1670090-2977/08/4003-0167 © 2008 Springer Science+Business Media, Inc.

Neurophysiology, Vol. 40, No. 3, 2008

Central Projections of the Lagena (The Third Otolith Endorgan of the Inner Ear) in the PigeonV. I. Khorevin1,2

Neirofiziologiya/Neurophysiology, Vol. 40, No. 3, pp. 199-210, May-June, 2008.

Received June 26, 2008.

Central projections of the lagena were studied in the pigeon using transport of biotinylated dextran amine (BDA) that was locally applied to the lagenar epithelium through the opened cochlear canal. Descending (dorsocaudal part) and superior (middle part) vestibular nuclei were the main rhombencephalon structures with the maximum density of labeled fibers and terminals. Lesser numbers of labeled fibers were observed in the ventral part of the lateral vestibular nucleus and also in the medial vestibular nucleus; single labeled fibers were found in the cochlear nuclei. In the cases where BDA diffused not only in the lagena but also on the basilar papilla after application of the marker to the cochlear canal, considerable numbers of labeled fibers were observed in the cochlear nuclei; apart from this, the pattern of distribution of labeled fibers in the vestibular nuclei did not differ in general from that described above (in the case of a sufficiently local application of BDA only to the lagena). Efferent lagenar neurons were localized ventrally with respect to the vestibular nuclei, in particular in the nucl. reticularis pontis caudalis.

Keywords: lagena, central projections, cochlear canal, vestibular nuclei, biotinylated dextran amine.

1 Bogomolets Institute of Physiology, National Academy of Sciences of Ukraine, Kyiv, Ukraine.

2 Washington University, St. Louis, USA. Correspondence should be addressed to V. I. Khorevin (e-mail: vkhor@biph.kiev.ua).

INTRODUCTION

The ability of animals to orient and move in space is to a considerable extent based on combined functioning of the otolith endorgans and semicircular canals whose receptor apparatus encodes information on the position and movement of an object in space with respect to certain axes and reference points. Despite the fact that the vestibular system in vertebrates is, from the evolutionary aspect, rather conservative, otolith endorgans in different animals demonstrate certain variability [1-3]. In most mammals, two otolith endorgans, the utriculus and the sacculus, are found, while in many vertebrates of other classes there is the third otolith endorgan (called the lagena). The receptor apparatus of the lagena includes a group of hair sensory cells localized in a certain cavity (chamber) of the membranous labyrinth of the inner ear and covered by the otoconium membrane [1-3]. Among vertebrates,

the lagena as an independent endorgan was described for the first time in elasmobranch cartilaginous fishes (sharks and rays) [4] in which this structure is considered to be the equilibrium organ [5]. It is believed that the lagena in bony fishes is related to the perception of vibrations of the body [6] and acoustic waves [7]. In amphibians, the lagena fulfills mostly the vestibular function per se and is to a lesser extent involved in perception of sound oscillations [8]. In amniotes, the lagena is localized in the cochlear canal more distally with respect to the auditory organ. In reptiles (crocodiles), birds [1, 2, 9], and monotremate mammals (platypus and echidna) [10], organization of the lagena demonstrates substantial similarity, despite the fact that sizes of the animal’s body and living conditions of representatives of the above-mentioned different classes of vertebrates are dissimilar. The functions of the lagena in amniotes, including birds, remain unclear in detail, although there are a number of hypotheses on the functional role of the sensory apparatus of this organ [3]. Our experimental study was aimed at elucidating the central projections of the lagena in birds; we believed that the data would help one to interpret the function of this sensory structure in a more scientifically grounded manner.

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METHODS

Experiments were carried out on pigeons, Columba livia (one to three years old), weighing 340 to 450 g. All procedures were performed according to the Guide for Care and Use of Laboratory Animals (National Institute of Health, NIH, publication, No. 86-23, Bethesda, 1985) and approved by the Institutional Animal Care and Use Committee of Washington University (St. Louis, USA).

Surgical preparation of the pigeons was performed under inhalation anesthesia (a mixture of 3-5% gaseous isoflurane and 95% oxygen). After cannulization of the trachea, the animal’s head was fixed in a special stereotaxic device with the help of ear holders placed in the external auditory canals. After opening of the scull surface by means of medisection, a special corrosion-resistant head bolt (length 12 to 15 mm) was then secured in the frontal bone using fast-hardening dental acrylic plastic. At the next stages of surgery, this bolt was used for rigid fixation of the skull, since the ear holder was removed for opening of the labyrinth on the surgical side.

