inferior nucleus, El3 and E14; and in the medial nucleus, E14. The early generation of neurons of the lateral vestibular nucleus may reflect the early differentiation of the circuit from the gravity receptors (utricle) to neurons of the spinal cord controlling postural balance. The later production of neurons of the superior vestibular nucleus may reflect the subsequent differentiation of the circuit from the rotational receptors (semicircular canals) to the neurons of the brain stem controlling eye movements. The generation time of neurons of the nucleus prepositus hypoglossi overlaps with that of the medial vestibular nucleus.

The neurons of the anteroventral and posteroventral cochlear nuclei are produced from days El3 to E17, with no temporal differences between the two nuclei. The neurons of the dorsal cochlear nucleus are generated over a very long time span, beginning on day E l 2 and extending into the postnatal period. There is a sequence in the production of neurons forming the different layers of the dorsal cochlear nucleus in the following order: pyramidal cells, cells of the inner layer, cells of the outer layer and, finally, cells of the granular layer. There is also a sequential production of neurons in four nuclei of the superior olivary complex. In the lateral trapezoid nucleus peak production time is day E12; in the medial superior olivary nucleus, day E13; in the medial trapezoid nucleus, day E15; and in the lateral superior olivary nucleus, day E16. This order yields a medial-to-lateral gradient in the dorsal aspect of the superior olivary complex, and a lateral-to- medial gradient ventrally. These mirror-image gradients were also seen intranu- clearly in the lateral superior olivary nucleus and the medial trapezoid nucleus. The cytogenetic gradients could not be related to tonotopic representation; however, they could be related to the lateral location of ipsilateral cochlear nucleus input to the lateral superior olivary nucleus and the medial location of the contralateral cochlear nucleus input to the medial trapezoid nucleus.

Cytogenesis in the upper medulla, excepting its vestibular and auditory nuclei, was the subject of the preceding paper (Altman and Bayer, '80b). This paper deals with cytogenesis in the medullary vestibular and auditory nu-

clei. The vestibular nuclei to be considered separately are the inferior, lateral, medial and superior nuclei, and the related nucleus prepositus hypoglossi. The auditory nuclei of the upper medulla are grouped into the cochlear

0021-9967/80/1944-0877$04.70 0 1980 ALAN R. LISS, INC.

878 JOSEPH ALTMAN AND SHIRLEY A. BAYER

nuclear complex and the superior olivary complex. Three components of the cochlear nucleus complex will be treated separately: the dorsal, posteroventral and anteroventral nuclei; and four components of the superior olivary complex: the medial and lateral trapezoid nuclei, and the medial and lateral superior olivary nuclei. The dorsal and ventral nuclei of the lateral lemniscus will be considered in the next paper of the series (Altman and Bayer, '80~) .

Previous radiographic datings in the rat of the time of origin of neurons of the vestibular complex and of the superior olivary complex are not known to us. Taber Pierce ('67) has provided a description of cytogenesis and histogenesis in the dorsal and ventral cochlear nuclei of the mouse, and there is a brief description of the postnatal origin of the granule cells of the cochlear nuclei from our laboratory (Altman and Das, '66). A correlation of radiographic dat- ing of the time of origin of neurons of the medullary vestibular and auditory nuclei with the embryonic development of this region will be attempted in a future publication.

MATERIALS AND METHODS

The autoradiographic material used in this paper is identical with that described in the first publication of this series (Altman and Bayer, '80a). The proportion of labeled cells was determined in the vestibular and auditory nuclei of the upper medulla, classifying a minimum of 100 cells in 6 animals each in all relevant injection groups from days E12-i-13 days E21+22. Statistical statements are based on Conover's sign test, as previously described (Altman and Bayer, '80a).

RESULTS The vestibular nuclear complex

Four nuclei are usually distinguished in the central vestibular complex: the lateral (nucleus of Deiters), the superior (nucleus of Bechterew), the medial (nucleus of Schwalbe) and the inferior (spinal, descending); Brodal('74) has described some additional smaller nuclei. According to Lorente de No's ('33a) study based on Golgi work, the lateral nucleus receives primary afferents from the utricle; the superior nucleus, from the semicircular canals. This pattern of projection was largely confirmed in recent experimental studies (Gacek, '69), although Stein and Carpenter ('67) also favor the termination of utricular fibers in the inferior nucleus. Fibers from the utricle and saccule also reach the medial nucleus. But the medial nucleus, and the inferior nucleus, are the projection fields mainly of the semicircular canals (Gacek, '69; Brodal, '74).

Several central nervous structures sup- ply secondary afferents to the vestibular nuclei: the cerebellar cortex (Dow, '36; Walberg and Jansen, '61; Angaut and Brodal, '67), the fastigial nucleus (Walberg et al., '62), and the reticular formation and spinal cord (Brodal, '74). Secondary visual input to the vestibular nuclei is indicated by physiological studies (Waespe and Henn, '77, '78).

The descending efferents of the lateral nucleus reach the spinal cord by way of the lateral vestibulospinal tract, whereas those of the medial nucleus arrive by way of the medial longitudinal fasciculus (Nyberg-Hansen, '64; McMasters et al., '66). Other efferents reach the cerebellum (Brodal and Torvik, '57; Kotchabhakdi and Walberg, '78) and several subdivisions of the pontine and medullary reticular formation (Ladpli and Brodal, '68). Ascending efferents, mainly from the superior nucleus and the rostra1 portion of the medial nucleus, reach, by way of the medial longitudinal fasciculus, the motor nuclei of the extraocular muscles, the intersititial nucleus of Cajal and the nucleus of Darkschewitsch (McMasters et al., '66; Stein and Carpenter, '67; Tarlov, '70; Graybiel and Hartwieg, '74). There is an orderly progression in cy-

togenesis in the four nuclei of the vestibular complex of the medulla. As a group, the neurons of the lateral vestibular nucleus are produced first, followed in succession by the neurons of the superior, inferior, and medial vestibular nuclei (Fig. 1A). In the lateral nucleus cytogenesis peaks on day E12, in the superior nucleus on day E 13; only a small proportion of cells arises in either structure on day E14, and these are all small cells. Peak generation time in the inferior nucleus is day E13, but a large proportion of cells arise on day E14; in the medial nucleus cell generation peaks on day El4 and 12% of the cells form on day E15. The differences between all pairs of vestibular nuclei were statistically significant (P<0.0127- 0.0001).

