1 Glial Cells

Early classical comparative studies brought to light the basic model of glial cytoarchitecture in the central nervous system (CNS) of vertebrates (Herrick 1948; Ramon y Cajal 1952). The studies of amphibian CNS highlighted the presence of glial cells whose cell bodies are located in the ependymal or periependymal layer. The long radially oriented processes of these cells pass through the neural wall to the meningeal surface where they form terminal expansions constituting the mambrana gliae limitans externa. These elements are found both in larvae and adults (Messenger and Warner 1989) and resemble the first glial cells appearing in the embryonic brain of amniotes (Ramon y Cajal 1952).

Radial glial cells have been identified as important elements for guidance of migrating neurons during development (see Rakic 1981; Hatten 1999). Moreover, radial glial cells are the source of neurons as well as other glial elements including astrocytes, oligodendrocytes, and ependymal cells (Campbell and Götz 2002; Weissman et al. 2003; Malatesta et al. 2008; Rowitch and Kriegstein 2010). During neurogenesis, radial glial cells descend directly from neuroepithelial cells (reviewed in Götz and Huttner 2005).

Adult amniotes show a different type of glial cell, called star-shaped astrocyte due to numerous cytoplasmic processes arising from the cell body. Star-shaped astrocytes coexist with prevailing radial glial cells in reptiles (Kalman and Pritz 2001; Lazzari and Franceschini 2001). On the contrary, star-shaped astrocytes are the main type of glial cells in the endothermic amniotes, birds and mammals (Elmquist et al. 1994; Kalman et al. 1998). The glial cytoarchitectures including star-shaped astrocytes appear functionally more complex and phylogenetically derived compared to patterns consisting of radial glial cells only.

Glial fibrillary acidic protein (GFAP), the major subunit of the gliofilaments belonging to the intermediate filament class (Eng 1985), has been recognized as a reliable marker of mature elements of the astroglial lineage (Dahl and Bignami 1985). The expression of GFAP has been reported in the glial cells of many vertebrates (Wasowicz et al. 1994; Wicht et al. 1994; Naujoks-Manteuffel and Meyer 1996). GFAP has a considerable stability in its molecular and antigenic properties throughout the vertebrate phylogenesis (see Eng et al. 2000).

Vimentin is another cytoskeletal protein of the intermediate filament group that is used in glial cell study. It was detected in not fully differentiated cells of the astroglial lineage in reptiles (Monzon-Mayor et al. 1990; Yanes et al. 1990) and mammals (Oudega and Marani 1991; Elmquist et al. 1994; Pulido-Caballero et al. 1994). It was also found in terminally differentiated glial cells of adult teleosts and amphibians (Zamora and Mutin 1988; Cardone and Roots 1990; Rubio et al. 1992).

Immunohistochemically detected GFAP abundance and vimentin scarcity can suggest a mature condition for the glial elements. Moreover, the relative abundance of radial elements in the glial pattern is morphologically indicative of an immature condition (Kalman et al. 1998; Kalman and Pritz 2001). The relationship between GFAP/vimentin expression and amount of radial elements in the glial pattern of vertebrates is still not fully clear. Exhaustive response to developmental and phylogenetic implications of specific intermediate filament molecular markers and specific glial cell types require additional studies.

The immunohistochemical detection of intermediate filament molecular markers of glial cells brought to light the cytoarchitecture of astroglial cell elements in the vertebrate CNS (Kalman 2002). The following sections describe the characteristics of astroglial cell organization in the main groups of tetrapods (Table 15.1).

Table 15.1 GFAP- and vimentin-immunopositive glial structure in the central nervous system of tetrapods and closest, extant relatives
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1.1 Lungfish

The distribution of glial intermediate filament molecular markers, GFAP and vimentin, was investigated in the CNS of the African lungfish Protopterus annectens using immunoperoxidase technique (Lazzari and Franceschini 2004). The Dipnoi (lungfishes) are a monophyletic group of osteichthyes (Schultze and Campbell 1986) and together with their sister group Crossopterygii (coelacanths) represent the unique surviving lobe-finned fish (Moy-Thomas and Miles 1971). Lungfishes are considered the closest extant relatives to terrestrial vertebrates (Forey 1986). Molecular analysis, mostly DNA sequencing, supports this assumption (Amemiya et al. 2013; Biscotti et al. 2016).