Access to the labyrinth was provided according to Schwartzkopff and Bremond [11]; opening of the lagena was performed using Harada’s technique [12] modified in our lab. A 25-mm-long incision of the skin was made about 5 mm caudally to the external auditory meatus, the muscle tissue was retracted, and the skull was cut under the horizontal semicircular canal. The trabeculae between the skull and bony labyrinth were removed; this allowed us to identify the utriculus and ampullae of the horizontal and posterior semicircular canals used as marks in the subsequent stages of surgery. In the pigeon, the lagena is localized on the base of skull, 2 to 3 mm from the midline at the blind distal end of the cochlear canal oriented toward the anterior and medial part of the vestibulum. The lagena was accessed via the labyrinth, to avoid injury of the adjacent essential vessels and nerves. Initially, a hole (1.5 mm in diameter) was made in the lateral wall of the bony labyrinth rostrally from the utriculus; after opening of the vestibulum, another hole was made in the bottom of the labyrinth. After this opening was expanded, the lagenar otoconia could be seen as a white strip. The bony capsule around the lagena was made thin and then pierced using a sharp needle. After aspiration of the perilymphatiс fluid, a marker, fast-diffusing biotinylated dextran amine (BDA, molecular mass 3,000 daltons, Molecular Probe, USA) was applied to the epithelium [13]. A day before surgery, the dose of the marker was dissolved in a depression slide in

distilled water. After the water evaporated from the solution and its consistency became paste-like, the tips of tungsten needles were immersed in this paste. Using such needles allowed us to apply BDA highly locally. On the 10th min after application, the opening in the skull was covered with a hemostatic sponge and bone wax, and the wound was sutured. Within the postoperation period, buprenex (0.4 mg/kg, i.m.) was injected in order to decrease pain sensations.

Six days after application of the marker, the pigeons were anesthetized with Nembutal (100 mg/kg, i.p.) and perfused transcardially, initially with 500 ml of a mixture of equal amounts of phosphate buffer and saline solution with the addition of 0.1% sodium nitrite (to provide vasodilation) and heparin (to prevent blood coagulation) at about 20°С. Then, perfusion was continued with a cold (4°С) fixative solution containing 2% glutaraldehyde and 1% phosphate-buffered paraformaldehyde. After perfusion of the labyrinth with the same fixative via openings in the horizontal and posterior semicircular canals, the pigeon’s head was isolated from the body and immersed in the fixative for 24 h. On the next day, a block of the brainstem tissues was made according to the orientation of the horizontal semicircular canal in the horizontal plane, and then all endorgans were isolated from the membranous labyrinth using a stereotaxic manipulator [14]. Then, the block was immersed in a 30% phosphate-buffered glucose solution, and, after cryoprotection was provided, coronal 50-µm-thick slices were made using a freezing microtome. To identify marker-containing fibers, we used a modified diaminobenzidine technique [14, 15]. After the blockade of the activity of endogenous nonspecific peroxidase, brain slices and endorgans were incubated for 24 h in 0.1 M phosphate buffer-based solution containing a complex of avidin D and horseradish peroxidase (10 μg/ml; Vector Laboratories Inc., USA) and 0.3% Triton Х-100. Labeled fibers were identified by means of histochemical staining with benzidine (0.5 mg/ml) with 1% solutions of nickel chloride and cobalt acetate added, according to the standard protocol. The brain slices obtained were mounted on slides. After air drying, the slices were counterstained with neutral red and coverslipped with Permount.

To identify regions of diffusion of the marker along the membranous labyrinth of the cochlear canal, transversal 10-μm-thick slices were made from the brain block containing the lagena and basilar papilla using a rotation microtome and Ralph’s glass knives. The slices were mounted on chrome alum subbed slides, stained by Richardson, and coverslipped with

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Permount. If stained fibers were observed only in the lagena and lagenar nerve, this situation was qualified as a successful local application. When stained fibers were found in the lagena, lagenar nerve, and between hair cells of the basilar papilla, we considered this pattern a result of diffusion of the marker not only to the lagena but also to the organ of hearing. Localization of the stained fibers on coronal cerebral slices was mapped according to contours of the vestibular nuclei whose boundaries were given in an atlas of the pigeon brain [16] and also in the study by Dickman and Fang [14]. To plot schemes of central projections of the lagena to the rhombencephalon structures using the data obtained with light microscopy, we used the histomorphometric software Neurolucida (MicroBrightfield, USA).