Most of the neurons of the lateral vestibular nucleus are large (Fig. 2B). A few of these large cells (Deiters neurons) were no longer labeled in the E12+13 group, indicating very early generation time. But many were still labeled in the E13-i-14 group, including the largest cells. In the E14+15 animals, the few remaining labeled cells were small ones. In the superior vestibular nucleus, the largest cells were smaller than the typical Deiters neurons; here cell acquisition began on day El2 and peaked later (Fig. 1). Again, the few labeled cells in the E14-i-15 group were small cells. Hence, large

VESTIBULAR AND AUDITORY CYTOGENESIS 879

Abbreviations CNa, anteroventral cochlear nucleus CNd, dorsal cochlear nucleus CNp, posteroventral cochlear nucleus gr, granular layer (cochlear nucleus) LV, ventral nucleus of the lateral lemniscus me, medial VI, abducens nucleus MLF, medial longitudinal fasciculus pl, pars lateralis pm, pars medialis PR, nucleus prepositus hypoglossi Sol, lateral superior olivary nucleus SOm, medial superior olivary nucleus TRl, lateral trapezoid nucleus TRm, medial trapezoid nucleus

v4, fourth ventricle 111, oculomotor nucleus IV, trochlear nucleus VM, motor nucleus of the trigeminal Vo, nucleus oralis of the trigeminal

W g , genu of facial nerve VIIIi, inferior vestibular nucleus VIIIm, medial vestibular nucleus VIIIl, lateral vestibular nucleus VIIIs, superior vestibular nucleus Xd, dorsal motor nucleus of vagus XII, hypoglossal nucleus

neurons are generated before small neurons in these two nuclei. However, in the inferior vestibular nucleus, large neurons comparable in size to those of the superior nucleus were still labeled in high proportion in the E14+15 rats (Fig. 3). Moreover, the neurons of the inferior nucleus that were labeled in E15+16 animals included the largest cells. Similarly, in the medial vestibular nucleus, in which neurons range in size from intermediate to small (Fig. 4), there was no relationship between cell size and labeling: for instance, the labeled cells in the E15+16 animals included all cell sizes. A weak ventrolateral-to-dorsomedial cytogenetic gradient was noted in the medial vestibular nucleus.

The nucleus prepositus hypoglossi The nucleus prepositus lies anterior to

the hypoglossal nucleus and merges inper- ceptibly into the nucleus of Roller caudally (Altman and Bayer, '80a: Fig. 2) and the medial vestibular nucleus rostrally (Altman and Bayer, '80b: Fig. 1). It has two parts, a caudal portion with many large cells, some of which approximate in size the neurons of the hypoglossal nucleus (Fig. 5 ) and a rostra1 and lateral portion of smaller cells that resemble the cells of the medial vestibular nucleus.

The prepositus nucleus was classified in the past as part of the perihypoglossal group of nuclei (Brodal, '52) on the assumption that its functions are related to the control of the tongue. It was subsequently shown that the nucleus projects to the vestibular cerebellum (Torvik and Brodal, '54) and receives fibers from the fastigial nucleus (Walberg, '61). More recent anatomical and physiological studies indicate that the prepositus nucleus is involved in the control of eye movements. In addition to cerebellar connections (Alley et al., '75; Kotchabhakdi et al., '78), projections from the prepositus nucleus have been described to the vestibular nuclei (Baker and Ber- thoz, '751, and the oculomotor (Graybiel

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EMBRYONIC DAY Fig. 1. Time of origin of neurons in the four vestibular

nuclei and the nucleus prepositus hypoglossi.

and Hartwieg, '74), the abducens (Maciewicz et al., '77) and the trochlear (Baker et al., '77) nuclei. Moreover, evidence is accumulating that neuronal activity in the prepositus nucleus is correlated with eye movements (Baker et al., '76; Hikosaka et al., '78). The large neurons of the prepositus nucleus,

which are concentrated in the posteromedial portion of the nucleus and are contiguous with


Fig. 2. A. Superior vestibular nucleus. B. Lateral vestibular nucleus. Arrows point to neurons with labeled nuclei. From a rat injected on days E13+14. Scale, 50 pm.

VESTIBULAR AND AUDITORY CYTOGENESIS aai

Fig. 3. A. Inferior vestibular nucleus. Area in rectangle shown at higher magnification in B. Note the labeled large neurons and some labeled smaller cells (arrows). From a rat injected on days E14+15. Scales: A, 100 pm; B, 50 pm.


Fig. 4. Medial vestibular nucleus, from a rat injected on days E14+ 15. Rectangular area in A shown at higher magnification in B. Scales: A, 200 Fm; B, 50 pm.


8.5


Fig. 6. A. Cochlear nuclear complex in sagittal section from a rat injected on days E13f14. Dorsal cochlear nucleus (rectangle b) is magnified in b; anteroventral cochlear nucleus in c; and posteroventral cochlear nucleus in d. Arrow ind is an unlabeled octopus cell. Scales: A, 200 pm; b d , 50 pm.