GFAP immunohistochemistry revealed a glial architecture. The cell bodies of radial cells are located at the ventricular surface (the epithelial-like monostratified layer separating the neural tissue from the cavities constituting the ventricular system of the CNS). The cell body gives rise to a long radial process, which ramify into finer fibers. These fibers form the submeningeal and perivascular glial layers. In the different cerebral regions, radial glial cells show a low degree of heterogeneity in staining intensity for GFAP. In the spinal cord, the cell bodies of the glial cells are placed in the periependymal zone. GFAP immunostaining does not reveal star-shaped astrocytes in the CNS of Protopterus. Vimentin immunopositivity is scarce in the nervous tissue of this lungfish.

1.2 Urodeles

GFAP and vimentin have been used for an immunohistochemical study of glial elements in the CNS of two urodele specie, Ambystoma mexicanum (Ambystomatidae) and Triturus carnifex (Salamandridae) (Lazzari et al. 1997). In A. mexicanum brain, GFAP-immunopositivity appears only in the ependymal cells showing a tanycytic feature (Fig. 15.1a). GFAP-positive radial processes give rise to variously extended endfeet forming the perivascular and submeningeal glial layers (Fig. 15.1b). In the spinal cord, GFAP-immunopositive radial glial cells show their cell bodies only in periependymal position. In A. mexicanum CNS, no vimentin immunopositive glial structures are present. In T. carnifex CNS, the distribution pattern of GFAP and vimentin results quite the opposite of the A. mexicanum condition.

Fig. 15.1
figure 1

GFAP-immunopositive structures in the brain of tetrapods. (a) In Ambystoma mexicanum telencephalon, radially oriented processes originate from ependymal radial glia (tanycytes) (arrows). (b) Radially oriented glial processes reach the submeningeal surface of A. mexicanum telencephalon where enlarged endfeet form (arrows). (c) In Trionyx sinensis telencephalon, tanycytes (arrows) mark the ventricular surface. Radial processes go through the neural wall. (d) In T. sinensis brain, glial processes give rise to perivascular glial covering (arrows). (e) In Podarcis sicula telencephalon, tanycytes (arrows) line the ventricular surface; glial endfeet (arrowheads) mark out the meningeal layer. (f) In P. sicula optic tectum, some star-shaped astrocytes (arrows) are intermingled with radial glial processes reaching the meningeal surface. Bars: 50 μm (a, b, d), 100 μm (e, f), 200 μm (c)

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1.3 Turtles

Among living animals, turtles are widely accepted as the most closely related extant vertebrates to the “progenitor” amniotes that represent the common ancestor of both reptiles and mammals (Gans and Parsons 1981; Bar et al. 2000).

The glial cytoarchitecture in the CNS of the soft-shell turtle Trionyx sinensis was investigated by immunoperoxidase detection of GFAP and vimentin intermediate filaments (Lazzari and Franceschini 2006). In this turtle, GFAP immunostaining reveals clear positivity in both the brain and spinal cord. The stained elements consist of long radial fibers coming from cell bodies placed in the ependymal layer displaying their tanycytic nature (Fig. 15.1c, d). In the brain, star-shaped or proper astrocytes are not detected by immunostaining; on the contrary, they appear in the spinal cord. According to the more complex turtle brain compared to amphibian condition, the ependymal radial glia of the turtle show some regional specializations concerning different size and the intensity of the immunohistochemical staining, which are comparable with the results in Emydidae (Kriegstein et al. 1986; Kalman et al. 1994, 1997).

In the spinal cord, both gray and white matters appear intensely GFAP immunopositive. The ependymal layer turns out to be immunonegative, whereas evident radial astrocytes have their cell bodies in the periependymal zone. A few GFAP-positive star-shaped astrocytes are found in the lateral column of the posterior intumescence (an enlargement of the spinal cord at the level of hind limbs). Anti-vimentin immunohistochemistry reveals no positive glial structures in the CNS of T. sinensis.