RESULTS

To estimate the level of specificity of applications of the marker, we examined all other five endorgans (cristae of all the three semicircular canals and maculae of the utriculus and sacculus), and also nerve trunks innervating these structures. No cases where labeled nerve fibers could be observed in the above endorgans and in the corresponding nerve trunks (including the sacculus most closely adjacent to the cochlear canal) were observed. In five preparations, marker-containing fibers were observed only in the lagena. In five other cases, we found somewhat more extended diffusion of the marker (not only within the lagena but also to the distal part of the cochlear basilar papilla).

Marker-containing fibers were localized in the anterior part of the inferior vestibular ganglion; their localization corresponded to the boundary between the superior and inferior vestibular nerves. Labeled bodies of first-order sensory neurons were observed mostly in the lateral part of the inferior vestibular ganglion, while labeled cells in the superior vestibular ganglion were sparse, and their localization was relatively uniform. The number of marker-containing neuronal somata in the inferior vestibular ganglion was four to six times greater than that in the superior ganglion. Ninety percent of the labeled cells were of oval shape; their dimensions (along the greater axis) were 20 to 25 μm. The shape of the other cells was round, and their diameters were greater (35 to 40 μm). Marker-containing fibers came to the vestibular nuclear complex at the site of contact of the lateral and descending vestibular nuclei (LVN and LVN and LVN DVN, respectively), and then divided into ascending and descending branches.

DVN.DVN.DVN Lagenar fibers in the DVN corresponded to DVN corresponded to DVNthe more caudal part of central lagenar projections. At the level of the nucl. cuneatus externus, they were localized in the dorsal part of the DVN (Fig. 1). The DVN (Fig. 1). The DVNdiameter of more than 90% of the marker-containing fibers was about 2 μm. Thick fibers (diameter 3-4 μm) were observed only in the dorsal parts of this nucleus, while thin fibers were found both in the dorsal and ventral divisions of this structure. The density of the terminals (appearing as swellings or thickenings) was greater in the dorsal parts of this nucleus than in the ventral ones. At the level of the nucl. laminaris, thedensity of labeled fibers decreased; they were localized mostly in the center of the DVN around large-sized DVN around large-sized DVNneurons. In the rostral parts of this nucleus, only single lagenar fibers were observed. The number of terminals gradually decreased in the caudo-rostral direction in a parallel manner with a decrease in dimensions of cross-sections of the nucleus.

Nucl. tangentialis is the internal nucleus of the vestibular complex in birds. It possesses an almond-shaped form and is easily differentiated from other cellular structures by large-sized intensely labeled cell somata localized medially with respect to the restiform bodies and laterally to the site of bifurcation of vestibular fibers [17]. Lagenar fibers crossing the nucl. tangentialis along its long axis were, in the most cases, thin (diameter about 2 μm). The dorsocaudal part of the nucleus contained single thick fibers that perhaps were the so-called giant fibers forming electrical contacts [17]. In the nucl. tangentialis, we observed, on average, five to seven giant fibers in three to four slices.

Medial Vestibular Nucleus (MVN).MVN).MVN Labeled fibers were more numerous in the caudal parts of this nucleus at the level of the medullary auditory nuclei; the rostral parts of this nucleus contained only single marked fibers. The density of lagenar fibers in the caudal parts of the MVN was about two times smaller MVN was about two times smaller MVNthan that in analogous parts of the DVN. At the level of the nucl. laminaris, labeled lagenar fibers in the MVN looked like short thin segments. They should MVN looked like short thin segments. They should MVNbe characterized as fragments of small-diameter (less than 2 μm) fibers.

Superior Vestibular Nucleus (SVN)SVN)SVN is the second (after the DVN) structure by the mean density of DVN) structure by the mean density of DVNmarked fibers in the vestibular nuclear complex. The main part of lagenar fibers was localized in the middle part of the SVN, somewhat rostrally with respect to the nucl. laminaris. Labeled fibers that extended for several tens of micrometers along the slice plane were typical of the SVN. The densities of the lagenar

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fibers and terminals in this part of the SVN were the SVN were the SVNmaximum, as compared with those in the vestibular nuclear complex, and only somewhat smaller than in the caudal parts of the DVN. Most labeled fibers had a diameter close to 1 μm, and only about 10% of such fibers were somewhat thicker (2 to 3 μm). Thick fibers were observed only in the medial parts of this

nucleus. The density of terminals was the maximum in the central region of the middle SVN part; they were SVN part; they were SVNconcentrated around large neurons.