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the large neurons of the hypoglossal nucleus, are distinguishable from the latter in au- toradiograms by their labeling pattern (Fig. 5). Hypoglossal neuron production peaks and ends on day E l2 (Altman and Bayer, '80a: Fig. 4), whereas the generation of prepositus neurons peaks on day E l 4 and ends on day E l 5 (Fig. 1B). The difference was significant at the P <0.0001 level. There was no apparent difference in the time course of production of cells of different sizes, and the latest forming neurons seen in the E15+ 16 group included the largest prepositus neurons. As shown in Figure lB, daily cell acquisition in the prepositus nucleus closely resembled that of the medial vestibular nucleus, into which it blends rostrally. The statistical test showed that the generation of prepositus neurons lagged slightly behind those of the medial vestibular neurons (P <0.0490).

The cochlear nuclear complex Ramon y Cajal ('11) divided the cochlear nu-

cleus into two parts, which are today referred to as the dorsal and the ventral cochlear nuclei. On the basis of extensive Golgi studies, Lorente de NO ('33b) devised a classification into 13 regions. Because these regions cannot all be identified in Nissl-stained material, modern investigators usually distinguish three nuclei: the dorsal, the posteroventral, and the anteroventral (Fig. 6); sometimes a fourth is added, the interstitial or ventral nucleus (Fernandez and Karapas, '67; Harrison and Howe, '74). The axons of the acoustic nerve enter the ventral nucleus and bifurcate into ascending and descending branches. The ascending axons terminate with large synaptic endings (the bulbs of Held) in the anteroventral cochlear nucleus; the descending axons end in pericellular nests in the posteroventral cochlear nucleus and in diffusely ramifying terminals in the dorsal cochlear nucleus.

In quantifying labeled cells in the cochlear nucleus (Fig. 7) we made no distinction between cell types and did not include the lightly labeled, and partially postnatally forming (Altman and Das, '66), granule cells. With this approach, there was no statistically significant difference in the birth dates of the neurons of the three cochlear nuclei. The time of origin of different types of cochlear nucleus neurons was deduced from qualitative observations.

The dorsal cochlear nucleus The dorsal cochlear nucleus has been di-

vided into three layers or regions (Ramon y Cajal, '11; Lorente de NO '33b). The outer (plexiform or molecular) layer contains a

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EMBRYONIC DAY

Fig. 7. Time of origin of neurons in the three components of the cochlear nucleus.

few globular cells. The intermediate layer contains pyramidal (or fusiform) cells whose dendrites are oriented toward the outer and inner layers. The inner layer, also called the central region, contains some giant cells and many small cells. The axons of the dorsal cochlear nucleus form the crossed, dorsal acoustic stria (the stria of Monakow). These fiber terminate in the ventral and dorsal nuclei of the lateral lemniscus (Lorente de NO, '33b) and, in addition, in some accessory nuclei of the superior olive and in the inferior colliculus (Fernandez and Karapas, '67). The cells of origin of this pathway are probably the pyramidal cells (Osen, '72; Beyerl, '78). Neuron production in the dorsal cochlear nu-

cleus begins on day E12, continues through the embryonic period (Fig. 71, and ends in the postnatal period, when an undertermined proportion of the granule cells are produced (Altman and Das, '66). In its extended generation time, and also in the sequential production of its large, intermediate and small neurons, the neurogenesis of the dorsal cochlear nucleus re- sembles that of the cerebellum (Altman and Bayer, '78a: Fig. 20).

The pyramidal cells are generated first. In some regions most of the pyramidal cells were


Fig. 8. Dorsal cochlear nucleus from a rat injected on days E13f14. Arrows point to unlabeled pyramidal cells. Scale, 50 w.

no longer labeled in animals injected on days E13+14 (Fig. 8), but in other regions many of the pyramidal cells were still labeled in this group (Fig. 9). Labeled pyramidal cells were absent (Fig. 10A) or reduced to a few in the E14+15 group; hence it was concluded that pyramidal cells are generated between days El2 and E14. The typical, relatively small cells of the inner layer was consistently tagged in the E14+15 rats, but the proportion of unlabeled cells increased from the E15+16 (Fig. l ld ) to the E16+ 17 group, and very few, if any,

were labeled in the E17+18 group. Thus, the predominant cell type of the inner layer is generated between days El4 andE16. However, an unidentified small cell type (one that is larger and stains less intensely than granule cells) wasoftenfoundwithlabel intheE17+18group and sometimes in the E18+19 rats. This cell resembled the globular cells of the outer layer, which typically were labeled in the latter group and in later-injected animals. The globular cells constituted the majority of neurons forming between days El7 and El9 (Fig. 7).


Fig. 10. Thecochlear nuclearcomplex from a rat injected on days E14+15. A, dorsal nucleus; B, posteroventral nucleus; C, anteroventral nucleus. In A, arrows point to unlabeled pyramidal cells. Scale, 50 pm.

In summary, the observations indicate that first (days E12+E14), the predominant cell of the prolonged production of dorsal cochlear nu- the inner layer next (peak on day E15; Fig. 7), cleus neurons represents the sequential gem and the small cells of the inner layer and the eration of its cell types. Pyramidal cells arise globular cells of the outer layer thereafter (days


Fig. 11. The cochlear nuclear complex from a rat injected on days E15+16. The subdivisions (areas in rectangles magnified in figures with corresponding letters) are: b, anteroventral nucleus; c, posteroventral nucleus; d, dorsal nucleus. Region in e may be part of the psteroventral nucleus. Scales: A, 500 pm; b e , 100 pm.

El7 to E19). The granule cells that are embed- ded in the dorsal cochlear nucleus and the granule cells that form a separate layer were all lightly labeled in the latest embryonic injection groups. An undetermined proportion of granule cells is generated postnatally.

