Discrimination of conspecific sex and reproductive condition using chemical cues in axolotls ( Ambystoma mexicanum).

Chemosensory cues play an important role in the daily lives of salamanders, mediating foraging, conspecific recognition, and territorial advertising. We investigated the behavioral effects of conspecific whole-body odorants in axolotls, Ambystoma mexicanum, a salamander species that is fully aquatic. We found that males increased general activity when exposed to female odorants, but that activity levels in females were not affected by conspecific odorants. Although males showed no difference in courtship displays across testing conditions, females performed courtship displays only in response to male odorants. We also found that electro-olfactogram responses from the olfactory and vomeronasal epithelia were larger in response to whole-body odorants from the opposite sex than from the same sex. In males, odorants from gravid and recently spawned females evoked different electro-olfactogram responses at some locations in the olfactory and vomeronasal epithelia; in general, however, few consistent differences between the olfactory and vomeronasal epithelia were observed. Finally, post hoc analyses indicate that experience with opposite-sex conspecifics affects some behavioral and electrophysiological responses. Overall, our data indicate that chemical cues from conspecifics affect general activity and courtship behavior in axolotls, and that both the olfactory and vomeronasal systems may be involved in discriminating the sex and reproductive condition of conspecifics.

Presence of the vomeronasal system in aquatic salamanders.

Previous reports have indicated that members of the proteid family of salamanders lack a vomeronasal system, and this absence has been interpreted as representing the ancestral condition for aquatic amphibians. I examined the anatomy of the nasal cavities, nasal epithelia, and forebrains of members of the proteid family, mudpuppies (Necturus maculosus), as well as members of the amphiumid and sirenid families (Amphiuma tridactylum and Siren intermedia). Using a combination of light and transmission electron microscopy, I found no evidence that mudpuppies possess a vomeronasal system, but found that amphiuma and sirens possess both vomeronasal and olfactory systems. Amphiumids and sirenids are considered to be outgroups relative to proteids; therefore, these data indicate that the vomeronasal system is generally present in salamanders and has been lost in mudpuppies. Given that the vomeronasal system is generally present in aquatic amphibians, and that the last common ancestor of amphibians and amniotes is believed to have been fully aquatic, I conclude that the vomeronasal system arose in aquatic tetrapods and did not originate as an adaptation to terrestrial life. This conclusion has important implications for the hypothesis that the vomeronasal organ is specialized for detection of non-volatile compounds.

Neuromodulatory effects of gonadotropin releasing hormone on olfactory receptor neurons.

The terminal nerve is an anterior cranial nerve that innervates the lamina propria of the chemosensory epithelia of the nasal cavity. The function of the terminal nerve is ambiguous, but it has been suggested to serve a neuromodulatory role. We tested this hypothesis by exposing olfactory receptor neurons from mudpuppies (Necturus maculosus) to a peptide, gonadotropin releasing hormone (GnRH), that is found in cells and fibers of the terminal nerve. We used voltage-clamped whole-cell recordings to examine the effects of 0. 5-50 micrometer GnRH on voltage-activated currents in olfactory receptor neurons from epithelial slices. We found that GnRH increases the magnitude, but does not alter the kinetics, of a tetrodotoxin-sensitive inward current. This increase in magnitude generally begins 5-10 min after initial exposure to GnRH, is sustained for at least 60 min during GnRH exposure, and recovers to baseline within 5 min after GnRH is washed off. This effect occurred in almost 60% of the total number of olfactory receptor neurons examined and appeared to be seasonal: approximately 67% of neurons responded to GnRH during the courtship and mating season, compared with approximately 33% during the summer, when the sexes separate. GnRH also appears to alter an outward current in the same cells. Taken together, these data suggest that GnRH increases the excitability of olfactory receptor neurons and that the terminal nerve functions to modulate the odorant sensitivity of olfactory receptor neurons.

Evolution of vertebrate olfactory systems.

