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. 2010 Jan 28;216(4):510–517. doi: 10.1111/j.1469-7580.2009.01208.x

The nasolacrimal duct of anuran amphibians: suggestions on its functional role in vomeronasal perception

Christine Nowack 1, Angela Wöhrmann-Repenning 1
PMCID: PMC2849528  PMID: 20136666

Abstract

Tear secretions discharged by the Harderian gland are suggested to function as a solvent for molecules sensed by the vomeronasal organ (VNO) in anurans. It has been assumed that chemical stimuli are absorbed at the surface of the eye to be carried – together with the lacrimal fluid – into the nasal cavity via the nasolacrimal duct. In the study presented herein, we examined the intranasal anatomy of 10 different anuran species to analyse the opening region of the nasolacrimal duct and its functional relationship with the VNO and the external naris. In addition, vital staining of the nasal cavities was conducted. Our results indicate that stimuli reaching the VNO are more likely to be ingested through the nostril than via the eye. In many cases the intranasal orifice of the nasolacrimal duct shows a close proximity to the external naris and simultaneously we observed a noticeable distance to the VNO. We suggest that the secretions of the Harderian gland are carried to the external naris by the nasolacrimal duct, where they bind chemical stimuli that are subsequently actively transported into the VNO. In some of the investigated species the opening region of the tear duct was situated in a more caudal part of the nasal cavity and closer to the VNO. In these cases a conspicuous system of channels can be found, which is suspected to carry the intruding medium of smell from the nostril to the nasolacrimal aperture.

Keywords: external naris, functional anatomy, Harderian gland secretion, olfaction, vital staining, vomeronasal organ

Introduction

In amphibians, the functional importance of the ductus nasolacrimalis (nasolacrimal duct) is an almost unanswered question. It was Gaupp (1904) who first described the radially arranged muscle fibres surrounding the duct in Rana esculenta (Anura, Amphibia). He concluded that constriction of these fibres would lead to a dilatation of the duct’s lumen and therefore draw in lacrimal fluid from the eye. Gaupp (1904) also assumed that these tear secretions, released by the Harderian gland, the only orbital gland in anurans, serve to lubricate the movements of the nictitating membrane and were plainly eliminated via the nasolacrimal canal into the nasal cavity. The paired nasal cavity of anurans consists of several inter-related chambers, containing at least two different chemosensory organs, the olfactory organ and the vomeronasal organ (VNO). The external naris and the choana connect the nasal sac with the environment and the oral cavity, respectively. A third access is established by the nasolacrimal duct, leading from the nasal cavity towards the eye.

Today many authors still assume that the main functional role of the Harderian gland in anurans is the lubrication of the eye (Shirama et al. 1982; Chieffi et al. 1996; De Rienzo et al. 2002). However, there are some investigations pointing to other tasks of this secretory organ. For mammals, several functions have been discussed, such as thermoregulation and photoprotection, as well as a source of saliva, growth factors or pheromones (for review see Payne, 1994; Chieffi et al. 1996). In some anurans the Harderian gland shows seasonal changes (Minucci et al. 1990; Chieffi Baccari et al. 1991, 1992) as well as sexual dimorphism (Minucci et al. 1989; Varriale & Chieffi, 1997; Serino et al. 2007). Minucci et al. (1989) observed lipid secretions occurring only in female individuals of Bufo viridis and supposed that they could act as a pheromonal stimulus.

Hillenius et al. (2001) discovered that in different anurans (Rana pipiens and Rana catesbeiana) the fluid flow leading from the eye through the nasolacrimal duct into the nasal cavity also enters the VNO. They believe that these tear duct fluids are involved in olfactory perception by the VNO as has been proposed for other tetrapods like snakes (Rehorek et al. 2000; Mason et al. 2006) and mammals (Wöhrmann-Repenning, 1992; Rossie & Smith, 2007). Furthermore, it was assumed that the stimuli activating the VNO in anurans were collected on the cornea of the eye, where they dissolve in the secretion of the Harderian gland and are then carried into the VNO by the nasolacrimal duct (Hillenius et al. 2001; Hillenius & Rehorek, 2005).

The results of the present study lead to a new theory concerning the role of Harderian gland secretions in vomeronasal olfaction in Anura, based on a new interpretation of the nasal anatomy regarding the opening region of the nasolacrimal duct and its relation to the VNO. In addition, a vital staining assay was carried out to support the anatomical results.

