THE JOURNAL OF COMPARATIVE NEUROLOGY 2975383-297 ( 1990)

Vestibulo-Ocular Projections in the 11-Day Chicken Embryo: Pathway Specificity GUDRUN PBTURSDOTTIR Institute of Physiology, University of Oslo, Oslo, Norway

ABSTRACT The organization of vestibulo-ocular projections and their spatial relationship to vestibulospinal projections were ascertained in the 11-day chicken embryo through retrograde tracing experiments. An in vitro preparation of the brainstem facilitated precisely localized application of tracers. Unilateral labelling of the ascending medial longitudinal fascicle (MLF), just caudal to the trochlear nucleus, labelled coherent groups of vestibular neurons that had an asymmetrical distribution on the two sides of the brainstem. Axons originating from either side followed one of four trajectories, and the groups of neurons could therefore be identified on the basis of their position and their projection pathway. Labelling each side of the MLF with a different tracer showed that although neurons projecting in the ipsi- and contralateral MLF were intermingled in some areas, individual neurons projected on one side only. Labelling the ascending MLF and the high cervical spinal cord with different tracers showed that vestibulo-ocular neurons had a wider rostro-caudal distribution than vestibulospinal neurons. Some vestibulospinal and vestibulo-ocular groups were spatially segregated, others were intermingled. Very few (in most preparations no) neurons were found to project to both targets. Together with our previous study on vestibulospinal projections in the 11-day chicken embryo (Glover and PBtursdbttir, J. Comp. Neurol. 270:25-38, '88) the results show that vestibular neurons in different regions project in characteristic subsets of the available pathways. Key words: avian brainstem, MLF, retrograde tracing

Connectivity in the nervous system is in many cases characterized by a spatiotopic organization of projections. One of the classic examples is the projection from coherent motor pools in the spinal cord to specific muscles (Romanes, '64; Landmesser, '78). Experimental manipulations indicate that such projection patterns are established by directed axonal outgrowth (Lance-Jones and Landmesser, '81a,b; Ferguson, '83). We have been studying the projection pattern of a particular class of premotor neurons: the vestibular nuclei. An in vitro preparation of the brainstem and spinal cord of the chicken embryo has enabled us to make well-localized applications of neuronal tracers and label specific vestibular projections completely. In a previous study using this preparation, we showed that three distinct, coherent groups of vestibular neurons project to the spinal cord along three separate pathways (Glover and PBtursdbttir, '88). The vestibular nuclei also project to other major targets, notably the oculomotor centers. These projections constitute a part of the vestibule-ocular reflexes which serve to stabilize the visual image on the retina during head movements. Physiological and anatomical studies have revealed o 1990 WILEY-LISS, INC.

much of the neuronal connectivity underlying vestibuloocular reflexes. An important component of this connectivity is the defined relationship between the semicircular canals and the various eye muscles (see, e.g., Precht, '79; Graf and Baker, '85, and references therein). The different eye motor nuclei receive semicircular canal information via characteristic regions of the vestibular nuclei (Gacek, '71; Mitsacos et al., '83) and single neurons in the vestibular nuclei have been shown to innervate the motoneurons of both muscles in a yoked pair producing vertical eye movements (Uchino et al., '82, '83;Graf et al., '83;Graf and Baker, '85; Graf and Ezure, '86). The vestibular neurons have been shown to mediate excitation to the extra-ocular motoneurons mainly via contralateral pathways while inhibition is mediated ipsilaterally (It0 et al., '73a,b, '76a,b). Eye movements have been most extensively studied in animals with frontally positioned eyes, and comparatively little research has been carried out on the neuronal circuitry Accepted February 7,1990. Gudrun PQtursd6ttir'spresent address is Department of Anatomy, University of Iceland, Vatnsmyrarvegur 16, Reykjavik 101, Iceland.

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of eye movements in birds. Mapping the vestibulo-ocular projections in the chicken embryo should provide insight into both developmental and comparative aspects of the vestibulo-ocular system. Our main aim by studying the development of the vestibulo-ocular and vestibulospinal projections, however, is to be able to elucidate the mechanisms whereby neurons establish specific projections to different targets. The chicken embryo is well suited for this purpose both because it allows experimental manipulations during embryonic development and because an in vitro preparation of the embryonic brainstem and spinal cord (Glover and Petursdbttir, '88) allows neuronal projections to be traced with the necessary precision. In this study, I have used retrograde tracers to map the vestibulo-ocular cell bodies and their axonal pathways. By using two different tracers, I have been able to compare the distribution of ipsiversus contralaterally projecting vestibulo-ocular neurons as well as vestibulo-ocular versus vestibulospinal projections. Together with our previous study the results illustrate the characteristic spatial organization of vestibular neurons projecting through different pathways.

descending projections from the brainstem of the 11-day chicken embryo (Glover and Pktursdbttir, '88), we ensured that tracer transport was restricted to a particular side or tract by transecting the tracts we did not wish to label and letting them seal up before the tracer was applied. This proved to be unnecessary in the case of the vestibulo-ocular projections. At the injection site the MLF has a medial position, but is well enough separated on the two sides that i t can be labelled completely on one side without contaminating the other side (Fig. 1).This was verified by inspecting horizontal sections, where individual labelled axons can easily be spotted. The three types of tracers rendered similar results. The same neuron groups and similar numbers of neurons were labelled regardless of which dye was used. Axons and cell bodies were heavily labelled, and the axon bundles could be traced easily in serial sections from the injection site to each group. Within single sections containing axon bundles and cell groups, individual axons could generally be followed to their cell bodies; this required careful focusing through the thick sections. Dendritic morphology was also revealed by all of the dyes.