LVN.LVN.LVN Labeled fibers were observed in the LVNmostly in its lateral part, while in the medial parts their number was small. Separate terminal swellings were found in the lateral part of the LVN.

Fig. 1. Schemes of distribution of labeled fibers in the pigeon medulla oblongata and ventral part of the cerebellum after application of biotinylated dextran amine to the lagenar epithelium in the cochlear canal exclusively. 1-10) Successive frontal 50-μm-thick sections (shift in the rostro-caudal direction; the distance between neighboring sections is 200 μm). Dots indicate localization of labeled fibers. A) Cell group A, An is the angular nucleus, B) Cell group B, CbL is the lateral nucleus of the cerebellum, VeD is the descending vestibular nucleus (DVN), DVN), DVN IX-X are the nuclei of IX-X are the nuclei of IX-XIX and X craniocerebral nerves, VeL is the lateral vestibular nucleus, La is the nucl. laminaris, LoCis the locus coeruleus, VeM is the medial vestibular VeM is the medial vestibular VeMnucleus, VeS is the superior vestibular nucleus, VeS is the superior vestibular nucleus, VeSMCC is the cochlear magnocellular nucleus, MCC is the cochlear magnocellular nucleus, MCC PVis the principal nucleus of the Vth cranial nerve (trigeminus), ST is the solitary tract nucleus, ST is the solitary tract nucleus, ST Tais the tangential nucleus, V is the nucleus of the V is the nucleus of the Vtrigeminal nerve, VI is the nucleus of the VI is the nucleus of the VI nervus abducens, XII is the nucleus of the hypoglossal XII is the nucleus of the hypoglossal XIInerve, and РН is the РН is the РН adjacent nucleus of the hypoglossal nerve. Abbreviations of the brainstem nuclei are given according to the stereotaxic atlas of the pigeon’s brain [16].

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Except for the vestibular nuclear complex, a great number of labeled lagenar fibers were also found in some brainstem structures belonging to other afferent or ascending neuronal systems. These fibers were

observed in the bulbar auditory nuclei, structures of the somatosensory system (nucl. cuneatus externusand descending nucleus of the trigeminal nerve), and noradrenergic locus coeruleus.

Fig. 2. Schemes of distribution of labeled fibers in the medulla oblongata and ventral part of the cerebellum of the pigeon after application of biotinylated dextran amine in the cochlear canal to the region of lagenar epithelium and distal part of the basilar papilla. Designations are the same as in Fig. 1.

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In birds, the medullary auditory nuclei include the nucl. angularis localized laterally and also the nucl. magnocellularis localized medially. These nuclei are considered to be homologs of the dorsal cochlear nucleus and anterior part of the ventral cochlear nucleus, respectively, in mammals [18, 19]. As was already mentioned, application of the marker to the cochlear canal was in some cases strictly limited only

by the lagenar epithelium, while in others the region of diffusion of the marker included not only the lagena but also 100 to 200 μm of the most distal part of the basilar papilla, an important structure of the organ of hearing in birds. If the marker was applied only to the lagena, labeled fibers were observed in all vestibular nuclei (as was already described). A few marker-containing fibers were also found in the cochlear

Fig. 3. Total schemes of distribution of labeled efferent neurons in the brainstem in the cases where applications of biotinylated dextran amine to the cochlear canal were limited by only the lagenar epithelium. 1-8) Successive frontal 50-μm-thick sections of the brain (shift in the rostro-caudal direction, the distance between neighboring sections is 100 μm). Asterisks indicate localization of labeled cells. GCt is the central gray, GCt is the central gray, GCt SСdСdС is the dorsal d is the dorsal dlocus coeruleus, IV is the nucleus of the IVth cranial IV is the nucleus of the IVth cranial IVnerve, V is the trigeminal nerve nucleus, V is the trigeminal nerve nucleus, V VII is the facial VII is the facial VIInerve nucleus, RP is the nucl. reticularis pontis caudalis, Rgc is the nucl. reticularis gigantocellularis lateralis, MLF is the medial longitudinal fascicle, MLF is the medial longitudinal fascicle, MLF TTD is the descending nucleus and tract of the trigeminal nerve. Other designations are the same as in Fig. 1.