The posterovental cochlear nucleus The posteroventral cochlear nucleus

may have several subdivisions (Lorente de NO, '33b; Harrison and Irving, '66b), but they are not easy to delineate in Nissl- stained sections. The central part of the nucleus is composed of cells that receive endings with bulbs of Held from the acoustic nerve and send axons to the trapezoid body. The large multipolar cell of the nucleus, the octopus cells of Osen ('721, con-

tribute large diameter fibers to the intermediate acoustic stria (the stria of Held) and terminate primarily in the ipsilateral superior olivary complex (Harrison and Howe, '74). All neurons were labeled in this nucleus in

the E13+ 14 group, except a few of the largest, the presumed octopus cells (Fig. 6d). Some of these largest cells were still labeled in the E14+15 animals but not in later injected groups, suggesting that octopus cells are produced between days El2 and E14. The dominant cell type of the posteroventral nucleus, which ranges widely in size, was consistently labeled in the E14+15 rats (Fig. lOB), but many of them were no longer labeled in the E15+16 group (Figs. llc,e). In the E16+17 animals, only the smaller cells were labeled.

891 VESTIBULAR AND AUDITORY CYTOGENESIS

The peak cell acquisition on day E l5 (Fig. 7) undoubtedly represents the bulk of the dominant cell type of this nucleus. It was not possi- ble to discern a directional gradient in the nucleus, and the small proportion of late- generated neurons appeared to be distributed irregularly.

The anteroventral cochlear nucleus In the anteroventral cochlear nucleus,

the preponderant neurons are the spherical cells. It has been observed that the larger spherical cells are situated dorsally and that they decrease in size ventrally (Harrison and Irving, '66b; Osen, '72). The spherical cells receive acoustic input by way of the bulbs of Held, and their efferents join the trapezoid body (the ventral acoustic stria). The large spherical cells are believed to project to the medial superior olivary nucleus; the small spherical cells, to the lateral superior olivary nucleus (Harrison and Howe, '74). According to another classification (Brawer and Morest, '75), the two cell types of the anteroventral nucleus are the bushy and the stellate cells. In our material, the cells of the anterovental

nucleus were slightly smaller than the dominant cells of the posteroventral nucleus and were more uniform in size and shape (Figs. 6c, 9b, lOC), with only a few scattered small cells. All the neurons were labeled in the E 13 + 14 group (Fig. 6c) and nearly all in the E14+15 group (Fig. 1 0 0 . The proportion of unlabeled cells increased in the E15+16 animals (Fig. llb), and only a few scattered small cells were labeled in the E16+17 and the E17+18 animals. The quantitative data showed (Fig. 7) that the bulk of the neurons of the anteroventral nucleus leave the proliferative compart- ment on days E l4 and E15, representing undoubtedly the dominant cell type.

The superior olivery nuclear complex The superior olivary complex occupies the

ventral protuberance of the upper medulla and is composed of several discrete, and some less clearly delineated, nuclei. Most prominent of these nuclei are the lateral and medial superior olivary nuclei and the medial nucleus of the trapezoid body (Fig. 12). In addition, there are several periolivary nuclei whose size, shape and location may vary in different species (Harrison and Howe, '74). We have examined only one of the latter nuclei, what we refer to as the lateral trapezoid nucleus.

The lateral superior olivary nucleus In the rat, the lateral superior olivary

nucleus has a convoluted shape, with two ovoid masses, the pars lateralis and the pars medialis (Fig. 12A). It is composed of elongated cells whose dendrites and axons are arranged radially (Harrison and Howe, '74). Aff'erents reach the nucleus from the ipsilateral cochlear nucleus (Stotler, '53; Powell and Cowan, '62; Harrison and Ir- ving, '66a; Strominger and Strominger, '71). The neurons of the nucleus are all excited by stimulation of the ipsilateral ear, and most of them are inhibited by contralateral stimulation (Galambos et al., '59; Guinan et al., '72; Tsuchitani, '77). The efferents of the lateral nucleus pass bilaterally in the lateral lemniscus and terminate in the central nucleus of the inferior colliculus (Stotler, '53; Elverland, '78). For quantitative purposes cells were counted

in the pars lateralis of the lateral superior olivary nucleus. The results indicate a prolonged acquisition time, with a peak on day El6 (Fig. 13). As shown in Figures 12b and 14, in rats injected on days E16+17 many neurons were labeled in the pars lateralis of the nucleus, but only few in the pars medalis. This medial-to- lateral intranuclear gradient within the lateral supraolivary nucleus was matched by a similar internuclear gradient between the lateral and medial superior olivary nuclei (see below).

The medial superior olivary nucleus The medial superior olive consists of

fusiform cells with medially and laterally directed dendrites (Stotler, '53). Each cell receives afferents from both the ipsilateral and the contralateral cochlear nucleus; afferents from the ipsilateral cochlear nucleus terminate on the lateral dendrite, whereas contralateral axons terminate on the medial dendrite (Stotler, '53; Lindsey, '75). The principal source of afferents is the anteroventral cochlear nucleus (Warr, '66; Osen, '69). Almost all the neurons of the medial nucleus respond to binaural stimulation (Goldberg and Brown, '68; Erulkar, '72). The efferents of the medial superior olivary nucleus enter only the ipsilateral lateral lemniscus (Stotler, '53; Elverland, '78) and terminate in the central nucleus of the inferior colliculus. The bulk of the neurons of the medial supe-

rior olive are generated on day E l3 (Fig. 131, apparently starting later than, but finishing ahead of, the neurons of the lateral superior


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EMBRYONIC DAY Fig. 13. Time of origin of neurons in four components of

the superior olivary nuclear complex. The difference between each pair was significant (P <0.07760.0001).

olive. Although cell counts were not made in the pars medialis of the lateral superior olivary nucleus, observations indicated that in the E16+17 group some of its cells were still labeled, but none were in the medial superior olive (Fig. 12A). The chronological difference in the generation of neurons of the medial and lateral superior olivary nuclei was significant (P <0.0108). The medial and lateral superior olivary nuclei may constitute a single cytogenetic system (see Discussion) in which earlier generated neurons settle medially and later produced neurons move progressively more laterally.