In the turtle, GFAP immunostaining shows a certain degree of heterogeneity significantly greater than what appears in dipnoans (Lazzari and Franceschini 2004) and urodeles (Lazzari et al. 1997). The glial pattern of turtle is simpler than the condition found in lizards (Lazzari and Franceschini 2001, 2005a, b), but clearly less complex than in birds (Kalman et al. 1993a, 1998) and mammals (Elmquist et al. 1994). According to Kalman and Pritz (2001), star-shaped astrocytes, prevailing in birds and mammalian CNS, are derived cells compared to radial glia. They give rise to a glial network which permits an increase of the neural wall thickness and addresses the needs of the regional neuronal environment (Kalman and Pritz 2001). Star-shaped astrocytes inside a predominant radial glial model may indicate the local, more complex nervous areas (Simard et al. 2003; Mulligan and MacVicar 2004; Benediktsson et al. 2005; Volterra and Meldolesi 2005). In the adult turtle brain, the radial glial pattern and the absence of star-shaped astrocytes may represent an ancient character, shared with the stem reptile and present at early developmental stages in the mammalian brain (Clinton et al. 2014).

A more recent study on the Emydidae Trachemys scripta elegans (Clinton et al. 2014) has shown that radial glia are neuronal precursors in the turtle telencephalon. These results show that in the adult, radial glia retain differentiating capabilities that are similar to the activity they carry out during development.

1.4 Lizards

The glial pattern of lizards has been studied in different species, Gallotia galloti (Lacertidae, Monzon-Mayor et al. 1990; Yanes et al. 1990), P. sicula (Lacertidae, Lazzari and Franceschini 2001), Agama impalearis (Agamidae), Eumeces algeriensis (Scincidae), Tarentola mauritanica (Gekkonidae, Ahboucha et al. 2003), Anolis sagrei (Polychrotidae, Lazzari and Franceschini 2005a), and Eublepharis macularius (Eublepharidae, Lazzari and Franceschini 2005b; McDonald and Vickaryous 2018).

In adult lizards, GFAP-immunocytochemistry demonstrates the presence of three different astroglial lineage cell type: (1) ependymal radial glia or tanycytes throughout the brain; (2) proper radial glia or radial astrocytes, in the spinal cord with their cell bodies displaced from the ependymal layer to a periependymal zone; and (3) star-shaped astrocytes in the optic tectum and spinal cord (Fig. 15.1e, f).

In the spinal cord, ependymal cells are clearly vimentin-immunopositive and GFAP-negative. Only in the brain can a few vimentin-positive radial glial fibers be observed (Monzon-Mayor et al. 1990; Lazzari and Franceschini 2001, 2005b). During development, vimentin-GFAP transition has also been observed in reptiles (Monzon-Mayor et al. 1990; Yanes et al. 1990).

The presence and arrangement of star-shaped astrocytes (true astrocytes) is another point that deserves consideration in the study of saurian glia. The glial pattern containing star-shaped astrocytes is considered more complex and a phylogenetically derived state compared to the model with only radial elements. For these reasons, the glial organization of lizards may be considered a derived character compared to that of turtles.

In lizard, the intensity of the GFAP immunostaining of glial cells turned out to be not uniform throughout the neural wall. This condition recalls the GFAP-staining heterogeneity found in turtle (Kalman et al. 1994; Lazzari and Franceschini 2006).

According to Kalman and Pritz (2001), uneven distribution and different size of the astroglial structure are the main causes of the differences in regional GFAP immunostaining of saurian brain. These authors consider differential GFAP expression less important in reptile than in birds and mammals.

Based on GFAP abundance and vimentin scarceness, the immunohistochemical reaction of adult lizard astroglial intermediate filaments is indicative of mature astroglial cell lineage. In contrast, the glial pattern of the lizard mostly based on radial elements is morphologically more immature than those found in crocodile, birds, and mammals (Kalman et al. 1998; Kalman and Pritz 2001).

However, the connections between GFAP/vimentin and radial glia/star-shaped astrocytes during CNS evolution and development are not fully clear. In lizards belonging to different families, morphology of GFAP-positive cells and their relative proportions exhibit remarkable differences (Ahboucha et al. 2003). Additional studies on the characteristic of the vertebrate astroglial elements are necessary to provide a comprehensive response.