Materials and methods

The nasal anatomy of 10 different anuran species was examined histologically. One fully metamorphosed individual from each species was investigated (Table 1). Animals were deeply anaesthetized by cooling (0–1 °C) and decapitated immediately. All animals were handled with permission from the Kassel Regional Administrative Authority (“Regierungspräsidium Kassel”, Tgb.-Nr. 378/05). Complete heads were fixed in 4% formalin. After 1 week of fixation, the tissue was decalcified in nitric acid (3.5%), dehydrated and embedded in paraffin. Histological sections of the nasal region were cut in a coronal plane (transverse sections, see Fig. 2) at 10 μm thickness. The series sections were stained alternating with Delafield’s haematoxylin and azan.

Table 1.

Specimens examined.

Species Developmental stage Snout-vent length (cm)
Trachycephalus resinifictrix Mid-age juvenile 2.5
Phyllomedusa bicolor Adult 6.0
Agalychnis callidryas Adult 4.9
Leptopelis vermicularis Late-age juvenile 3.2
Rhacophorus reinwardtii Adult 5.3
Hypopachus barberi Adult 2.8
Pelodytes punctatus Adult 2.1
Megophrys nasuta Mid-age juvenile 4.8
Bufo marinus Adult 12.0
Bombina orientalis Adult 4.0–5.0
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Fig. 2.

Fig. 2

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Dorsal view of the head of Phyllomedusa bicolor. The red line exemplarily indicates the sectional plane of the transverse sections presented in Figs 3, 4 and 5.

Schematized drawings were made from selected sections using enlarged prints of digital photographs of the stained slices and transparent paper, under microscopic control. Drawings were digitalized and processed with Adobe© Photoshop© CS2.

Vital staining was carried out following an approach described by Døving et al. (1993). Three adult specimens of Bombina orientalis were kept in a glass aquarium (base area: 20 × 30 cm) containing a 4.0-cm depth of the staining solution for 18 h. We used a 0.001% aqueous solution of Cresyl Violet, which produces a robust red fluorescence. After staining, the animals were processed as described above and heads were immediately frozen using liquid nitrogen. The tissue was surrounded with Jung Tissue Freezing Medium™ and cryostat sections (40 μm) of the nasal region were cut at −20 °C. The sections were mounted on glass slides, air dried, coverslipped with Entellan© and analysed with the aid of a fluorescence microscope.

Results

Anatomy of the nasal cavities

In summary, the paired nasal cavity itself consists of three inter-related chambers, the cavum principale, cavum medium and cavum inferius (Figs 1B and 3A–F). The cavum principale is the largest part of the nasal cavity and contains the main olfactory organ. At its anterior region the cavum principale opens via the dorsolaterally orientated external naris to the outside (Figs 1, 2 and 3A). Caudally it communicates with the oral cavity through the internal naris (Fig. 5C, schematic drawing). Below the cavum principale and directly connected to it lie the cavum medium and the cavum inferius. The flattened, broad cavum inferius contains the VNO. With the exception of Bombina orientalis, the VNO lies in a rostro-medially orientated blind appendix of the cavum inferius. In addition, this protruding recess of the VNO is bending dorsally (Figs 1B, 3 and 4B). In Bombina orientalis, the rostral part of the cavum inferius, containing the VNO, shows a more lateral orientation (Fig. 5). The smaller cavum medium is situated between the cavum principale and cavum inferius. The posterior area of the cavum medium establishes a connection between the VNO and the upper parts of the nasal cavity (Figs 3B,F and 4B). Moreover, at the lateral margin of the cavum medium the ductus nasolacrimalis has its nasal (anterior) opening (Figs 3A,E and 4A). From here the duct is running backwards towards the eye, where its forked-out posterior opening is found at the ventral margin of the lower eyelid (Fig. 1B).

Fig. 1.

Fig. 1

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Trachycephalus resinifictrix. (A) Position of the external naris in relation to the eye. Scale bar: 2.5 mm. (B) Schematic illustration of the left nasal cavity. Note the distance between the rostral opening of the nasolacrimal duct (red arrow) and the vomeronasal organ (VNO).

Fig. 3.