MATERIALS AND METHODS

Two tracer experiments

The procedures for producing in vitro brainstem-spinal cord preparations and for retrograde tracing with horseradish peroxidase (HRP, Sigma type VI) and fluorescein- or rhodamine-conjugated dextran amines (FDA and RDA, respectively, Molecular Probes, or prepared as per Gimlich and Braun, '85) are described in detail in Glover e t al. ('86) and Glover and Pktursdottir ('88). Briefly, white leghorn chicken eggs were incubated for 11 days, a t which time the embryos were anesthetized by cooling, decapitated, and dissected in physiological saline (137 mM NaC1, 5 mM KC1, 2 mM CaCl,, 1 mM MgCl,, 1 mM Na-phosphate, 5 mM Hepes, 11 mM glucose, pH 7.4). Retrograde tracers were applied to the isolated brainstem and upper cervical spinal cord. FDA and RDA were pressure-injected as 25% solutions through a glass micropipette, while H R P was recrystallized onto the tip of minuten pins. The preparations were incubated in vitro overnight at 24-28OC, fixed in 10% paraformaldehyde in phosphate buffer (FDA and RDA) or 2.5% glutaraldehyde in physiological saline (HRP),and cut into 70 Fm (FDA and RDA) or 100 pm (HRP) sections in the horizontal (n = 14), transverse (n = 21), or sagittal plane (n = 5 ) . The HRP sections were processed with metal intensified diaminobenzidine (DAB) (as per Adams, '81) to visualize the tracer. Thionine was used to counterstain H R P sections, and allowed for the identification of several landmark nuclei. The nuclear dye Hoechst 33258 was used to counterstain a few fluorescence sections, but was only marginally useful in identifying landmarks.

The relative positions of vestibulo-ocular neurons that projected in the ipsi- versus the contralateral MLF, and the possibility of single neurons extending axons on both sides of the brainstem, were examined by labelling the MLF with FDA on one side and RDA on the other. We had previously shown that these dyes can be used to double label neurons with multiple processes (Glover et al., '86). Each side was injected in the manner described above, and contamination between the twc injection sites was avoided by carefully and immediately removing any spillage of the tracers with a tissue paper wick, waiting an hour and covering the first injection site with vaseline before injecting the other site. Contamination between the two injection sites invariably resulted in labelling of one or both sides of the MLF with both dyes. The same precautions applied when vestibulo-ocular and vestibulospinal projections were labelled with different fluorescent dyes. The vestibulospinal projections were labelled by injecting the dye unilaterally a t the transition from medulla to spinal cord. For a detailed description of the procedure see Glover and Pbtursdbttir ('88). The two dyes behave similarly with regard to uptake, transport time (Glover et al., '86), and labelling results, and were therefore used interchangeably to label either the vestibulo-ocular or the vestibulospinal projections. The VIth (abducens) cranial nerve and the ascending MLF were labelled with different tracers to determine whether neurons projecting to the oculomotor centers from the abducens nucleus included motoneurons. The nerve was labelled by the following procedure: The dura was left. intact, covering the ventral brainstem and the nerves. The bath was partially drained. A piece of thin suture (9-0 Dermalon) was fastened to the distal end of the dura covering the nerve and used to pull the nerve through a small sleeve made of fine plastic tubing. The proximal end of the sleeve was coated very thinly with histoacryl and provided a watertight seal when pressed lightly against the dura covering the brainstem. The sleeve was filled with 25% RDA and the nerve cut inside the sleeve to increase tracer uptake. The preparation was partially submerged in oxygenated saline for an hour, then the RDA was removed from the

Labelling vestibulo-ocular neurons In order to label vestibular neurons projecting to the trochlear nucleus as well as the oculomotor nuclei, the retrograde tracers were injected into the medial longitudinal fascicle (MLF) caudal to the trochlear nucleus, just above the rostra1 wall of the IVth ventricle (Fig. 1). In a few preparations the injection was placed into the MLF adjacent to the trochlear nucleus; this did not affect the pattern of labelled cells. Multiple, small injections were made across the MLF, and fibers that remained intact were subsequently cut to increase uptake of the tracer. In most cases the aim was to label the MLF unilaterally. In previous studies on

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-Fig. 1. Labelled axons, terminals, and cell bodies resulting from a typical injection are shown a t 3 different levels, arranged in rostrocaudal sequence. a: Anterograde labelling of terminals in the oculomotor nuclei. Subdivisions of the oculomotor nuclear complex are made visible by the thionine counterstain and are abbreviated as follows: dorso-medial (drn), dorso-lateral (dl), ventro-medial (vm), and EdingerWestphal (ew). The fibers crossing into the contralateral oculomotor nucleus (see text) are not shown. b The injection site just caudal to the

trochlear nucleus, where HRP was injected into the MLF on the left side. c: Retrogradely labelled comrnissural axons and their cell bodies in the contralateral vestibular region. These neurons include interneurons in the abducens nucleus and the most medial vestibulo-ocular neurons. The initial trajectories of the axons can be traced from the cell bodies to the MLF (*). Sections are transverse and 100 pm thick. Scale bar, 200 pm (a,b),100 pm (c).

sleeve and the bath washed several times. Subsequently the ascending MLF was labelled bilaterally with FDA, and the preparation incubated overnight and processed as usual.

the labelled cells were counted. Although double counts cannot be excluded, the proportion of cells included in two adjacent sections should be small since the cell body diameters are small relative to the thickness of the sections (70 w).

Cell counts A few of the preparations that were labelled with two tracers contained doubly labelled cell bodies, despite no evidence of contamination. In order to get an idea of the proportion of doubly labelled cells in these preparations, all

Drawings Camera lucida drawings were made of preparations labelled with HRP, while an x - Y plotter (Hewlett Packard)

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List of preparations

TABLE 1. Correspondenceof Groups Defined by PositionPathway and by Cytoarchitectonics Groups defined by-