In a recent study on the neurogenesis in the medial cortex of E. macularius, immunohistochemistry identified proliferating pools of neural stem/progenitor cells within the telencephalon (McDonald and Vickaryous 2018). These cells, which express GFAP and vimentin and show a clear radial morphology, are identified as the radial glia involved in neurogenesis in adult.

1.5 Crocodilians

GFAP-immunopositive structures were studied in the brain of caiman, Caiman crocodilus, as representative of the order Crocodilia (Kalman and Pritz 2001). The caiman astroglia were clearly GFAP-immunopositive, and similar to other reptilians, the predominant elements were radial astrocytes. The glial architecture of crocodilian brain is characterized by a regional variability that is much greater than in the other reptiles studied so far, but smaller than in birds. Kalman and Pritz (2001) suggested that this condition is indicative of a more regional specialization of the glial cytoarchitecture in caiman compared to the other reptiles. The preeminent characteristic of caiman glial system compared to the other reptiles is represented by the widespread distribution of GFAP-immunopositive star-shaped astrocytes that are absent in turtles and scarce in lizards and snakes. Kalman and Pritz (2001) pointed out that, also in caiman, star-shaped astrocytes appear mixed together with radial fiber elements and do not represent the prevailing glial elements throughout the brain. Moreover, their presence appears not related to the brain wall thickness. Although Crocodilians represent the extant reptilian group most closely related to birds, Kalman and Pritz (2001) documented that the features of the caiman glial system are only partially shared by birds and mammals. This indicates the independent, parallel evolution from the diapsids or synapsids, different ancestral groups of stem reptiles.

1.6 Birds

The immunohistochemical detection of GFAP and vimentin was studied in the domestic chicken (Gallus domesticus) from hatching to adulthood (Kalman et al. 1993a, b, 1998). In the telencephalon, the day-old chicken shows a vimentin-immunopositive radial fiber system. Many of these radial elements persist until adulthood and during development undergo transition from vimentin to GFAP expression.

In the posthatch telencephalon, the distribution of star-shaped astrocytes resembles the condition in the adults, except in the visual center that shows a slow maturation rate. In the diencephalon (the rear region of the forebrain) and in the mesencephalic tegmentum (the ventral part of the mesencephalon), some GFAP-positive astrocytes are present at birth, and their number increases during the first two posthatching weeks. At hatching, these districts of the chicken brain contain only a few positive fibers. Therefore, a gradual and clear substitution of radial fibers by star-shaped astrocytes does not occur. On the contrary, this substitution is evident in mammals (Kalman et al. 1998). Whereas in reptiles the GFAP-positive radial glial system persists in optic tectum throughout life, in chicken it disappears from the tectal superficial layer that appears a derived character. At hatching, numerous GFAP-positive, vimentin-negative astrocytes accompany axons in the cerebral tracts. In posthatching birds, vimentin-positive glia are observed at the midline in decussation (medial areas of the CNS where nervous fibers pass from the right side to the left one and vice versa). Probably, these vimentin-positive elements are engaged in fiber-crossing regulation. In general, disappearance of radial fibers, dominance of astrocytes, and uneven distribution of GFAP-immunopositivity characterize glial development and organization in avian brain. Since these characters are found in phylogenetically distant mammals and only rudimentary outlined in reptiles, Kalman et al. (1998) suggested that they evolved independently along parallel paths.

1.7 Mammals

In mammals, radial glial cells have almost disappeared in adults. They represent the first developmental stage of astrocytes from which fibrous and protoplasmic astrocytes originate through shortening of their long processes and displacement of their cell body away from the ependymal layer (Levitt and Rakic 1980).

In adult mammals, GFAP is regarded as the principal intermediate filament protein in astrocytes (Voigt 1989), whereas vimentin is found in ependymal cells (Dahl et al. 1981; Bignami et al. 1982).

During development, vimentin predominates in early radial glia; afterward, this vimentin-like immunoreactivity decreases as radial cells mature into astrocytes (Oudega and Marani 1991; Elmquist et al. 1994; Moore and Oglesbee 2015; Luo et al. 2017). Therefore, in mammals, vimentin-immunoreactivity can be interpreted as marker of immature astrocytic cells and GFAP-immunoreactivity as marker of mature astrocytes. As the brain matures, radial glia become true astrocytes, and the change in intermediate filament protein expression from vimentin to GFAP may parallel this morphological transformation (Schnitzer et al. 1981; Elmquist et al. 1994). Embryonic astroglia of higher vertebrates resemble astrocytic cells of adult lower vertebrates.