Fig. 3

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Schematic drawings of serial transverse sections through the nasal region of Phyllomedusa bicolor (A–C) and Hypopachus barberi (D–F), representing the two possible types of relation between the nasolacrimal duct and the vomeronasal organ (VNO) (and the external naris, respectively) in anurans. In both cases the drawings were arranged from the most anteriorly (A and D) to the most posteriorly (C and F) located section, indicated by the large arrows at the sides. Each individual drawing represents only the left half of one transverse section; therefore, left is lateral and right is medial (smaller arrows at the bottom). (A) Level of the external naris (compare Fig. 2) and the opening of the nasolacrimal duct. (B) Section posterior to the external naris. (C) Caudal part of the VNO, where the bonding zone between the cavum medium and cavum inferius is situated. (D) Section showing the vestibular channel connecting the external naris (situated rostral to this slice) and the opening region of the nasolacrimal duct (E). (F) Region caudal to the orifice of the nasolacrimal duct. The cavum medium and cavum inferius are interconnected and thereby establish the connection to the VNO. Red, olfactory epithelium; hatched red, vomeronasal sensory epithelium; black, bone; dotted, cartilage.

Fig. 5.

Fig. 5

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Left side: fluorescent histological transverse sections of the nasal region of Bombina orientalis after the vital staining. Right side: matched schematic drawings of the sections. Only the right half of each section is shown; therefore, in each section left is medial and right is lateral. (A) Stained parts of the nasal cavity are the vomeronasal organ (VNO), the cavum medium and the vestibulum. The cavum principale remains uncoloured. (B) Opening region of the nasolacrimal duct. Note the intensive staining of the vestibular channel leading from the external naris to this region. (C) Section showing the well-stained nasolacrimal duct. Hatched, sensory epithelium. Scale bars: 480 μm.

Fig. 4.

Fig. 4

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Histological cross-sections through the nasal region of Agalychnis callidryas (A and B) and Megophrys nasuta (C and D) showing a more detailed picture of the two possible positions of the nasolacrimal aperture within the nasal cavity. Each photograph displays a detail of the left nasal cavity in a transverse plane. As in Fig. 3, left is lateral and right is medial. (A) Opening of the nasolacrimal duct (arrow) near the external naris. (B) More caudally located section of the same animal showing the vomeronasal organ (VNO) and the connection between the cavum medium and cavum inferius. (C) Section showing the vestibular channel (asterisk) connecting the external naris (situated rostral to this slice) and the opening region of the nasolacrimal duct (D). (D) Opening of the nasolacrimal duct (arrow). In this region the vestibular channel, the nasolacrimal duct and the opening between the cavum medium and cavum inferius meet each other. Scale bar: 160 μm. Staining: azan.

Opening region of the nasolacrimal duct

The anatomical arrangement of the anterior termination of the ductus nasolacrimalis, in contrast to other parts of the nasal cavity, varies considerably between the species investigated. Basically there are two main types developed, which differ especially in their formation concerning the relation between the intranasal orifice of the nasolacrimal duct and the external naris. The simpler construction is found in those cases where the nasolacrimal duct opens in close proximity to the external naris. This situation occurs in Phyllomedusa bicolor (Fig. 3A–C), Agalychnis callidryas (Fig. 4A,B), Leptopelis vermicularis, Rhacophorus reinwardtii and Trachycephalus resinifictrix. The opening is amenably exposed to the external naris, as the cavum medium in this area possesses an open connection to the cavum principale (Figs 3A and 4A). Furthermore, a large-scaled vestibulum, which in other species separates the external naris from the main nasal cavity, is missing. Whenever the anterior ending of the nasolacrimal duct obtains such a rostral position, the distance between this opening and the VNO increases. This phenomenon becomes exceedingly apparent in the case of A. callidryas, where the opening of the nasolacrimal duct is located at the anterior corner of the external naris, whereas the connection between the cavum medium and VNO has a much more posterior position, considerably behind the nostril (Fig. 4A,B).