Positiodpathway

Cytoarchitedonics

The subsequent description is based on the results from cDC (caudal part) descending descending,medial, tangential 40 out of 45 brainstems prepared. Five were discarded cDC (rcatral part) descending,tangential cVC (caudal part) because of poor histological quality, the others are divided cvc (rostral part) descending,medial descending,medial as follows: iVC dorsal Deiter's, superior Seventeen preparations were labelled with H R P to reveal iVR superior cDR, IDR the organization of vestibulo-ocular projections (10 with successful unilateral labelling of the MLF and 7 with slight contamination of the contralateral side). Six were sectioned horizontally, 9 were sectioned transversely, and 2 were duced silver stains which best reveal vestibular nuclear sectioned sagittally to provide views of the labelled groups boundaries in the chicken embryo (Peusner and Morest, '77) are poorly or not a t all compatible with the tracers I used. in all 3 dimensions. Twenty-three preparations were labelled with the fluores- The comparison should be reasonably accurate, however, as cent dyes: a) The two sides of the ascending MLF were I had direct access to the Bodian silver and iron-hematoxylabelled with different dyes (n = 3). b) Vestibulo-ocular lin stained transverse and sagittal series established by versus vestibulospinal projections were labelled with dif- Harkmark ('54), and I also referred to previously published ferent dyes (n = 16; 4 with bilateral labelling of the tracts, cytoarchitectonic studies (Wold, '76, '78a; Peusner and 11 with unilateral labelling of the tracts on the same side [8 Morest, '77). successfully and 3 with some contamination of the contralatDistribution of labelled neurons era1 MLF], and 1 with unilateral labelling of the tracts on opposite sides); c) The abducens nerve and the ascending Most of the neurons that projected in the ascending MLF MLF were labelled with different dyes (n = 2). d) Vestibulo- lay a t or dorsal to the level of the eighth nerve, that is, within ocular projections were labelled unilaterally (n = 2). the region of the vestibular nuclei in the adult chicken (Wold, '76). I refer to these neurons as "vestibulo-ocular," even though some of them may project to regions rostral to RESULTS the eye motor nuclei (Brodal and Pompeiano, '58; Carpenter Projections ascending from the vestibular nuclei in the and Hanna, '62; Schall and Delius, '86; Wild, '88). The 11-day embryo were labelled retrogradely by applying trac- labelled neurons extended from just caudal to the eighth ers to the MLF just caudal to the trochlear nucleus. The in nerve to the rostral border of the cerebellar peduncles (Fig. vitro brainstem preparation made possible a massive unilat- 2). No labelled cell bodies were observed further caudally in eral labelling of the MLF a t this point, with little or no the brainstem, even though axons in the MLF were contamination of the contralateral MLF. Except where labelled at least to the first cervical spinal segment in every mentioned, all injections referred to below were made a t this preparation. I t is therefore unlikely that any neurons caudal location. The three types of tracers used rendered compara- to the eighth nerve have axons ascending to the level of the ble results, and a very consistent picture was obtained in the trochlear nucleus in the 11-day embryo. The labelled de40 experiments on which the subsequent description is scending axons are most probably derived from the interstibased. tial nucleus of Cajal, which is located in the vicinity of the It was clear from comparison with cell stained material oculomotor nucleus and projects in the MLF to the spinal and with previous studies that the cell groupings revealed by cord (Okado and Oppenheim, '85; Glover and PCtursdottir, retrograde tracing did not correspond strictly to cytoarchi- in preparation). tectonic groupings (see Discussion). As the axonal trajectoVestibulo-ocular projections in mammals originate primaries could be traced to the cell groups from the injection site, rily from rostral parts of the vestibular nuclei (Carpenter the groups could nevertheless be identified uniquely on the and McMasters, '63; Carpenter and Strominger, '65; Gacek, basis of their position and projection pathway. T o keep the '71), whereas in the chicken embryo they were found to description simple, I thus found it convenient to apply a originate from caudal levels as well, A notable difference separate nomenclature which is based on these two criteria. between the distribution of vestibulo-ocular neurons in The neuron groups are named with respect to their location embryonic and adult chicken is that in the embryo vestibulowithin the vestibular complex in the rostro-caudal (R or C) ocular neurons extended further caudally than vestibulospiaxis, and their relative dorso-ventral (D or V) positions. In nal neurons, whereas the opposite is the case in the adult addition I refer to neurons with axons that ascend ipsilateral chicken (Wold, '78a; see below and Discussion). to the cell body as ipsilateral (i), while those with axons that Some neurons retrogradely labelled from the injection site cross the midline are called contralateral (c). For example, did not lie within the vestibular nuclei. These included the most dorsal group situated caudally within the vestibu- neurons near the abducens nucleus, which are shown below lar complex, and with axons that cross the midline, is called to be abducens interneurons. These neurons play an importhe cDC group. tant role in vestibulo-ocular reflexes (see Discussion). In In the Discussion I will relate the groups defined by this addition, however, there were labelled non-vestibular neunomenclature to groups defined on the basis of cytoarchitec- rons whose function may not be related to the vestibulotonic characteristics. T o assist those readers familiar with ocular reflex. In the rostral brainstem, just caudal to the the vestibular nuclei as defined cytoarchitectonically, how- injection site, a bilateral, sickle-shaped group of neurons ever, I include Table l, which shows the cytoarchitectonic extended from the floor of the fourth ventricle downwards equivalents of the groups defined by pathway and position. between the trigeminal motor and principle sensory nucleus These equivalents are necessarily tentative, since the re- to the ventro-lateral edge of t,he brainstem (Fig. 2a). They

VESTIBULO-OCULAR PROJECTIONS I N CHICKEN EMBRYO may correspond to a group of tegmental neurons that have been retrogradely labelled from the oculomotor complex in the cat (Graybiel and Hartwieg, '74). Other labelled neurons scattered ventral to the injection site appeared to be reticular neurons (not shown). Since vestibular neurons have not been described in these locations and these neurons do not correspond to any of the groups that Wold ('76) mentions in his description of the vestibular nuclei in the adult chicken, I will not include them in the account of the vestibulo-ocular projections.

The vestibulo-ocular projections Injection of tracer on one side of the MLF labelled axons in the MLF strictly ipsilaterally but labelled vestibuloocular neurons on both sides of the brainstem (Fig. 2). In general, neurons whose axons crossed the midline and projected contralaterally predominated in caudal regions while ipsilaterally projecting neurons predominated in rostral regions. It should be remembered that the classification of a projection as ipsi- or contralateral refers to the laterality of the cell bodies with respect to the axon. It does not refer to the ultimate target of the neurons within the oculomotor nuclei, where crossing axons or collaterals have been reported (McCrea et al., '87b, and see below). As is shown in Figure 2, ipsi- and contralaterally projecting neurons were spatially segregated in some regions but intermingled in others. Such intermingling raised the possibility that individual neurons might project on both sides, even though no sign of such collaterals was seen following retrograde labelling. To test this idea directly the two sides of the MLF were labelled with different fluorescent dyes (Fig. 3). In two of three such preparations, I found no doubly labelled cells, and in the third less than 1% of all the labelled cells contained both dyes ( 5 out of ca. 900). These five cells were found on the same side of the brainstem, which suggests that they were doubly labelled through contamination a t the injection site. Thus I conclude that individual vestibulo-ocular neurons project their axons on one side of the brainstem only.