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nuclei where their densities were at least 10 times smaller than in the neighboring DVN. If the region of application of the marker (except the lagena) involved also the basilar papilla (which was identified by the presence of labeled fibers around hair cells of this structure), the number of labeled fibers in the cochlear

nuclei was considerably greater. In the case where the marker was applied not only to the lagena but also to the basilar papilla, labeled fibers were observed in the ventral, rostral, and dorsal parts of the nucl. angularis and in the caudal and lateral parts of the nucl. magnocellularis. This observation agrees well

Fig. 4. Total schemes of distribution of labeled efferent neurons in the brainstem in the cases where application of biotinylated dextran amine to the cochlear canal spread from the regions of the lagenar epithelium to distal parts of the basilar papilla. Designations are the same as in Figs. 1 and 3.1 1 mm

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with the data obtained by other researchers; according to these data, apical parts of the basilar papilla project to the ventral part of the nucl. angularis and also to the lateral part of the nucl. magnocellularis [18, 20].It should be mentioned that when application of the marker was insufficiently local (both to the lagena and basilar papilla), labeled fibers in the vestibular nuclear complex were distributed in all vestibular nuclei (Fig. 2). In this complex, their distribution was, on the whole, similar to that described above under conditions of local lagenar applications.

Efferent lagenar neurons had somata of triangular or oval shape, and their diameters varied from 12 to 25 μm. Three to four dendrite trunks of such cells formed sparse perisomal branchings. Labeled axons could be observed in one slice at a distance of about 100 μm from the soma. Thin labeled fibers crossed by dendrites at different angles at distances of 50 to 70 μm from the soma of efferent neurons demonstrated in some cases swellings at the site of crossing. It seems probable that these terminals belong to lagenar afferents synaptically connected with dendrite branchings of the efferent neurons projecting their axons to the lagena. The BDA-labeled somata were localized bilaterally and rostrally with respect to the nucleus of the VIth cranial nerve and more medially with respect to the dorsal motor nucleus of the facial nerve (Fig. 3). It should be noted that efferent neurons sending their axons to the labyrinth of the inner ear and motoneurons of the facial nerve have common precursors; some efferent vestibular fibers come together with axons of the motoneurons of the facial nerve [21]. In addition, marker-filled somata were observed in some parts of the brainstem reticular formation, in particular in the nucl. reticularis pontis caudalis (RP) and nucl. reticularis gigantocellularis lateralis (Rgc). If the region of application of the marker was strictly limited by the lagenar macula, the labeled somata were distributed in the RP, a member of the so-called dorsal efferent group. If the marker was applied both to the lagena and basilar papilla, labeled somata of efferent cells were observed in theRP and Rgc (Fig. 4). In the latter case, the shift of the marked neurons from the midline (on average, 1,187 ± 16.2 μm) was noticeably greater (Р = 0.02) than in the cases of local applications limited only by the lagena (1,098 ± 33.7 μm). The main part of the labeled neurons during “pollution” of the sensory apparatus of the hearing organ by lagenar application was localized close to the dorsal motor nucleus of the facial nerve, and only 15% of the labeled cells were localized within the ventral nucleus of the facial nerve

(within the so-called ventral efferent group). These data allow us to hypothesize that lagenar efferent neurons are localized in the dorsal efferent group, while efferent neurons projecting their axons to the organ of hearing (basilar papilla) are localized in both the ventral and dorsal efferent groups.

DISCUSSION

The somata of primary sensory neurons innervating the lagena are localized mostly in the lateral parts of the inferior vestibular ganglion, while the superior vestibular ganglion contains four to six times less primary lagenar neurons distributed relatively uniformly. In mammals, the somata of primary sensory neurons innervating the sacculus and posterior semicircular canal [22-24] (in birds, innervating also the lagena [14]) are localized in the inferior vestibular ganglion, while the superior vestibular ganglion contains mostly primary sensory neurons innervating the utriculus and the two other semicircular canals (anterior and horizontal) [14, 22-24]. Most labeled ganglionic cells had a diameter of 12 to 25 μm, while about 10% of the primary lagenar neurons were larger (35 to 40 μm). Our data on heterogeneity of primary lagenar neurons agree with the findings obtained by other researchers (on the noticeable morphological heterogeneity of both vestibular ganglion neurons and primary vestibular afferents) [24].