The general features of the olfactory system are remarkably consistent across vertebrates. A phylogenetic analysis of central olfactory projections indicates that at least three distinct olfactory subsystems may be broadly present in vertebrates and that a fourth, the accessory olfactory or vomeronasal system, arose in tetrapods. The origin and function of the vomeronasal system have been the subject of much controversy, but some conclusions can be drawn. The vomeronasal system did not arise as an adaptation to terrestrial life, as indicated by the presence of a vomeronasal system in modern aquatic amphibians and the increasing paleontological evidence that the last common ancestor of amphibians and amniotes was aquatic. The vomeronasal system is involved in both foraging and reproductive behaviors in reptiles and has been shown to be involved in some pheromonally mediated behaviors in mammals. However, among mammals, some pheromonal responses are not mediated by the vomeronasal system, and the possible involvement of the vomeronasal system in other type of behaviors has not yet been investigated. Thus, the relative functions of the olfactory and vomeronasal systems of tetrapods remain unclear. Other hypotheses that features of the olfactory system are specialized for aquatic chemoreception or for pheromone detection are similarly insupportable. For example, the suggestion that members of the olfactory receptor family can be separated into two groups that function for transduction of air-borne or water-borne odorants is contradicted by the presence of both groups in aquatic amphibians and by a phylogenetic analysis of the sequences for these genes. Interestingly, the putative odorant receptors from the vomeronasal epithelium share little sequence similarity with those from the olfactory epithelium, indicating that these receptors may have been independently co-opted from the larger family of seven transmembrane domain receptors for use in odor transduction. A phylogenetic analysis of the distribution of olfactory receptor cell types indicates that microvillar olfactory receptor cells are widespread among vertebrates and are not restricted to aquatic animals or to the vomeronasal epithelium of tetrapods. Previous suggestions that all microvillar receptor cells are specialized for the detection of pheromones are not tenable. Attempts to recognize features of the olfactory system that are common to all vertebrates and might be specialized for the detection of pheromones vs. more general odorants, or for the detection of water-borne vs. air-borne odorants, are not supported by current evidence.

Silver lampreys (Ichthyomyzon unicuspis) lack a gonadotropin-releasing hormone- and FMRFamide-immunoreactive terminal nerve.

The terminal nerve is a ganglionated cranial nerve with peripheral processes that enter the nasal cavity and centrally directed processes that enter the forebrain. Members of all classes of gnathostomes have been found to possess a terminal nerve, some components of which demonstrate immunoreactivity to the peptides Phe-Met-Arg-Phe-NH2 (FMRFamide) and gonadotropin-releasing hormone (GnRH). To explore the possibility that lampreys possess a terminal nerve, we examined the distribution of these peptides in the silver lamprey, Ichthyomyzon unicuspis, by using antisera to FMRFamide and to four forms of GnRH. We found cells with FMRFamide-like immunoreactivity in the preoptic area and the isthmal gray region of the mesencephalon, and found labeled fibers throughout the preoptic-infundibular region. Occasional labeled fibers were scattered through many regions of the brain, including the optic nerve and olfactory bulb; however, unlike species that possess a terminal nerve, lampreys have no immunoreactive cells or fibers in the olfactory nerve or nasal epithelia. In addition, we observed GnRH-immunoreactive cell bodies in the preoptic area of all animals and in the ventral hypothalamus of one individual. Most of the labeled fibers extended ventrally to the hypothalamus, with other fibers extending throughout the striatum and hypothalamic-neurohypophyseal region. A few fibers in other regions, including the optic nerve, were also labeled; we detected no immunoreactivity in the olfactory bulb, olfactory nerve, or nasal epithelia. The use of different GnRH antisera resulted in remarkably similar patterns of labeling of both cells and fibers. In summary, we did not observe either GnRH or FMRFamide-like immunoreactivity in the olfactory regions that represent the typical path of terminal nerve fibers, nor were we able to locate a terminal nerve ganglion. We conclude that lampreys may lack a terminal nerve, and that the previously described fiber bundle extending from the nasal sac to the ventral forebrain may constitute an extra-bulbar olfactory pathway.

Anatomy and forebrain projections of the olfactory and vomeronasal organs in axolotls (Ambystoma mexicanum).