Contralateral projections predominate in caudal regions Caudal to the rostral border of nucleus laminaris, three groups of vestibulo-ocular neurons were labelled. Two of these were contralateral and one ipsilateral. The two contralateral caudal groups were very similar, differing only in their dorso-ventral positions and their caudal extents. The dorsal group appeared just caudal to nucleus laminaris. From here it extended rostrally, separated from the ventral border of nucleus laminaris by a relatively narrow sheath of neurons (Fig. 2c-e). Its mediolateral distribution varied along the rostro-caudal axis, becoming progressively more medial further rostrally. The density and shape of the neurons in this group varied along the medio-lateral axis (Fig. 4). The most lateral neurons were often oval in shape and had tufted dendrites aligned with the incoming fibers of the eighth nerve. More medially the neurons were stellate and loosely aggregated. I refer to this group as the contralateral dorso-caudal group (cDC). The other contralateral group lay ventral to the cDC group, separated from it by a zone that was free of labelled cells. I refer to this group as the contralateral ventro-caudal group (cVC) (Fig. 2d). It had a similar cytoarchitecture and roughly the same mediolateral extent as the cDC group, but did not extend as far caudally (Fig. 2c,d). The two groups

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fused medially near the rostral pole of nucleus laminaris, and then terminated at the rostral end of the abducens nucleus. The axons of the cDC and cVC neurons projected directly towards the midline, crossed it in a tight dorsal bundle, and ascended in the MLF. Thus, the cDC and cVC groups are distinguished primarily by position but are similar in cytoarchitecture and axonal trajectories. The most medial part of the fused cDC and cVC groups intermingled with labelled neurons in the region of the abducens nucleus. These were identified as interneurons of the abducens nucleus by labelling the abducens nerve and the MLF with different tracers. Two distinct ipsilateral groups of motoneurons were labelled via the abducens nerve: the abducens nucleus proper and the accessory abducens nucleus at the ventrolateral border of the brainstem. This is precisely the labelling obtained in the young chicken by Labandeira-Garcia et al. ('87). The neurons labelled from the MLF encircled and intermingled with the motoneurons in the rostral part of the abducens nucleus proper. The neurons were small and situated mainly near the dorso-lateral edge of the nucleus, although some were situated ventral to the nucleus (Fig. 2b). The position and morphology of these contralateral neurons match the description of abducens interneurons by Labandeira-Garcia et al. ('87), who labelled them by applying HRP to the oculomotor nucleus in the young chicken. Abducens interneurons projecting contralaterally to the oculomotor centers have also been described in mammals (Graybiel and Hartwieg, '74; Spencer and Sterling, '77; Steiger and Buttner-Ennever, '78; Buttner-Ennever and Akert, '81; Evinger and Baker, '83; McCrea et al., '861, and shown to relay excitation to the contralateral medial rectus motoneurons in cat (Baker and Highstein, '75; Highstein and Baker, '78; Nakao and Sasaki, '80).

Although predominated by these contralateral groups, the caudal vestibulo-ocular region contained a distinct ipsilateral element. Labelling the two sides of the MLF with different tracers revealed that this ipsilateral group had roughly the same rostro-caudal dimensions as and was to a large extent intermingled with the cVC group (Fig. 3b). I refer to it as the ipsilateral component of the ventro-caudal group (iVC). Caudally, it consisted of stellate cells that formed a round lateral cluster (Figs. 2d, 4).Further rostrally it extended in a loose aggregation towards the midline (Fig. 2c). Thus, although the ipsilateral and contralateral components of the VC group are intermingled, they can be distinguished both by their morphology and the laterality of their projections. The iVC neurons also extended into the "cell-free'' zone between the cVC and the cDC groups, but were not found in the area occupied by the cDC group (Fig. 3b). However, a few ipsilateral neurons were located dorsal to the cDC group, close to the ventral border of nucleus laminaris. The axons of these neurons coursed ventrally to join the main bundle of iVC axons before projecting to the MLF (Figs. 2d, 4).

Ipsilateral projections predominate in the rostral regions Rostra1 to nucleus laminaris, the vestibulo-ocular projections were bilateral, but predominantly ipsilateral. The main group of neurons was located dorsally and dorsolaterally, extending into the cerebellar peduncle. I refer to this group as the dorso-rostra1 (DR) vestibulo-ocular group (see iDR and cDR in Fig. 2a-c). It extended from about the

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rostral pole of nucleus laminaris to the rostral border of the cerebellar peduncle. The DR neurons were stellate or spindle shaped and loosely aggregated. In addition, a smaller, well-demarcated, dense cluster of stellate neurons was found ventral to the DR group on the ipsilateral side. This is the ventro-rostra1 (VR), vestibulo-oculomotor group. It appeared ventro-lateral to the rostral pole of nucleus laminaris, moving dorso-medially and increasing in circumference further rostrally, where it eventually merged with the iDR group (Fig. 2a-c).

The ascending tracts and terminals Labelled axons could be traced in bundles from the injection site to the various vestibulo-ocular groups. They followed two types of trajectories (Fig. 5). Most of the axons joined the MLF by an initial trajectory that was either directly mediad or slightly rostro-mediad from the cell body. Some of the axons from the most rostral and dorsal groups, however, coursed in a separate bundle along the dorsolateral wall of the fourth ventricle and joined the MLF just caudal to the trochlear nucleus. This trajectory is similar to that of vestibulo-ocular projections from the dorsal superior nucleus in mammals, which are described as running in the brachium conjunctivum (Yamamoto et al., ‘78; Highstein and Reisine, ’79; Lang et al., ’79). Axons were also labelled anterogradely in the MLF rostral to the injection site and could be followed to the oculomotor nucleus. Profuse terminals branched off into the ipsilateral trochlear and oculomotor nuclei (Fig. la). A few branches could be traced across the midline to the contralateral oculomotor nucleus as well (not shown). In addition, axons labelled further rostrally than the oculomotor nucleus appeared to be retrogradely labelled to neurons in the interstitial nucleus of Cajal (not shown). Fig. 3. Ipsi- and contralateral vestibulo-ocular neurons are spatially segregated in some regions and intermingled in others. The positions of ipsilateral neurons (open circles) versus contralateral neurons (filled circles) are plotted in two 70 pM transverse sections from a preparation in which the two sides of the ascending MLF were labelled with different fluorescent dyes. Each symbol represents one labeNed neuron. The level of section A corresponds to that of section B in Figure 2; the level of section B corresponds to that of section D in Figure 2. Abbreviations as in Figure 2. Scale bar, 200 pm.