The medial trapezoid nucleus The principal neuron of the medial nu-

cleus of the trapezoid body has an eccentric

nucleus and richly branching dendrites (Morest, '68). Large diameter axons of the trapezoid body from the contralateral ventral cochlear nucleus terminate here with single calices of Held (Held, 1893; Ramon y Cajal '11; Stotler, '53; Warr, '72). Virtually all neurons of this nucleus respond to contralateral stimulation (Galambos et al., '59) with short latency (Goldberg and Brown, '68). The axons of the nucleus pass to the ipsilateral lateral superior olivary nucleus (Harrison and Howe, '74; Elver- land, '78). Ninety percent of the neurons of the medial

trapezoid nucleus are generated on day El5 (Fig. 13). The nucleus has a pronounced lateral-to-medial intranuclear gradient (the opposite of that in the lateral superior olivary nucleus), and the small complement of cells generated on day E l6 (Fig. 13) are situated consistently in the medial aspect of the nucleus (Figs. 12, 15). No gradient could be discerned along the anteroposterior extent of the nucleus (Fig. 16).

The lateral trapezoid nucleus There are several additional nuclei in

the superior olivary nuclear complex, var- iably referred to in generic terms as the periolivary, preolivary and retro-olivary nuclei, and by such specific terms as the lateral, ventral and dorsal trapezoid nuclei, etc. We have examined only one of these, a nucleus that is made conspicuous in Nissl-stained sections by the orientation and tight packing of its cells and, in au- toradiograms, by the singularly early ces- sation of labeling of its neurons.

This nucleus is spindle- or coma-shaped, and is oriented vertically (Altman and Bayer, '80b: fig. 2B) between the anterior halves of the lateral and medial superior olivary nuclei. There must be considerable species differences in the shape and, perhaps, position, of this nucleus (Brown and Howlett, '72). It may correspond in the cat t o the nucleus referred to as the ventral trapezoid nucleus (Taber, '61), the retrool- ivary nucleus (Rasmussen, '60) or the medial periolivary nucleus (Fernandez and Karapas, '67; Goldberg and Brown, '68), and in the rat, as the dorsal trapezoid nucleus (Brown and Howlett, '72). We shall refer to it as the lateral trapezoid nucleus. The region of this nucleus has been identified in other studies as the site of origin (or one of the sites of origin) of the efferent


Fig. 14. A. Sagittal section through the ventral aspect of the upper medulla with the pars lateralis of the lateral superior olivary nucleus. Region in rectangle enlarged in B. From a rat injected on days E16+17. Scales: A, 200 Fm; B, 50 Fm.


Fig. 15. A and B. Coronal sections through the medial trapezoid nucleus from a rat injected on days E16+17. Note that labeled cells (arrows) are medially situated. Scales: A, 100 pm; B, 50 pm.


Fig. 16. Sagittal sections through the medial trapezoid nucleus from lateral (A) to medial (B) from a rat injected on days E16+17. The cells in the medial aspect of the nucleus are labeled, but there is no gradient along the anteroposterior axis. Scale: 100 pm.


Fig. 17. A. Coronal section showing the unlabeled cells of the lateral trapezoid nucleus from a rat injected on days E 14+ 15. The nucleus is enlarged in B. In this animal all the cells of the medial trapezoid nucleus and medial superior olivary nucleus are labeled. Scales: A, 100 pm; B, 50 pm.

olivocochlear bundle (Rasmussen, '60; Shute and Lewis, '60; Osen and Rother, '69; Warr, '75). As a group, the neurons of the lateral

trapezoid nucleus are the earliest produced cells in the auditory nuclei of the upper medulla. All comparisons with the other auditory nuclei were highly significant (P <0.0001). The bulk of the cell population of the lateral trapezoid nucleus is generated on day E 12, with

some cells forming before and afterwards (Fig. 13). In some regions a few labeled cells were seenin theanimalsinjectedondaysE13+14; in other regions all cells remained unlabeled (Fig. 17). Because of the relatively small size of the nucleus, the existence of an intranuclear gradient could not be determined. But if the identification of the nucleus as a component of the trapezoid nuclear subsystem is correct, then there is an internuclear gradient from the


early-generated lateral trapezoid nucleus to the late-generated medial trapezoid nucleus. Moreover, as in the case of the superior olivary nuclei, there is a match between the di- rectionality of the intra- and internuclear gradients: medial-to-lateral in the case of the two superior olivary nuclei, lateral-to-medial in the case of the two trapezoid nuclei (Fig. 18).

DISCUSSION

The vestibular nuclear complex Our datings indicate an orderly progression

in the generation of neurons of the four vestibular nuclei: the lateral nucleus first, followed by the superior and inferior nuclei, and the medial nucleus last. This suggests two cytogenetic gradients (Fig. 19); a lateral-to-medial gradient (from the lateral to the superior nucleus, and from the inferior to the medial nucleus) and a rostral-to-caudal gradient (from the lateral and superior nuclei to the inferior and medial nuclei). The lateral-to-medial (outside-in) gradient is the opposite of that seen in the other sensory nuclei of the medulla. We noted previously (Altman and Bayer, ’80a) a medial-to-lat- era1 (inside-out) gradient in the caudal nucleus of the trigeminal complex, and in the dorsal column nuclei in relation t o the external cuneate nucleus. Moreover, the neurons of the vestibular nuclei, as a group, are generated somewhat earlier (mainly between days E l 2 and 14) than the neurons of the other relay nuclei (mainly between days E l 3 and 15). These differences justify the tentative classification of the vestibular nuclei as part of a separate cytogenetic system’ designated zone VE (Fig. 19).