In the adult mammalian CNS, radial glial cells are restricted to few locations: the retina (Müller cells), the cerebellum (Bergmann glia), and the hypothalamus where they are called tanycytes (Sild and Ruthazer 2011). New information derives from Mack et al. (2018) who investigated the astroglial cells in the cerebral cortex of the lesser hedgehog tenrec, Echinops telfairi. These animals belong to the group of Afrotheria representing a clade which is possibly a sister group of all other placental mammals (O’Leary et al. 2013; Averianov and Lopatin 2014). Lesser hedgehog tenrecs are considered to have the lowest encephalization index with relatively little neocortex compared to other placental mammals (Stephan et al. 1991; Krubitzer et al. 1997). The adult lesser hedgehog tenrec brain shows also continued cell proliferation (Alpar et al. 2010), reminiscent of the continued cell addition in anamniotes.

The authors concluded that the radial glia in the adult lesser hedgehog tenrec represents an immature form of astroglia that persists in these animals throughout life. It is noteworthy that radial glial structures occur at locations with continued neurogenesis (e.g., dentate gyrus and the paleocortex). Therefore, it is possible to imagine that the radial glial fibers of the adult are involved in the differentiation of new neurons. Radial glial fibers can lead differentiating neurons to appropriate locations. In adults, this activity is similar to the role of radial glia during development. The coincidence of radial glia and continued growth is found also in the brains of many anamniotes.

1.8 Ensheathing Glial Cells

GFAP and vimentin, together with other cellular markers, are useful tools also in characterizing olfactory ensheathing cells (OECs) that represent a specific type of glial cells (Pellitteri et al. 2010). Throughout life, the regeneration ability of olfactory sensory neurons (OSNs) is connected to OECs that cover olfactory axons in their route from the basal lamina of the olfactory epithelium to the glomeruli in the olfactory bulb (Pellitteri et al. 2010). In recent years, growing attention was given to OECs as they appeared able to promote and support regeneration of injured CNS (Raisman 2001; Barnett and Riddell 2004; Chung et al. 2004). In particular, studies underlined their potential role in cell-based therapy for spinal cord injury repair (Lu et al. 2002; Mackay-Sim 2005).

Recent studies are focused on OECs of anamniotes (Lazzari et al. 2013, 2014, 2016). A comparative immunohistochemical study on microsmatic (Poecilia reticulata) and macrosmatic fish (Carassius auratus, Danio rerio) showed some differences between the two types of olfactory organization and some similarities between fish (anamniotes) and mammals (amniotes) OSNs (Lazzari et al. 2007, 2013, 2014; Bettini et al. 2009). D. rerio is a good model also in olfaction-related research. In fact, its olfactory system is histologically comparable to that of other vertebrates, and the cells of its olfactory neuroepithelium can be clearly characterized by immunological approaches (Germanà et al. 2007; Gayoso et al. 2011; Braubach et al. 2012; Lazzari et al. 2017, 2019). The immunohistochemical characterization of OECs was also studied in A. mexicanum (Lazzari et al. 2016). Urodeles have a relatively simple olfactory system and represent a good model for studying OECs in extant representatives of basal tetrapods. The olfactory pathways of A. mexicanum, representative of basal terrestrial anamniotes, share characteristics with both fish (aquatic anamniotes) and mammals (amniotes). It remains speculative whether these similarities are basal features conserved during evolution or evolved independently in different vertebrate groups. So far, the functional role of the regional and interspecific differences displayed by OEC immunolocalization remains unknown.

2 Olfactory Crypt Neurons

In addition to ciliated (cOSNs) and microvillous (mOSNs) neurons, the olfactory organ of fish is peculiarly composed of a third sensory cell type, called crypt cell. The crypt cells were observed for the first time in the olfactory neuroepithelium of zebrafish, Danio rerio (Hansen and Zeiske 1998). The cell body of the crypt cell is egg-shaped, and it is located in the upper layer of the epithelium. The most striking feature of the crypt cell is an invagination, called the “crypt,” located in the apical part of the neuron. The border of the crypt is surrounded by microvilli, while short cilia grow in its lumen. The large nucleus of the cell resides in the lower third of the soma. A thin axon emerges from the basal portion of the neuron. The last histological characteristic of crypt cells is that they are surrounded by specialized supporting cells which are particularly electron-lucent (Fig. 15.2). Although crypt cells are randomly distributed in all the lamellae of the olfactory organ, their number is lower than that of ciliated and microvillous cells.