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Fig. 2. An overview of the vestibulo-ocular groups and their initial axon trajectories as labelled after injection of HRP into the MLF on the right side. Camera lucida drawings were made of individual 100 pM transverse sections, that were all from the same preparation. Because only individual sections are shown, the full trajectory from the cell bodies to the MLF is not apparent for some groups. The level of each section and the injection site are indicated on the profile of the horizontal brainstem at lower right. The arrows in A indicate the “sickle-ahaped group,” which is presumed not be vestibular because it lies well outside the vestibular region (see text). Scale bar, 600 pm. Abbreviations used cDC, group contralateral dorso-caudal group; cDR, group contralateral dorso-rostral group; iDR, group ipsilateral dorsorostral group; cVC, group contralateral ventro-caudal group; iVC, group ipsilateral ventro-caudal group; iVR, group ipsilateral ventro-rostra1 group; MLF, medial longitudinal fascicle; n. VI, nucleus abducens; n. VI int., nucleus abducens interneurons; n. VII, nucleus facialis; n. lam, nucleus laminaris; n. mc, nucleus magnocellularis; g. V, trigeminal ganglion.

Vestibulospinal versus vestibule-ocular projections The distribution of vestibulo-ocular neurons was compared with that of vestibulospinal neurons (previously described in the 11-day chicken embryo, Glover and PBtursdbttir, ’88), by labelling the spinal cord and the ascending MLF with different tracers in the same preparation. The subsequent description is based on the results from 16 such preparations, of which 5 were sectioned horizontally, 8 transversely, and 3 sagittally. Vestibulo-ocular neurons had a greater rostro-caudal distribution than vestibulospinal neurons (Fig. 6). Unilateral injections into each target on the same side revealed a difference in the degree of overlap between vestibulo-ocular and vestibulospinal neurons ipsi- and contralaterally. Ipsilaterally, they were spatially segregated to a large extent (Fig. 6a). The iVC group lay ventral, medial, and caudal to the vestibulospinal neurons, while the iVR group lay dorsal to the vestibulospinal neurons (Fig. 6b). Contralaterally, vestibulo-ocular and vestibulospinal neurons were intermingled (Fig. 6a,b). The well-defined contralateral vestibulospinal group (Glover and Phtursdbttir, ’88) occupied the same area as the cVC group. As previously mentioned, ipsilateral vestibulo-ocular neurons are also found in this location (the iVC group, see Fig. 3b). No doubly labelled neurons were observed in 14 of the 16 preparations. In the remaining two, fewer than 1% of the labelled cells were doubly labelled (19 of about 2,000 neurons). I conclude that in the 11-day chicken embryo very

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Fig. 4. Dendritic architecture varies within certain vestibulo-ocular groups. This is a camera lucida drawing of a 100 pm transverse section showing the caudal vestibulo-ocular groups, located a t the caudal border of the auditory/vestibular nerve (between sections D and E in Fig. 2),

retrogradely labelled after injection of HRP into the right MLF. The insets provide more detailed drawings of the dendritic structure of selected neurons in different regions. Scale bar, 200 pm for the whole section and 100 pm for the insets.

few, if any, vestibulo-ocular neurons have collaterals that reach the spinal cord.

tibulo-ocular axons follow two types of trajectories, either joining the MLF directly (rostro)mediad, or running rostrally along the edge of the fourth ventricle to join the MLF close to the injection site. Ipsilaterally and contralaterally projecting neurons are intermingled in some groups, but individual neurons project strictly unilaterally. Vestibulo-ocular neurons have a wider rostro-caudal distribution than vestibulospinal neurons. Ipsilaterally projecting vestibulospinal and vestibulo-ocular neurons are spatially segregated, while contralaterally projecting vestibulospinal and vestibulo-ocular neurons are intermingled. Very few (in most preparations no) neurons project to both targets.

DISCUSSION This study describes the spatial organization of vestibuloocular projections in the 11-day chicken embryo. It shows that the projections originate from coherent groups of neurons that can be distinguished on the basis of position and projection pathway. Projections from the caudal vestibular region are predominantly crossed, while those from the rostra1 vestibular region are predominantly ipsilateral. Ves-