Assuming that the four vestibular nuclei constitue a single cytogenetic system, the noted internuclear gradients allow certain infer- ences. The larger neurons of both the lateral and superior nuclei are produced before the

‘A specific set of letters designating a “cytogenetic system’’ or “cytagenetic Zone” (Altman and Bayer, ’80a) refers to related structures that either have similar patterns of cytogenesis or jointly constitute a continuous cytogenetic gradient. The assumption is made that neurons of such a cytogenetic system derive from a shared germinal source in the embryonic neuroepithelium. The birth dates ofneuron production in such a cytogenetic system provide the clue as to when (what embryonic ages) the corresponding neuroepithelial sites should show a high level of mitotic activity; the cytogenetic gradient, as a “directional m o w ” of the migratory route of the settling cells, may provide hints as to where (what embryonic sites) the specific germinal site should be found. An example of such an attempt was the probable identification, in the neuroepithelium of the third ventricle (Altman and Bayer, ’784, of the sites of origin of the neurons of the supraoptic and paraventricular nuclei, constitutingone cytogenetic system, and of the dorsomedial and ventromedial nuclei, constituting another. In the case of the distally settling neurons of the supraoptic nucleus, spindle- shaped (apparently migrating1 cells could he traced from the presumed production site, a t the future location ofthe paraventricular nucleus, to their final destination (Altman and Bayer, ‘78c: Figs. 7, 81.

1 II

II

Fig. 18. Schematic diagram of intranuclear gradients (light arrows) and internuclear gradients (heavy arrows) in the superior olivary nuclear complex. In the superior olivary nuclei the gradients are medial-to-lateral; in the trapezoid nuclei the gradients are lateral-to-medial. Number refers to approximate peak embryonic days of neuron production.

me

Fig. 19. Schematic diagram of the sequential order (from 1 to 4) in the production of neurons of the four vestibular nuclei. The lateral-to-medial, and the rostral-to-caudal gradients are indicated.

smaller cells, and the very large Deiters neurons of the lateral nucleus are generated before the next-sized neurons of the superior nucleus. According to the available evidence, the efferents of the Deiters neurons descend ipsilater- ally the entire length of the spinal cord by way of the lateral vestibulospinal tract (Nyberg- Hansen, ’64; McMasters et al., ’661, whereas the efferents of the superior nucleus ascend by way of the ipsilateral medial longitudinal fasciculus and terminate bilaterally in the nuclei of the extraocular muscles and some related structures (Tarlov, ’70; Graybiel and Hartwieg, ’74; Brodal, ’74). If the larger neurons within these structures are the main sources of efferents, it would follow that the neurons with descending efferents are generated before the neurons with ascending efferents. Moreover, insofar as the lateral nucleus receives primar-

JOSEPH ALTMAN AND SHIRLEY A. BAYER

Wilson, '72; Brodal, '74) but also from the utricle and saccule (Stein and Carpenter, '67). Un- like the outflow from the lateral nucleus, which is descending by way of the lateral vestibulospinal tract, the outflow from the inferior nucleus is mostly reticular and commissural (Brodal, '74). Ascending efferents from the inferior nucleus are few (McMasters et al., '66) or absent (Tarlov, '70; Graybiel and Hartwieg, '74; Brodal, '741, resembling the lateral nucleus in this respect. The medial vestibular nucleus receives semicircular afferents (Stein and Car- penter, '67; Gacek, '69; Brodal, '74), as does the medially situated superior nucleus. In addition to its ascending fibers in the medial longitudinal fasciculus <McMasters et al., '66; Farlov, '70; Graybiel and Hartwieg, '74), the medial nucleus also provides descending efferents to the cervical and upper thoracic spinal cord (Nyberg-Hansen, '66). However, these descend-

3 ing efferents travel by way of the "medial vestibulospinal tract" (Nyberg-Hansen, '66; Wil- son, '721, and the physiological effects of these axons are different from those of the lateral vestibular axons traveling in the lateral vestibulospinal tract. The latter have an excitatory effect on extensor motor neurons, whereas the former have an inhibitory influence (Wilson, '72). Brodal ('74) suggested that these shorter descending fibers are involved in the integ-ration of eye and neck movements.

n (r 0 0 1

_I

2 $

a J 1 ; Y E cn

me

w

Fig. 20. The sequential production of neurons of the four vestibular nuclei (1 to 4, as in Fig. 20) in relation to the major input and output relations of the nuclei. The ipsilateral ascending MLF outflow of the superior nucleus, and contralat- era1 outflow of the medial nucleus is based on Tarlov ( ' 70 fig. 6). See text for details.

ily utricular afferents and the superior nucleus semicircular afferents (Lorente de NO, '33a; Stein and Carpenter, '67; Gacek, '69; Wilson, '72; Brodal, '74), it would follow that the relay neurons in the utricular-spinal cord circuit related to gravity receptors and the righting re- flexes are generated before neurons of the semicircular-oculomotor circuit related to the vestibular control of eye movements (Fig. 20).

The relationships observed in the lateral and superior vestibular nuclei seem to hold only partially for the later arising inferior and medial vestibular nuclei. First, the sequential production of large and small neurons is absent in these two nuclei; in the medial nucleus some of the larger neurons originate as late as day E15. Moreover, both input and output from these nuclei are more heterogeneous. The inferior nucleus appears to receive afferents not only from the semicircular canals (Gacek, '69;

In summary, the earliest generated neurons of the vestibular nuclei are the Deiters neurons that receive afferents from the gravity receptors and have an excitatory influence on the extensor (antigravity) motoneurons of the spinal cord. The neurons of the superior nucleus are produced next; they receive afferents from the semicircular canals and, in turn, influence the nuclei concerned with eye movements. The later generated neurons of the inferior and medial nuclei also appear to be associated with the oculomotor component of the vestibular system, providing shorter descending fibers, commissural fibers and fibers that ascend in the contralateral medial longitudinal fasciculus (Fig. 20). It will remain for future research to establish whether the order of the outgrowth of efferents from these nuclei matches the order in which the neurons are generated. The regional differences in the order of generation of large and small neurons will be discussed in a future publication.