Despite the fact that the glutamatergic transmitter nature of primary vestibular afferents in different classes of vertebrates has been clearly identified [25, 26], the first-order relay vestibular neurons and their central processes are, perhaps, heterogeneous in their physiological properties. The majority of glutamatergic neurons in the Scarpa’s ganglion are small-sized cells. In rats and frogs, about 10 to 20% of the statoacoustic ganglion cells had greater diameters and contain glutamate and glycine [27]. The dissimilar chemical nature of primary vestibular sensory neurons can be related to different physiological functions of these cells. For example, in amphibians, primary vestibular afferents of large diameters activate NMDA and non-NMDA receptors on the membrane of relay cells, while thin primary vestibular afferents activate only non-NMDA receptors [28]. It seems probable that a small number of larger primary vestibular neurons can innervate the striolar region of the lagena where, similarly to the other otolith endorgans, the calyx-like afferents are numerous and have greater diameters [29, 30]. At the same time, the main part of smaller

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primary vestibular neurons innervates the extrastriolar region of the lagena by their thin axons with bouton-like terminals.

We found that the general course of lagenar fibers along the brainstem to the vestibular nuclear complex is to a considerable extent similar to that of vestibular afferents. This statement agrees with the data of previous studies [14, 31, 32]. On the whole, the vestibular nuclear complex in all the main classes of vertebrates [33], including fishes [34], amphibians [35], birds [14], and mammals [36], has a rather similar structure. Based on the peculiarities of cytoarchitectonics in this complex, four main nuclei (DVN, MVN, SVN, and LVN) and several smaller LVN) and several smaller LVNcellular groups are usually classified. The results of earlier studies performed on mammals using transport of markers, as well as recent investigations carried out on different amniotes using modern morphological techniques, showed that features of hodological organization of vestibular fibers found in birds do not differ fundamentally from those in other vertebrates [37]. In all classes of vertebrates, including amphibians [38], birds [32], and mammals [39], it was found that, although each vestibular endorgan projects to all four vestibular nuclei, the otolith afferents terminate more laterally and dorsally, while the canal afferents terminate more medially and ventrally, with certain overlapping of the projections in intermediate parts [1-3]. Afferent fibers from the otolith endorgans (sacculus and utriculus) innervate mostly the DVN, MVN, and SVN; afferents from the semicircular canals project to the SVN, ventral part of the LVN, and nucl. tangentialis; the latter in birds is a homolog of the interstitial nucleus of the vestibular nerve in mammals [14, 31, 32].

According to our own data, the main targets for lagenar fibers in the vestibular nuclear complex are its caudal (DVN) and rostral (DVN) and rostral (DVN SVN) parts. At the same time, SVN) parts. At the same time, SVNless numerous marked fibers (in the MVN) come to other MVN) come to other MVNparts of the vestibular nuclear complex or pass through the brainstem nuclei practically with no terminals (in the ventral LVN part and nucl. tangentialis). In the DVN and DVN and DVN SVN, we observed numerous labeled fibers coming from the lagena, the third otolith endorgan in non-mammals. These findings differ somewhat from the data obtained by other authors on mammals; in these animals, projections of the otolith endorgans to the above nuclei of the vestibular-nuclear complex are relatively sparse. For example, only 16.3 and 9.0% of cells responding to the corresponding stimulation (19 and 11 neurons, respectively) of 117 neurons tested by stimulation of the utricular and saccular nerves in the

cat were localized in the DVN and DVN and DVN SVN, respectively [40]. It seems probable that the presence of abundant projections formed by primary lagenar afferents in the DVN and DVN and DVN SVN reflects the specificity of organization SVN reflects the specificity of organization SVNof the vestibular system in birds.

In the LVN, lagenar fibers were observed in the dorsolateral region, which corresponds to the ventral part of this nucleus adjacent to the ventrolateral part of the SVN (where labeled fibers were numerous). In SVN (where labeled fibers were numerous). In SVNthe dorsal LVN part, giant Deiter’s cells (sources of LVN part, giant Deiter’s cells (sources of LVNdescending vestibulo-spinal fibers) are present; in this region, only single labeled fibers crossing the nucleus and forming no terminals were found. These findings agree with the published data on the absence of direct primary vestibular inputs to giant Deiter’s cells [14].