VESTIBULO-OCULAR PROJECTIONS IN CHICKEN EMBRYO

Technical limitations Common to studies with retrograde tracers is the problem of defining the area where axons have taken up the tracer. In this respect the in vitro preparation has the advantage of allowing a controlled and precisely located application of tracers. Axonal uptake of the tracers used is greatly dependent on axonal damage (Mesulam, '82, and references therein; Glover et al., '86). By injecting moderate amounts of tracers into the MLF on one side and subsequently cutting the axons to maximize the uptake, I consistently managed to label heavily that side of the MLF without contaminating the other side. It was difficult to avoid axonal damage just ventral to the MLF, however, and in most preparations labelled neurons were found in the ventral brainstem close to the injection site. These neurons are presumably not vestibular (cf. Wold, '76). Another question common to studies of this kind is whether all axons projecting to the injection site have been labelled. The cross-sectional area of the MLF was well delineated in counterstained material. Comparison of the labelled and unlabelled sides suggested complete labelling. Moreover, the results were very reproducible. Similar numbers of neurons were labelled in all the preparations and the pattern of labelled neurons was the same with the three different tracers used. In this study it was not possible to define the labelled neurons on the basis of their target, which in addition to the trochlear and oculomotor nuclei may include more rostral areas (Schall and Delius, '86; Wild, '88) and other parts of the brainstem reached by axon collaterals. Such collaterals, especially to eye movement related areas, have been observed in other species (Graf et al., '83; Graf and Ezure, '86; MacCrea et al., '87a,b). The results do show that the axons ascend either ipsilaterally or contralaterally as far as the injection site. Since the trochlear and oculomotor nuclei were dense with labelled terminals only on the side of the injection, this suggests that most axons terminate on eye motor neurons on the same side as they project in the MLF. Crossed terminal branches were found only in the oculomotor nuclei. The situation is similar in other species (Graf and Ezure, '86; McCrea et al., '87b). It should be possible in the in vitro preparation to label selectively the crossed terminal branches to the oculomotor nuclei and identify the vestibuloocular groups responsible for that projection (Jansen et al., work in progress). Finally, one may ask how well the projection pattern at embryonic day 11 represents the adult pattern. All the vestibulo-ocular projections reported in the adult chicken were present in my material. The main discrepancy between the embryonic and adult patterns is the caudal extension of vestibulospinal neurons reported in the adult but not found in the embryo (see below). Since in embryos more complete labelling is obtained in vitro than in vivo, a quantitative comparison between embryonic (in vitro) and adult (in vivo) material is probably not warranted.

Comparison with cytoarchitectonic studies Before I compare my results with other studies where the vestibulo-ocular projections have been labelled specifically, I will consider the location of vestibulo-ocular groups labelled in this study in the context of the normal anatomy of vestibular nuclei in chicken as reported by Wold ('76), (for the relevant description in pigeon see Karten and Hodos, '67).

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The caudalmost neurons, judging from their locations both ipsi- and contralaterally, lie within the descending nucleus. They lie too far laterally to be a part of the medial nucleus, which is the only other vestibular nucleus at this caudal level. Further rostrally, the caudal groups span the medio-lateral width of the brainstem, and probably consist a t that level of neurons from the tangential, descending, and medial nuclei. The most lateral neurons are oval in shape with dendrites that are aligned with the incoming fibers of the eighth nerve. These are morphologically similar to the principal neurons of the tangential nucleus as described by Peusner and Morest ('77). By contrast, none of the ipsilaterally projecting caudal neurons had this morphology. The VR group, although very densely packed, cannot be recognized as a separate entity in thionine-stained material, being surrounded by neurons of a similar size and packing density. From its position, the caudal part may belong to the dorsal Deiter's nucleus, while rostrally it lies within the superior vestibular nucleus. Finally, the dorsalmost rostral vestibulo-ocular group (DR group), which was found bilaterally and contains a loose aggregation of vestibulo-ocular neurons, is situated within and perhaps extends rostral to the superior vestibular nucleus. That part of it which lies close to the fourth ventricle rostrally and projects only ipsilaterally may correspond to cell group A in Wold's ('76) description: a densely packed cluster of neurons that is found between the rostral superior nucleus and the wall of the fourth ventricle. Our studies of the vestibulospinal projections (Glover and PCtursdbttir, '88) showed that groups defined by cytoarchitecture do not coincide with groups defined by projection pathway. This is also true in the present study. Vestibuloocular neurons projecting in the same pathway have highly different shapes, sizes, and clustering densities, and are spread among different cytoarchitectonically defined nuclei. For example, the cVC neurons are found in the tangential, descending, and ventral part of the medial vestibular nuclei (Wold's nomenclature). The converse is also true. Thus, the principal cells of the tangential nucleus, the morphologically most distinct type of neuron in a particularly well-defined cytoarchitectonic group (Peusner and Morest, '77), project through multiple pathways: partly through the contralateral ascending MLF and partly through two vestibulospinal pathways (Glover and PCtursdbttir, '88). The functional significance of cytoarchitectonic clustering is therefore far from obvious, at least with respect to efferent projections. That the vestibulospinal groups projecting in different pathways are spatially segregated suggested that different functions might be spatially segregated (Glover and PBtursdbttir, '88). However, inclusion of the vestibulo-ocular system complicates the picture. For example, the region occupied by the cVC group contains not only vestibulo-ocular neurons projecting through the contralateral ascending MLF, but also neurons projecting by at least two other pathways: ipsilaterally through the ascending MLF (iVC group) and contralaterally through the descending MLF (the contralateral vestibulospinal group; Glover and P6tursdbttir, '88). Moreover, the degree of functional overlap may be appreciably greater than suggested by the localization of the cell bodies, since the dendritic branches may extend over considerable distances. Despite this complexity, there seem to be some examples of functionally specific regions. The VR group of the present study consists of a sharply circumscribed, very dense cluster of neurons. At its caudal end even the dendritic arbors seem largely confined within

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VESTIBULO-OCULAR PROJECTIONS IN CHICKEN EMBRYO the cluster. Only at its dorsal and rostral end does the structure of the group loosen as it merges with the more scattered neurons of the DR group. The efferent pathway of the VR group is exclusively along the ipsilateral ascending MLF and its dense packing and the morphological uniformity of the neurons suggest a uniform function.