The nucleus prepositus hypoglossi The prepositus nucleus is continuous cau-

dally with the region of the hypoglossal nu-


cleus. There is also an apparent continuity between the large cells of this nucleus and the motor neurons of the hypoglossal nucleus. However, our results indicate that these two nuclei differ from one another both in terms of the birth dates of their neurons and the temporal patterns of their cytogenesis. The bulk of hypoglossal neurons form on a single day very early (E12; Altman and Bayer, '80a: Fig. 4A), whereas the prepositus neurons form later over an extended period (days E12-15, with a peak on day 14; Fig. 1). Our earlier results indicated that the cytogenesis of prepositus neurons resembled that of the nucleus of Roller, with which it is continuous caudally (Altman and Bayer, '80a: Fig. 2). The nucleus of Roller and prepositus nucleus. which have been classified together as the perihypoglossal nuclei (Brodal, '52), may constitute a single cytogenetic system; we have designated it zone PR (Altman and Bayer, '80a: Fig. 26).

The accumulating experimental evidence shows that the perihypoglossal nuclei are not functionally related to the hypoglossal nucleus but rather to the cerebellum (Torvik and Brodal, '54; Walberg, '61; Alley et al., '75; Kotchabhakdi et al., '78), the vestibular nuclei (Baker and Berthoz, '75) and the motor nuclei of the extraocular muslces (Graybiel and Hartwieg, '74; Maciewicz et al., '77; Baker et al., '77). It is of interest that the time of origin and pattern of neurogenesis in the prepositus nucleus is similar to that of the medial vestibular nucleus (Fig. l), with which it merges with- out evident boundaries rostrally. It is conceiv- able that the prepositus nucleus constitutes the fifth of the large medullary vestibular nuclei.

The cochlear nuclear complex The anteroventral and posteroventral

cochlear nuclei differ in several respects. The anteroventral nucleus receives the ascending branches of the bifurcating primary acoustic fibers, and its efferents form the ventral acoustic stria (the trapezoid body). The posteroventral nucleus received the descending branches of the primary acoustic fibers, and its efferents form the intermediate acoustic stria (the stria of Held). In our material there was also a size difference in the dominant cell types of the two regions: the typical cells of the posteroventral nucleus tended to be somewhat larger than those of the anteroventral nucleus. However, there were no corresponding differences in the generation times of neurons in these two nuclei. Moreover, we could not detect any cytogenetic gradients that might correspond to

the gradient that was observed in the spiral ganglion of the mouse (Ruben, '671, the earliest produced primary neurons being situated in the basal portion of the cochlea; the latest neurons, in the apical portion. The only cytogenetic sequence that did emerge was between large and small cells. In the posteroventral nucleus the largest neurons, what may be the octopus cells, were produced between days El2 and E14, the dominant intermediate-size cells (which contributed to the peak in Fig. 7) were produced on day E15. The generation of the dominant cells in the anteroventral nucleus peaked, likewise, on day E15. In both regions, small neurons were still produced on days El6 and E17. We conclude tentatively that these two components of the cochlear nuclear complex are cytogenetically similar.

The dorsal cochlear nucleus differs in many respects from the other two nuclei of the complex. Its efferents, which form the dorsal acoustic stria (the stria of Monakow), are directed rostrally and terminate in the ventral and dorsal nuclei of the lateral lemniscus (Lorente de NO, '33b), in contrast to the anteroventral and posteroventral nuclei whose efferents are directed ventrally and reach various components of the superior olivary complex (Harrison and Howe, '74). There is also a cytological difference: the dorsal cochlear nucleus is composed of a greater variety of cell types and it has a laminar organization. The heterogeneity of cellular composition of the dorsal nucleus is matched by an extremely protracted neurogenesis that begins on day El2 (Fig. 7) and ends postnatally (Altman and Das, '66). The earliest produced cells are the large pyramidal or fusiform cells, which may be the source of the efferents passing in the stria of Monakow. The pyramidal cells are generated on days El2 and 13, a few of them on day E14. The predominant cells of the inner layer are produced mostly on days ElPE16, with some unidentified cells being generated later. The globular cells of the outer layer are produced next, mostly between days El6 and 19. The last arisingcells are the granule cells that are found scattered in the dorsal nucleus and in a separate layer. This sequence results in a complex lamination pattern (Fig. 211, with the latest produced cells forming the external and inter- nal boundaries of the nucleus.

The cytogenesis of the dorsal cochlear nucleus bears some resemblance to that of the cerebellum (Altman and Bayer, '78a). This includes the prolonged production of neurons and a sequence from large output cells to smaller


Fig. 21. Schematic summary of cytogenetic lamination pattern in the dorsal cochlear nucleus.

intrinsic neurons. At least one component of both structures, the granule cells, may derive from a shared source issuing from the germinal trigone of the recess of the fourth ventricle (Altman and Bayer, '78a). If it is correct that some cells of the cochlear nuclear complex share the germinal matrix of the cerebellum, and considering the great cellular heterogeneity of a t least the dorsal nucleus, it is pos- sible that the cells of the cochlear nucleus complex are derived from more than one cytogenetic zone. This possibility will be examined in embryonic material.

The superior olivary nuclear complex There are considerable species differences in

the size, configuration and location of some of the nuclei of this system (Harrison and Howe, '74). We found it convenient to distinguish in the rat two horizontally arranged set of nuclei on the basis of cytogenetic gradients (Fig. 18). The major components of the dorsal tier nuclei, the medial and lateral superior olivary nuclei, have a medial-to-lateral gradient. The two components of the ventral tier nuclei, the medial and lateral trapezoid nuclei, have a lateral-to-medial gradient. According to electrophysiological studies in the cat (Guinan et al., '721, the tonotopic organization in both the lat-

eral superior olivary nucleus and the medial trapezoid nucleus is from high frequency medially to low frequency laterally (Fig. 22). Since in these nuclei tonotopic representations are in register, whereas cytogenetic gradients are mirror images, the cytogenetic gradient cannot be related to tonotopic organization.