We examined the anatomy of the nasal cavity and forebrain in the axolotl (Ambystoma mexicanum) to determine whether the olfactory and vomeronasal systems are present in this neotenic aquatic salamander. The current study was motivated by two considerations: (a) little is known of the anatomy of the vomeronasal system in aquatic vertebrates, and (b) the presence of both olfactory and vomeronasal systems in larval amphibians has broad implications for the evaluation of these systems in vertebrates. From cresyl-violet-stained sections of snouts we determined that the nasal cavity of axolotls is much like that of terrestrial salamanders. The main chamber of the nasal cavity contains an olfactory epithelium, which is confined to grooves between longitudinal ridges of connective tissue covered in a nonsensory epithelium which lacks goblet cells. Using transmission electron microscopy, we found morphologically distinct olfactory receptor cells: many receptor cells terminate in microvillar dendrites, and fewer terminate in motile cilia with the 9 + 2 microtubule array typical of vertebrate olfactory receptor cells. The ciliated and microvillar cells occur in clusters with little intermingling. Horseradish peroxidase labeling revealed that axons of the olfactory receptor cells terminate in large glomeruli in the main olfactory bulb at the rostral end of the telencephalon. Lateral to the main chamber of the nasal cavity is a diverticulum that is entirely lined with a vomeronasal epithelium containing basal cells, microvillar receptor cells, sustentacular cells that lack specialized processes on the apical surface, and large ciliated cells that may function to move fluid across the vomeronasal epithelium. Unlike the olfactory epithelium, the vomeronasal epithelium lacks Bowman's glands. Using horseradish peroxidase, we determined that the axons of the vomeronasal receptor cells project to the accessory olfactory bulb, a distinct structure dorsal and caudal to the main olfactory bulb. The presence of both olfactory and vomeronasal systems in axolotls and other neotenic salamanders implies that both systems are pleiomorphic in larval amphibians; we therefore suggest that the vomeronasal system may not have originated as an adaptation to terrestrial life.

Phylogeny of the vomeronasal system and of receptor cell types in the olfactory and vomeronasal epithelia of vertebrates.

In this paper, the evolutionary origin of the vomeronasal system as a discrete sensory system separate from olfaction is examined. The presence of a discrete vomeronasal system appears to be a derived character in tetrapods, and its presence in larval amphibians indicates that the system did not arise as a terrestrial adaptation. The vomeronasal system has been lost independently in several taxa, including crocodilians, some bats, cetaceans, and some primates. The presence of microvillar receptor cells in the vomeronasal epithelium appears to be the ancestral condition for tetrapods, and alternative hypotheses concerning the ancestral condition for receptor cell types in the vertebrate olfactory epithelium are discussed. Finally, the possibility that the vomeronasal system is present in some fishes in a form that has not been recognized is discussed in relation to the phylogenetic distribution of receptor cell types in vertebrates.

Chemosensory conditioning of Hermissenda crassicornis.

Bite-strike responses of Hermissenda crassicornis, elicited by chemosensory stimulation of the lips, were found to be modified when food extracts were paired with rotation-produced stimulation of the statocysts. Animals that received repeated pairings of an extract of 1 food (conditioned stimulus, CS) with rotation exhibited suppressed bite-strike responses to that food for up to 48 hr after training. This suppression was usually specific to the trained food and was pairing-specific as well. Discriminative conditioning was also demonstrated. Animals trained with 1 CS paired with rotation and a second CS that was unpaired (CS-) showed suppressed bite-strike responses to the first CS. The results demonstrate that Hermissenda can learn to avoid foods that reliably signal an aversive event and may allow an analysis of higher order conditioning phenomena.

Behavioral responses of male guinea pigs to conspecific chemical signals following neonatal vomeronasal organ removal.

Following vomeronasal organ removal or sham surgery at 4-7 days of age, male guinea pigs were tested for responsiveness to conspecific chemical uses as infants and again as adults. In the first experiment, vocalizations in response to soiled home cage bedding and male bedding were monitored twice prior to surgery and twice weekly for four weeks. Home cage cues elicited more vocalizations than did male bedding in both groups; however, there was no effect of vomeronasal organ removal. When tested as adults in a second experiment, animals without vomeronasal organs exhibited depressed investigative responsiveness and vocalizations to female genital smears. The data from the first experiment fail to indicate a role for the vomeronasal organ in infantile response to conspecific odor. However, the second experiment demonstrates that adult responses to similar odors are substantially depressed by an absence of the vomeronasal organ.