Another situation emerges if the anterior termination of the ductus nasolacrimalis is situated caudal to the external naris. On the one hand, the contact between the opening of the duct and the VNO becomes closer. On the other hand, the aperture itself shows a much more complicated constellation. Differently designed channels running backwards from the nostril join the opening region. For example in Hypopachus barberi, a horizontally orientated groove at the lateral aspect of the cavum principale leads from the vestibulum to the rostral opening of the ductus nasolacrimalis (Fig. 3D, vestibular channel). The floor of this groove continues caudally as a lateral ridge on the wall of the nasolacrimal duct (Fig. 3E). A quite similar situation can be found in Pelodytes punctatus, Megophrys nasuta (Fig. 4C,D) and Bufo marinus. In Bombina orientalis, the passage leading towards the opening of the nasolacrimal duct shows a slight difference, as it is orientated vertically (Fig. 5B). In all cases a sort of vestibular prolongation establishes a contact channel between the external naris and the intranasal opening of the nasolacrimal duct. This canal system is usually surrounded by conspicuous supporting skeletal structures, predominantly the septomaxillare.

Vital staining

As a result of the vital staining, epithelial surfaces that had been in intensive contact with the staining solution exhibited a strong red fluorescence. Areas with little or no staining were hardly or not at all observable under the fluorescence microscope. The epithelium of the cavum principale remained almost uncoloured, whereas the other aspects of the nasal cavity were clearly stained (Fig. 5): the VNO and the cavum medium, as well as the external naris, the vestibulum and the vestibular channel leading to the opening of the ductus nasolacrimalis. The nasolacrimal duct itself was also stained (Fig. 5C), as was the cornea of the eye (not shown). The strongest staining, however, could be observed in the rostral parts of the nose, especially in the opening region of the nasolacrimal duct and the VNO (Fig. 5A,B). More caudally the staining within the nasal region decreased; the internal naris and oral cavity were practically uncoloured (Fig. 5C).

Discussion

The gross intranasal anatomy of all species investigated in this study is quite similar, as described elsewhere in detail (Gaupp, 1904; Helling, 1938; Døving et al. 1993; Nowack & Wöhrmann-Repenning, 2009). Nonetheless, the anatomical results presented in this study lead to a new perspective regarding the functional relation between the tear secretions delivered by the nasolacrimal duct and the VNO in anurans. The anuran VNO, as in other tetrapods, is fluid-filled (Sbarbati et al. 1991; Døving et al. 1993). Therefore, it is necessary that olfactory stimuli detected by the VNO need to be dissolved in the fluid before they can reach the vomeronasal sensory epithelium. In mammals it has been revealed that specific carrier molecules, derived from nasal gland secretions, are used to transport the stimuli into the VNO (Pelosi & Maida, 1990; Rama Krishna et al. 1995; Ohno et al. 1996). The orbital Harderian gland also seems to be a possible source of such odorant-binding proteins. It has been suggested before that an interaction between the fluids of this gland and the vomeronasal system is a common and even primitive character within tetrapods (Hillenius & Rehorek, 2005). Secretions of the Harderian gland are proven to reach the VNO in snakes via the nasolacrimal duct (Rehorek et al. 2000). There is strong evidence that these secretions also contain binding molecules that provide the transport of olfactory stimuli into the VNO (Huang et al. 2006; Mason et al. 2006). Furthermore, it could be shown that in different orders of amphibians (Gymnophiona: Schmidt & Wake, 1990; Anura: Hillenius et al. 2001) the nasolacrimal duct is also capable of transferring fluids into the VNO. Hillenius et al. (2001) found that india ink, applied to the surface of the eye, could later appear in the VNO. The authors concluded from their results that, in anurans, olfactory stimuli detected by the VNO are usually collected via the cornea and conjunctiva of the eye. Thus, the stimuli should dissolve in the secretion of the Harderian gland, spread over the cornea and move to the VNO through the nasolacrimal duct. An advancement of this hypothesis led to the statement that this ‘corneal/conjunctival mechanism’ (Hillenius & Rehorek, 2005) is a primordial amphibian specialization of the vomeronasal system that later got lost in terrestrial reptiles and mammals, where the stimuli were absorbed on other surfaces like the tongue or the rhinarium (Halpern & Kubie, 1980; Wysocki et al. 1980; Wöhrmann-Repenning, 1993; Meredith, 1994). Rossie & Smith (2007) similarly recognize a difference between the mammalian situation and that of more primitive tetrapods. They conclude that, e.g. in amphibians, the VNO receives a direct flow of fluids from the nasolacrimal duct, whereas in some mammals the secretions were delivered to the external naris, where they spread over the rhinarium and reach the VNO through the oral cavity.