In terms of available information of cochlear cytogenesis in the mouse (Ruben, '671, there are two gradients: the production of hair cells and supporting cells proceeds from apical to basal (low to high frequencies), corresponding to that of the neurons of the medial trapezoid nucleus, whereas in the spiral ganglion the gradient is from base to apex (high to low frequency), corresponding to that of the lateral superior olivary nucleus. But insofar as the nuclei of the superior olivary complex receive second-order fibers from the anteroventral and posteroventral cochlear nuclei, and we have not detected cytogenetic gradients in the latter, it is not likely that the two gradients in the superior olivary complex are related to those in the cochlea.

Another way to conceptualize the observed cytogenetic gradients is to view the two earliest generated nuclei, the medial superior olivary nucleus and the lateral trapezoid nucleus (or medial periolivary nucleus), as the axial com-

VESTIBULAR AND AUDITORY CYTOGENESIS 90 1

Fig. 22. A. Cytogenetic gradients ofhair cells and spiral ganglion cells of the mouse cochlea (based on Ruben, '67). B. An alternative interpretation of cytogenetic gradients in the superior olivary complex to that shown in Figure 19. Tonotopic representation in the medial trapezoid nucleus and the lateral superior olivary nucleus are in register, but the cytogenetic gradients are not. The latter, instead, seem to point to the distal source (contralateral vs. ipsilateral) of the afferents. Arrows do not imply a statement about the actual course of fibers.

ponents of the system to which are added the medially settling neurons of the medial trapezoid nucleus and the laterally settling neurons of the lateral superior olivary nucleus

(Fig. 22). In the medial superior olive of the cat (Guinan et al., '72) and the dog (Goldberg and Brown, '68) the tonotopic representation is from dorsal (low frequency) to ventral (high


frequency). According to Goldberg and Brown ('68), almost all the cells of the medial superior olive of the dog are binaural. This is in line with Stotler's (1953) original finding of an ipsilat- era1 and contralateral projection to the lateral and medial dendrites, respectively, of neurons of the medial superior olivary nucleus. Goldberg and Brown also found that virtually all the neurons of the medial trapezoid nucleus are excited by contralateral stimulation. This result has been confirmed and extended by the findings of Guinan et al. ('72) in the cat. They concluded that, with few exceptions, units in the region ventromedial to the medial superior olive (which includes the medial trapezoid nucleus) are excited by contralateral tones, and ventromedially situated units (which include the lateral superior olive) are excited by ipsilateral tones. Similar results were obtained by Tsuchitani ('77); all superior olivary units lo- cated lateral to the medial superior olive were excited by stimulation of the ipsilateral ear, and most of them were inhibited by stimulation of the contralateral ear. Essentially the same results were obtained with anatomical techniques by Strominger and Strominger ('71) in the rhesus monkey.

The germinal source of the neurons of the superior olivary complex remains to be determined, but it is likely that, as afferent relay cells, they are produced in the dorsal aspect of the medullary neuroepithelium and, like the neurons of the inferior olive and pontine gray (Altman and Bayer, '78b), they migrate some distance to their ventral position. The earliest generated neurons of the midline strip of the system (Fig. 22) could arrive first. If so, the medial superior olive neurons would be the first elements to receive cochlear nucleus input, and they settle a t a midpoint in relation to the ipsilateral and contralateral inputs. The first complement of the subsequently generated neurons of the lateral superior olive and medial trapezoid nucleus settle on either side of this midpoint ("stimulopetal center"?, Ariens Kap- pers, '17) in such a way that the later generated (and arriving?) elements are added on either side facing the sources of the ipsilateral (lateral superior olive) and contralateral (medial trapezoid nucleus) fiber sources, respectively. Embryological investigations in progress may shed light on this hypothesis.

The other component of the superior olivary nuclear complex to be considered is the medial trapezoid nucleus. Its neurons are the earliest forming cells of the auditory system of the medulla. This nucleus is probably identical to

the medial periolivary nucleus of the cat. Ac- cording to Goldberg and Brown ('681, units in the latter region differ from the other elements of the superior olivary complex in having low discharge rates, resembling the afferent fibers of the olivocochlear bundle. In terms of its position, the medial trapezoid nucleus of our description appears to be a part of what Brown and Howlett ('72) call in the rat the ventral and dorsal trapezoid nuclei. According to their thiocholine study, this region is the source of the cholinergic efferents of the olivocochlear bundle. Possibly, the early generation of the neurons of this region is related to the cir- cumstance that they are efferent elements, in contradistinction to other components of the system, which are afferent relay neurons. In summary, the neurons of the auditory nuclei of the upper medulla constitute a heterogeneous system, and additional research on embryological material will be neccessary to determine the sources (neuroepithelial zones) of i ts neurons.

ACKNOWLEDGMENTS

This research project is supported by grants from the Public Health Service and the Na- tional Science Foundation. Excellent technical assistance was provided by Ronald Bradford, Sharon Evander, Carol Landon, Kathy Shuster and Mary Ward.

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NOTE ADDED IN PROOF

An extension of this study to rostra1 components of the auditory system (J. Altman and S.A. Bayer, Time of origin of neurons of the rat inferior colliculus and the relations between cytogenesis and tonotopic order in the auditory pathway, Exp. Brain Res. [in press]) has re- vealed that tonotopic order (high-to-low frequency) and cytogenetic gradient (early-to-late cell production) are in register in the following structures: lateral superior olivary nucleus, inferior colliculus, medial geniculate body and auditory cortex. Tonotopic order and cytogenetic gradient are aligned, but in reverse order (as defined), in the medial trapezoid nucleus and the dorsal nucleus of the lateral lemniscus. The latter two structures receive contralateral input from the cochlear nuclei.

Development of the brain stem in the rat. III. Thymidine ...Bayer JCN 194 3.pdf · THE JOURNAL OF...

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