Fig. 15.2
figure 2

Crypt cell morphology. (a) Schematic drawing of the crypt cell (modified from Camacho et al. 2010); cilia reside in the apical invagination, called “crypt” (arrow). (b) An ovoid crypt cell surrounded by specific electron lucent sustentacular cells (arrowheads) in the olfactory epithelium of P. reticulata (modified from Lazzari et al. 2007). (c) Magnification of the crypt showing cilia (black arrows) and microvilli (white arrow). Bars: 5 µm (b), 2 µm (c)

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2.1 Crypt Cell Phyletic Distribution and Evolutionary Considerations

Since their first description, crypt neurons have been observed in many actinopterygian species (Hansen et al. 2003; Hamdani and Døving 2006; Lazzari et al. 2007; Camacho et al. 2010), including freshwater and marine fish (see Hansen and Finger 2000 and Hansen and Zielinski 2005 for a review of species examined, and crypt cell distribution). Moreover, crypt cells were discovered in Chondrichthyes, both in sharks (Scyliorhinus canicula, Ferrando et al. 2006) and batoids (Raja clavata, Ferrando et al. 2007). Interestingly, no evidence exists about the presence of this cell type in lungfish, which suggests that the crypt neurons are ancestral traits evolved before actinopterygian radiation, but lost at the actinopterygian-sarcopterygian transition.

The evolution of air breathing in vertebrates could be a possible event that influenced the disappearance of this cells in tetrapods. Ultrastructural, histological, and ontogenetic studies on the olfactory organ of amphibians, the tetrapods most closely related to lungfish, did not reveal crypt cells in either terrestrial or aquatic adult species (see Hansen and Finger 2000 for a list of publications). The developmental analysis from early larval stage to post-metamorphic juveniles in different species of anurans (Benzekri and Reiss 2012; Jungblut et al. 2009, 2017; Quinzio and Reiss 2018) and urodeles (Franceschini et al. 2003; Saito et al. 2003; Nakada et al. 2014) with gill-bearing larvae did not report the presence of crypt cells. The olfactory system of the anuran model system Xenopus laevis, has two cavities, one of which is devoted to aquatic olfaction and the other to aerial olfaction, but no crypt cells have been detected in any of them (Hansen et al. 1998; Oikawa et al. 1998).

The most characteristic anatomical difference in the olfactory system between fish and amphibians is the presence in the latters of a vomeronasal system. The vomeronasal system is an accessory olfactory system possessed by most tetrapods, except birds, aquatic reptiles, aquatic mammals, and higher primates (including man) in which it is vestigial or lost (Bertmar 1981). It is composed by a vomeronasal organ (VNO), containing only mOSNs (while the main olfactory organ is exclusively composed by cOSNs) projecting to an accessory olfactory bulb (AOB) in the rostral telencephalon (D’Aniello et al. 2017). Interestingly, a recent study (Gonzales et al. 2010) shows that the African lungfish Protopterus dolloi possesses, at the base of the olfactory lamellae, the epithelial crypts expressing markers of vomeronasal receptors. Cells localized in the epithelial crypts project their axons to an AOB identified in the lateral olfactory bulb, revealed also by lectin binding in a previous work (Franceschini et al. 2000). During evolution from lungfish to tetrapods, the formation of a vomeronasal organ was accompanied by a redistribution of morphological olfactory cells subtypes, with cOSNs segregated to the main olfactory organ and mOSNs to the vomeronasal organ, and crypt cells disappeared. Possible causes of crypt cell disappearance may reside in the function of the newly developed/evolving vomeronasal organ.

2.2 Crypt Cells Have Similarities with the Vomeronasal Organ of Tetrapods

Crypt cells seem to be ancestral olfactory neurons, which became replaced by microvillous cells of the vomeronasal organ during the evolutionary divergence of tetrapods (Fig. 15.3). This shift was possible probably because these two cell types show common features. In fact in D. rerio (Oka et al. 2012), the crypt neurons express only the Gαi-coupled receptor gene ora4 belonging to the Vomeronasal-1-Receptor (V1R)-like family.