A question of the involvement of lagenar fibers in the formation of the afferent input to the cochlear nuclei in birds has been discussed in the literature for a long time. Most authors believed that lagenar fibers project to the medullary auditory nuclei [14, 18, 31, 32, 41], while other researchers insisted that the presence of labeled fibers in the cochlear nuclei (when a degeneration technique was used) is a result of destruction of not only lagenar fibers but also afferents coming from the basilar papilla. Such a conclusion can be made in the case where marker techniques did not allow researchers to apply the marker with a sufficient accuracy (the marker spread from the lagena to the basilar papilla) [20].

As was found in our study, in the case where the marker application zone covered only the lagena (which was confirmed by the absence of stained fibers among hair cells of the basilar papilla), the above-described pattern of distribution of lagenar fibers in the vestibular nuclear complex was supplemented by the presence of labeled fibers in the medullary auditory nuclei. However, the density of BDA-filled fibers in the cochlear nuclei was, in this case, significantly (at least 10 times) smaller than that in the neighboring DVN, i.e., such fibers were in fact sporadic.

In the experiments where the marker diffused to the basilar papilla (which was confirmed by the presence of labeled fibers among hair cells of this structure), the pattern of distribution of labeled fibers in the vestibular nuclear complex was in general similar to that described for conditions of strictly local lagenar applications. The density of labeled fibers in the cochlear nuclei was in the former cases considerably higher; however, localization of these fibers was analogous to that observed in experiments using degeneration techniques (where fibers from apical regions of the basilar papilla were injured) [18].

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The presence of the marker-filled fibers in experiments with a sufficient locality of applications of BDA to the lagena can be explained by the following. One of the reasons is that the lagena, from the evolutionary aspect, is a derivative of the sacculus, and primary afferents of the latter structure in birds and mammals form projections not only to the vestibular nuclear apparatus but also to the cochlear nuclei [42, 43]. It seems probable that the transport of BDA observed in our experiments on birds occurred along such afferent pathways formed in the course of evolution of the labyrinth of the inner ear in vertebrates and providing connections of the otolith endorgans with more dorsally situated regions of the rhombencephalon (corresponding to the cochlear nuclei in amniotes). Nevertheless, a defect of the technique used, i.e., spreading of small amounts of the marker along the cochlear canal (insufficient for labeling of afferent fibers among hair cells of the basilar papilla but capable of being accumulated in neurons of the inferior vestibular ganglion from which it is transported to the cochlear nuclei), can be another reason for labeling of the cochlear fibers. To rule out such a possibility, control studies should perhaps be carried out (e.g., iontophoretic application of BDAx to the region of the lagenar epithelium, but not mechanical application of the marker).

Efferent fibers coming along the VІІІth cranial nerve are observed in vertebrates of all classes [1-3]. In fishes and amphibians, efferent vestibular neurons are localized ipsilaterally in the medial reticular formation together with auditory vestibular neurons [44]. In amniotes, efferent neurons whose axons come along the statoacoustic nerve are localized bilaterally in the medial reticular formation between the nuclei of the abducent and facial nerves [44]. According to our data, lagenar efferent neurons in birds are localized bilaterally in the dorsal efferent group, in particular in the pontine caudal reticular nucleus. They are localized somewhat more ventrally and medially with respect to the site of location of utricular and canal efferent neurons [45]. Efferent neurons projecting their axons to the basilar papilla adjacent to the lagena are localized bilaterally in the nucl. reticularis gigantocellularis lateralis, in the so-called ventral efferent group. They are localized at a somewhat greater distance from the midline than the “lagenar” efferent cells. The presence of labeled processes of lagenar afferents in close proximity to the labeled dendrites and somata of efferent and reticular neurons can be indicative of the fact that functioning of the lagena is modulated by the brainstem efferent system;

this system, in turn, is under the control of primary lagenar afferents.

Acknowledgments. The author is grateful to Prof. J. D. Dickman who kindly gave me a chance to carry out this study, to Dr. M. Zakir for help in the development of techniques for access to the lagena, and to Dr. D. Huss for collaboration in some investigations.

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Central Projections of the Lagena in Pigeons