Comparison with studies on vestibulo-ocular projections in avians Neuronal tracers have been used to label avian vestibuloocular projections in a few studies (Wold, '78a,b; Correia et al., '83;Evinger and Erichsen, '86; Labandeira-Garcia et al., '89). These studies report labelling consistent with my results in most respects. Wold ('78a) applied HRP to the oculomotor region of the adult chicken in vivo. Unfortunately, the uptake of HRP could not be restricted to one side of the brainstem, which precludes any conclusions about the laterality of the projections, and limits a comparison with my results. He reported that the bulk of vestibulooculomotor projections arises from the superior, tangential, and the rostral part of the medial nucleus. In addition, he found smaller projections from cell group A and the rostrolateral part of the descending nucleus. Evinger and Erichsen ('86) labelled the vestibulotrochlear projection in adult pigeon trans-synaptically by injecting fragment C of tetanus toxin into the superior oblique muscle of the eye, and compared the results with the labelling obtained by a unilateral injection of HRP into the trochlear nucleus. They found labelling ipsilaterally in rostral regions (corresponding to the iDR and iVR groups), and bilaterally in caudal regions, with a distribution very similar to that of the iVC, cVC, and cDC groups of the 11-day chicken embryo. Labandeira-Garcia et al. ('89) injected HRP unilaterally into the oculomotor nucleus of the 8-week-old chick in vivo, and report a labelling pattern which is essentially similar to that reported here. Neurons were labelled bilaterally in the superior nucleus (with a compact ipsilateral cluster reminiscent of the iVR group), in a contralateral band covering the tangential, descending, and medial vestibular nuclei (thus similar to the cDC and cVC groups), in an ipsilateral cluster at about the same level but more restricted medio-laterally (thus similar to the iVC group), and in and around the contralateral abducens nucleus (abducens interneurons, Labandeira-Garcia et al., '87). The labelling obtained in these studies is consistent with my results in most respects; one discrepancy lies in the relative caudal extents of vestibulo-ocular and vestibulospinal neurons. Wold ('78a,b) indicates that vestibulo-ocular neurons are not found caudal to vestibulospinal neurons. He describes the vestibulo-ocular projections as arising from the rostral parts of the descending and medial nuclei and the vestibulospinal as arising from the caudal parts (Wold,

___ Fig. 5. A schematic overview of the vestibulo-ocular groups and their axon trajectories (a) and photographs of some of the trajectories (h,c) are shown in the horizontal plane. FDA was injected into the right MLF. In b the axonal trajectories of the iVC group (on the right) and the abducens interneurons (on the left) join the MLF. c: shows iDR neurons with axons ascending (between arrows) along the wall of the fourth ventricle (dotted line). Section thickness, 70 p M . Scale bars, 100 pm. The photographs have the same orientation as the overview. g.VIII, auditory/vestibular ganglion; inj., site of injection; n.111, oculomotor nucleus; n.IV, trochlear nucleus; VI, abducens nucleus interneurons; All other abbreviations as defined in the legend of Figure 2.

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'78a,b). In contrast, my two-tracer experiments show that a considerable fraction of the vestibulo-ocular neurons are situated further caudally than the vestibulospinal neurons (Fig. 6). This discrepancy may reflect developmental modifications whereby the caudal embryonic vestibulo-ocular projections disappear either by cell death or withdrawal of the axons. Alternatively, the caudal vestibulospinal projections that Wold ('78b) reports may not yet be established by day 11of embryonic life. Clearly, further experiments need to be done to resolve this issue. A fourth neuronal tracing study in birds has some bearing on the tracts of the vestibulo-ocular projections. Correia et al. ('83) used anterograde transneuronal autoradiography to label the multisynaptic pathway from the labyrinthine end organs to the eye motor and spinal motor nuclei in pigeon. Since all neurons receiving afferents from the labyrinth may have been labelled, the study does not reveal which of the vestibular nuclei give rise to the vestibulo-ocular projections. Nevertheless, the pathways of these projections were revealed. Fibers ascending towards the oculomotor centers were shown to run bilaterally, but predominantly contralaterally in the MLF, while no labelling was observed in the brachium conjunctivum or the area occupied by the ascending tract of Deiter's in mammals. The demonstration of a possible homologue to the brachium conjunctivum in the present study may reflect a difference between avian species, but is probably rather due to a difference in the resolution of the methods employed.

Comparison with vestibulo-ocular projections in other vertebrate classes A comparison of my results with those obtained in non-avian species is limited by differences in methodology and in the anatomy of the vestibular nuclei. In reptiles, vestibulo-ocular projections are predominantly ipsilateral from rostral vestibular regions and contralateral or bilateral from caudal regions, similar to the chicken embryo. This is the pattern reported in the turtle by Kunzle ('85), and by Bangma and ten Donkelaar ('83), who traced an ipsilateral ascending projection from the dorsolateral vestibular nucleus and a contralateral projection that arose mainly in the medial vestibular nucleus. Similarly, in the lizard ascending projections from the dorso-lateral vestibular nucleus are mainly ipsilateral, while the ventromedial, ventro-lateral, and descending vestibular nuclei give rise mainly to contralateral ascending projections (ten Donkelaar et al., '85). In mammals, it is generally agreed that strong vestibuloocular projections arise in the superior nucleus and the rostral part of the medial nucleus. Ascending projections from the cell group "Y" are also well documented, while the extent to which other parts of the vestibular nuclei contribute is unclear. Ascending projections from the lateral nucleus and the rostral part of the descending or inferior nuclei have been reported, but seem less prominent than the ones from the superior and medial nuclei (Brodal and Pompeiano, '58; McMasters et al., '66; Gacek, '71, '79; Tarlov, '72; Graybiel and Hartwieg, '74; Steiger and Buttner-Ennever, '79; Yamamoto et al., '78; Carleton and Carpenter, '83; Carpenter and Cowie, '85). Thus, studies on mammals suggest that most of the vestibulo-ocular projections arise from rostral parts of the vestibular complex, while in the chicken embryo I found prominent ascending vestibular projections from caudal levels as well.

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Fig. 6. T h e spatial relationship between vestibulo-ocular and vestibulospinal neurons is different on the sides ipsi- and contralateral to the injections. Plots were made from 70 pm sections of two different two-tracer preparations (see text)-one cut sagittally (A), the other cut transversely (B).Each panel represents a single section and each symbol a single neuron; filled symbols represent vestibulo-ocular and open symbols vestibulospinal neurons. T h e parasagittal sections in A illustrate the segregation of ipsilaterally projecting vestibulo-ocular and vestibulospinal neurons ( u p p e r panel), in contrast to the almost complete intermingling of their contralaterally projecting counterparts (lower p a n e l ) . T h e arrows highlight the difference in the distributions,

filled arrows symbolizing the vestibulo-ocular and open arrowheads the vestibulospinal neuron groups. Note i hat ipsilaterally the vestibulospinal neurons are sandwiched by the vestibulo-ocular along an axis oblique to t h e longitudinal. B shows these relationships in more detail (see text for description), and also shows the greater rostro-caudal extent of vestibulo-ocular neurons with respect to vestibulospinal. In B the open arrowheads indicate the regions containing vestibulospinal neurons, while vestibulo-ocular neurons are labelled as in Figure 2. The level of each section and the injection sites are indicated on the horizontal profile ( l o w e r l e f t ) . Scale bars, 500 pm.