Fig. 15.3
figure 3

Cladogram reporting the presence of crypt cells and vomeronasal organ in the main vertebrate groups. “Yes”: the presence has been confirmed in at least one species; “Yes”: an accessory olfactory system with segregated projections has been observed in different studies, but the organ is unique; “?”: the group has never been examined

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Moreover, in sexually mature Oncorhynchus mykiss, crypt cells preferentially respond to gonadal extract and pheromones from the opposite sex (Bazáes and Schmachtenberg 2012), and the vomeronasal organ is presumed to detect pheromones (Halpern and Martinez-Marcos 2003). However, the vomeronasal organ can respond to a variety of other odorants with different behavioral significance in all groups of tetrapods. Recently, zebrafish crypt cells were proposed to be involved in detecting kin odor-related signal (Biechl et al. 2016). However, which chemical compounds are detected by crypt cells remain elusive because of different results obtained in various species (Vielma et al. 2008).

Important clues supporting an evolutionary relationship between crypt cells and the vomeronasal organ come from their pathways to glomerular targets in the central nervous system.

In crucian carp (Hamdani and Døving 2006) and channel catfish (Hansen et al. 2003), olfactory bulb backtracing experiments suggested a medioventral position of the crypt neuron target region where a population of secondary neurons specific for sex pheromones are located (Lastein et al. 2006). Medial glomeruli in the bulb are connected with the medial olfactory tract, mediating reproductive behavior. Interestingly, in zebrafish, axons of crypt cells project in one single glomerulus within the mediodorsal area of the olfactory bulb (Ahuja et al. 2013), a region connected with medial amygdala (Biechl et al. 2017) that in tetrapods receives input from the vomeronasal organ. This finding supports the hypothesis that a primordial accessory olfactory system, which originates in crypt olfactory sensory cells, may exist in teleosts.

All together, these data reinforce the hypothesis that the crypt cells may be related to the reproductive behavior and, in general, similarly to the vomeronasal organ, to the conspecific recognition (Liu et al. 2014).

2.3 Developmental Studies Give Useful Information in Determining a Possible Role of Crypt Cells

Camacho et al. (2010) proposed a sequence of stages in crypt cell development, characterized by morphological profiles observed with TEM: (1) at first, immature cells appear close to the epithelial basal lamina; (2) then they differentiate into cells similar to the crypt neurons (absence of knob, envelopment by supporting cells, etc.) but with cilia still remaining on their non-invaginated apical surface; and (3) the formation of the crypt would be the final step of their differentiation.

In vertebrates, the olfactory epithelium exhibits neurogenesis throughout the life of the animal, from the embryonic/juvenile development to the adulthood. This continuous turnover is required to restore an intact epithelium after chemical or physical injuries. However, the growth dynamics of the crypt cell population is different than cOSNs and mOSNs. In Acipenser naccarii (Camacho et al. 2010) and zebrafish (Sandulescu et al. 2011), the crypt cell differentiation occurs in the early days of embryonic life, but later than differentiation of cOSNs and mOSNs. Hamdani et al. (2008) demonstrated that the number of crypt cells in the olfactory epithelium of the adult crucian carp Carassius carassius varies dramatically throughout the year, with seasonal regression in winter and renewal during the summer spawning months. Interestingly, in Poecilia reticulata, a species with a year-round breeding season (Houde 1997), crypt cell density remains invariant after 90 days of life, and it is different between males and females (Bettini et al. 2012). Moreover, crypt cells reach their adult density, through sex-specific dynamics, at the end of gonad development. These findings may suggest that crypt neurons are involved in mediating behavior related to reproduction and confirm their possible sensitivity to sex pheromones. However, the onset of crypt cell differentiation occurs very early (Bettini et al. 2012; Camacho et al. 2010; Ferrando et al. 2007; Sandulescu et al. 2011) so they might be involved also in the nonreproductive functions, perhaps in association with cOSNs and mOSNs.