Our anatomical results, however, indicate that in Anura, due to the topological arrangement of the opening region of the nasolacrimal duct, the tear duct fluids often do not reach the VNO directly but are first directed towards the external naris. Thereby the secretions of the Harderian gland would accomplish a notable detour, assuming that their purpose is to bind olfactory stimuli at the eye and lead them to the VNO as suggested by Hillenius & Rehorek (2005). In some of the species investigated in this study (e.g. A. callidryas, Fig. 4A,B) this indirection would be remarkably extended as the nasolacrimal duct opens at the anterior corner of the external naris, whereas the access to the VNO is located a considerable distance behind the nostril. So why is this indirection made? The only reasonable interpretation of the anatomical facts is that the secretions of the Harderian glands have to perform a special task at the nostril. One likely explanation would be that olfactory stimuli reaching the nasal cavity via the external naris are solved in the glandular secretions and are then led into the VNO. As mentioned before, Harderian gland secretions of snakes are suspected to contain specific binding molecules that help to carry the vomeronasal stimuli from the tips of the tongue into the VNO (Huang et al. 2006; Mason et al. 2006). In anurans these glandular secretions might be responsible for capturing the relevant stimuli as they pass the entrance of the nasal cavity. The recently described anuran intranasal pumping mechanism (Nowack & Wöhrmann-Repenning, 2009) should play an important role in the further transport of the stimuli-loaded fluid into the VNO. In addition, some molecules might also be bound on the surface of the eye and moved into the VNO but the direct absorption of the stimuli at the external naris presumably represents the more relevant source, as this route provides a shorter and thereby faster entry into the VNO. Furthermore, the active influx of olfactory medium into the nasal cavity provides a larger and more concerted uptake of odorous substances than the mainly passive contact between the eye and the environment.

The second group of anuran species investigated in this study possesses a more caudally located opening of the nasolacrimal duct (Figs 3E and 4D) and, according to this, the entry to the VNO is in closer proximity. In these cases the opening region is associated with an exceptional system of channels (Figs 3D, 4C and 5B) leading to the external naris. Hitherto this ductwork has not been functionally analysed. It seems to bridge the gap between the nostril and the orifice of the nasolacrimal duct. In this manner the olfactory stimuli could be carried in a targeted way from the external naris to the opening of the tear duct, where they again are bound to the secretions of the Harderian gland. An interesting fact is that in animals with such a channel system the orifice of the nasolacrimal duct itself also shows a very special morphology. Often in cooperation with the cavum medium an enlarged estuary cavern is formed (Fig. 3E,F), building a potential room to collect the incoming lacrimal fluid. This cavern simultaneously constitutes a place to bring the olfactory stimuli in contact with the gland secretions. The arising mixture could then be carried towards the VNO.

The conclusions that we have made so far were supported by the results of the vital staining experiments. The examined species (Bombina orientalis) featured a more caudally located opening of the nasolacrimal duct. According to this the mentioned vestibular channel connecting this opening to the external naris is well developed. It is exactly this region of the nasal cavity that shows the most intense Cresyl Violet staining. The stain dispersal in the enlarged estuary cavern including the opening of the nasolacrimal duct is shown in Fig. 5B. Assuming that the staining pattern represents the movements of stained fluid within the intranasal structures, the channel system connecting the external naris to the nasolacrimal opening and the VNO seems to be the main route. The weaker colouration of the cornea and the nasolacrimal duct indicates that fluids are also carried between the eye and the nasal cavity but to a lesser degree.

According to our results, there seems to be a strong functional inter-relation between the Harderian gland, the nasolacrimal duct, the external naris, the inward leading vestibular channel and the VNO. Further studies are needed to investigate the exact binding mechanisms and the character of the chemical stimuli received via the external naris for vomeronasal detection.

Acknowledgments

The authors would like to thank Rudolf Wicker (Frankfurt Zoo), Gunther Köhler (Senckenberg Research Institute), Herbert Nigl (Aquarium Dietzenbach) and Jörg-Peter Ewert (Department of Neurobiology, University of Kassel) for kindly providing us with the material examined in this study. We also thank Sue Hayes (University of Bristol) for proofreading the manuscript.

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