Three different tracts containing vestibulo-ocular fibers have been described in mammals: the MLF, the brachium conjunctivum (BC), and the ascending tract of Deiter's (ATD) (Highstein and Reisine, '79). The MLF is reported to contain a strictly ipsilateral projection from the superior vestibular nucleus, while the other vestibular nuclei are reported to give rise to crossed and/or uncrossed MLF fibers (see McMasters et al., '66; Tarlov, '72; Brodal, '74; Gacek, '79; Carleton and Carpenter, '83). The BC has been shown to contain a vestibulo-ocular projection that arises in the dorsal superior vestibular nucleus and crosses to the contralateral oculomotor nucleus in rabbit, cat, and monkey (Yamamoto et al., '78; Highstein and Reisine, '79; Lang et

al., '79; Mitsacos et al., '83). Thus the dorsal and rostral parts of the superior nucleus project in the BC, while more central parts project in the MLF (Yamamoto et al., '78; Highstein and Reisine, '79). This organization is similar in the chicken embryo, where the projection arising in the most rostral and dorsal part of the superior vestibular nucleus and coursing rostromedially along the dorsal wall of the fourth ventricle may be a homologue of the BC projection in mammals (Fig. 5). A difference is that in the chicken embryo this projection joins the MLF caudal to the trochlear nucleus, whereas in mammals it crosses over as an independent tract ventral to the oculomotor nucleus (Lang et al., '79). The third vestibulo-ocular pathway, the ATD, has been

VESTIBULO-OCULAR PROJECTIONS I N CHICKEN EMBRYO most thoroughly described in the cat, where it courses just lateral and parallel to the MLF and terminates in the oculomotor nucleus. Muskens ('14) originally described it as arising from the dorsal Deiter's nucleus, but later studies have shown that it originates in the ventral part of lateral (Gacek, '71), and medial vestibular nuclei, and conveys excitation to the motoneurons of the ipsilateral medial rectus (Highstein and Reisine, '79; McCrea et al., '87a). I have not found any homologue to this tract in my material. However, my injections were restricted to the MLF caudal to the trochlear nucleus and the ATD, if present, may therefore have escaped labelling.

Vestibulo-ocularversus vestibulospinal projections and dichotomizing axons Only a couple of studies, on the turtle and the rabbit, have employed retrograde labelling with two tracers to map vestibulo-ocular versus vestibulospinal neurons (tenDonkelaar et al., '85, and an unpublished reference therein). tenDonkelaar et al. ('85) injected Fast Blue in the rostra1 mesencephalic tegmentum and Nuclear Yellow in the rostral spinal cord of ten turtles. Spatial segregation between neurons giving rise to ascending versus descending projections was found to be greater on the side ipsilateral to the injections, which is in accord with my results. In both this study and the referenced unpublished study on rabbits that employed similar methods, neurons with both ascending and descending projections were found in all the vestibular nuclei but preferentially in the ventromedial vestibular nucleus. This is a clear discrepancy from the situation in the chicken embryo, where such dichotomizing projections were virtually nonexistent. Vestibular neurons in mammals that project axon collaterals both in the ascending MLF (to trochlear and/or oculomotor nuclei) and in the descending MLF have also been identified by combining electrophysiological recordings with intracellular labelling. While in some studies the descending collaterals were only followed into the medulla (Graf et al., '83; Graf and Baker, '85; Graf and Ezure, '86), others have shown such descending collaterals reaching the upper levels of the spinal cord (Isu and Yokota, '83; Uchino and Hirai, '84;Boyle et al., '88). Quantitative estimates of such bifurcating axons vary, but are generally much higher than the less than 1%found in the chicken embryo. The discrepancy between my findings and those of others may represent a species difference, as has been described among mammals. For example, the prevalence of vestibulo-ocular neurons with descending collaterals appears to be higher in rabbit and cat (McCrea et al., '81; Isu and Yokota, '83; Uchino and Hirai, '84; Graf et al., '83; Graf and Ezure, '86) than in the squirrel monkey (McCrea et al., '87a,b). Alternatively, the discrepancy may reflect the possibility that descending collaterals have not yet reached the spinal cord in the 11-day chicken embryo. It should also be noted that my results do not rule out collaterals from vestibulospinal axons into the abducens nucleus. Thus, it is certainly possible that there exist in avians vestibular neurons participating directly in both vestibulospinal and vestibulo-ocular reflexes.

Functional implications A semicircular canal may produce an excitatory or an inhibitory vestibulo-ocular effect, depending on the direction of the head rotation. The pathways involved, from the second-order vestibular neuron to the oculomotor nuclei, are asymmetrical. In rabbit, excitation is transmitted mainly

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contralaterally (an exception is the excitatory ipsilateral ATD), while inhibition is transmitted ipsilaterally (Ito, '73a,b, '76a,b). My results show that in the chicken embryo the spatial organization of ipsi- versus contralaterally projecting neurons is asymmetrical. Some of the neurons form spatially segregated groups on the basis of this laterality. If the laterality of the projection reflects functional role in the chicken as it does in the rabbit, this would indicate a spatial segregation of excitatory and inhibitory neurons. However, only some of the vestibulo-ocular neurons were spatially organized into unilateral groups. Intermingling of ipsi- and contralaterally projecting neurons occurred in the ventrocaudal part of the vestibular complex (the cVC and iVC groups) and in the dorsal part of the superior nucleus (the DR groups which project in the putative homologue of the BC). Determining the functional modality of the various pathway-defined vestibulo-ocular groups will be important for understanding the significance of their spatial organization.

ACKNOWLEDGMENTS I thank my colleagues in Oslo for being such delightful and supportive company. I am deeply indebted to Dr. Jan Jansen and my closest collaborator, Dr. Joel Glover, for their help with both theoretical and practical aspects of this work. I also thank HBvard Tbnnesen for superb technical assistance and Dr. Eric Rinvik for useful comments on the manuscript. Financial support from the Nordic Council and the Icelandic Council of Science is gratefully acknowledged.

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