2.4 Crypt Cell Markers

In all fish examined in the literature, crypt cell morphology has been fully described by TEM studies, and in various species (e.g., channel catfish, crucian carp, sturgeon, zebrafish), they have been visualized also by retrograde transport of fluorescent dye (Hansen et al. 2003; Hamdani and Døving 2006; Camacho et al. 2010; Ahuja et al. 2013). The use of specific immunohistochemical staining in the olfactory system is important for numerous fields of research, for example, environmental studies on the differential effects of pollutants on olfactory neuronal populations (Bettini et al. 2016; Lazzari et al. 2017, 2019). However, only in the zebrafish model antigenic properties of crypt neurons have been studied.

2.5 The Zebrafish Model

Germanà et al. (2004) reported that crypt neurons in zebrafish olfactory lamellae are specifically immunostained by antiserum against S100, a neural calcium-binding protein that stimulates cell proliferation and migration and inhibits apoptosis and differentiation (Donato et al. 2013). Thus, the anti-S100 serum was used in several subsequent studies (Gayoso et al. 2011, 2012; Sato et al. 2005). However, in the last decade, some studies (Ahuja et al. 2013; Kress et al. 2015; Oka et al. 2012) showed that the specificity of anti-S100 varies according to tissue processing: in fresh-frozen sections, the cells with crypt-neuron morphology appeared specifically stained, while, in fixed conditions, other cell types, possibly microvillous neurons, were also stained. Moreover, while anti-S100-positive microvillous cells express the s100z gene, anti-S100-positive crypt cells do not express S100 genes, raising some doubts on which crypt cell antigen is recognized by the antiserum. In addition, the dorsomedial glomerular field of zebrafish olfactory bulb receives afferents from the crypt cells, as revealed by DiI retrolabeling (Gayoso et al. 2012), but their axons are S100-negative (Gayoso et al. 2011). This uncertainty precludes the use of S100-like immunoreactivity in various studies and procedures, including visualization of axonal terminal regions.

The first molecular characterization of crypt cells was made by Catania et al. (2003), who discovered that anti-TrkA, specific for a neurotrophin receptor, labeled only zebrafish crypt cells in the olfactory epithelium, but this antibody was not investigated further at the time. Ten years later, Ahuja et al. (2013) demonstrated that anti-TrkA-immunohistochemistry constitutes a robust and sensitive marker for zebrafish crypt cells, and nowadays, it is the only marker with such specificity.

2.6 What About Other Fish?

In scientific research, the use of only one animal model (zebrafish, in this case) allows direct comparisons of data, and this facilitates the determination of cause and effect relationship and the formulation of hypothesis. On the other hand, the characteristics described in the model could not be extended to the majority of other species. For this reason, recently, for the first time, the antigenic properties of crypt cells in another fish, P. reticulata (guppy) was examined (Bettini et al. 2017). P. reticulata possesses some anatomical and histological characteristics of the olfactory organ different from zebrafish (Lazzari et al. 2007). Crypt cell markers in the guppy appeared to overlap only partially with zebrafish markers (Fig. 15.4). While no olfactory neuron was labeled by anti-TrkA, the anti-S100 immunohistochemistry gave a pattern comparable to what has been reported in zebrafish. In addition, in the gyppy, the calretinin, a known marker of zebrafish cOSNs and mOSNs (Germanà et al. 2007), was expressed also by some S100-positive crypt neurons. However, other anti-S100-labeled crypt cells remained immunonegative. This revealed two distinct subpopulations of the same morphotype recognizable for their protein content (calretinin-positive and calretinin-negative crypt neurons). It is possible that this heterogeneity in protein expression also reflects some functional differences. All that considered, this recent study revealed that the molecular features of olfactory crypt neurons described in the fish model D. rerio cannot be extended to all teleosts, and the investigation of other phyletic taxa is necessary to provide more conclusive answers on the evolution of olfactory crypt cells in fish.

Fig. 15.4
figure 4

Comparison between TrkA (a–b) and S100 (c–d) immunohistochemistry in D. rerio (a, c) and P. reticulata (b, d). TrkA is a specific marker for crypt cell (arrows) in zebrafish but not in P. reticulata; S100-immunohistochemistry stains crypt cells (arrows) and microvillous cells with short dendrites in the medial layer of the epithelium (arrowheads) in both species. Bar: 20 μm

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