GABAergic modulation of olfactomotor transmission in lampreys.

Odor-guided behaviors, including homing, predator avoidance, or food and mate searching, are ubiquitous in animals. It is only recently that the neural substrate underlying olfactomotor behaviors in vertebrates was uncovered in lampreys. It consists of a neural pathway extending from the medial part of the olfactory bulb (medOB) to locomotor control centers in the brainstem via a single relay in the caudal diencephalon. This hardwired olfactomotor pathway is present throughout life and may be responsible for the olfactory-induced motor behaviors seen at all life stages. We investigated modulatory mechanisms acting on this pathway by conducting anatomical (tract tracing and immunohistochemistry) and physiological (intracellular recordings and calcium imaging) experiments on lamprey brain preparations. We show that the GABAergic circuitry of the olfactory bulb (OB) acts as a gatekeeper of this hardwired sensorimotor pathway. We also demonstrate the presence of a novel olfactomotor pathway that originates in the non-medOB and consists of a projection to the lateral pallium (LPal) that, in turn, projects to the caudal diencephalon and to the mesencephalic locomotor region (MLR). Our results indicate that olfactory inputs can induce behavioral responses by activating brain locomotor centers via two distinct pathways that are strongly modulated by GABA in the OB. The existence of segregated olfactory subsystems in lampreys suggests that the organization of the olfactory system in functional clusters may be a common ancestral trait of vertebrates.

Odorant organization in the olfactory bulb of the sea lamprey.

Olfactory sensory neurons innervate the olfactory bulb, where responses to different odorants generate a chemotopic map of increased neural activity within different bulbar regions. In this study, insight into the basal pattern of neural organization of the vertebrate olfactory bulb was gained by investigating the lamprey. Retrograde labelling established that lateral and dorsal bulbar territories receive the axons of sensory neurons broadly distributed in the main olfactory epithelium and that the medial region receives sensory neuron input only from neurons projecting from the accessory olfactory organ. The response duration for local field potential recordings was similar in the lateral and dorsal regions, and both were longer than medial responses. All three regions responded to amino acid odorants. The dorsal and medial regions, but not the lateral region, responded to steroids. These findings show evidence for olfactory streams in the sea lamprey olfactory bulb: the lateral region responds to amino acids from sensory input in the main olfactory epithelium, the dorsal region responds to steroids (taurocholic acid and pheromones) and to amino acids from sensory input in the main olfactory epithelium, and the medial bulbar region responds to amino acids and steroids stimulating the accessory olfactory organ. These findings indicate that olfactory subsystems are present at the base of vertebrate evolution and that regionality in the lamprey olfactory bulb has some aspects previously seen in other vertebrate species.

Chemical cues and pheromones in the sea lamprey (Petromyzon marinus).

Chemical cues and pheromones guide decisions in organisms throughout the animal kingdom. The neurobiology, function, and evolution of olfaction are particularly well described in insects, and resulting concepts have driven novel approaches to pest control. However, aside from several exceptions, the olfactory biology of vertebrates remains poorly understood. One exception is the sea lamprey (Petromyzon marinus), which relies heavily upon olfaction during reproduction. Here, we provide a broad review of the chemical cues and pheromones used by the sea lamprey during reproduction, including overviews of the sea lamprey olfactory system, chemical cues and pheromones, and potential applications to population management. The critical role of olfaction in mediating the sea lamprey life cycle is evident by a well-developed olfactory system. Sea lamprey use chemical cues and pheromones to identify productive spawning habitat, coordinate spawning behaviors, and avoid risk. Manipulation of olfactory biology offers opportunities for management of populations in the Laurentian Great Lakes, where the sea lamprey is a destructive invader. We suggest that the sea lamprey is a broadly useful organism with which to study vertebrate olfaction because of its simple but well-developed olfactory organ, the dominant role of olfaction in guiding behaviors during reproduction, and the direct implications for vertebrate pest management.

Responses of round goby (Neogobius melanostomus) olfactory epithelium to steroids released by reproductive males.

The wild perciform teleost Neogobius melanostomus (the round goby) originated from the Ponto-Caspian region and is now a highly successful invasive species in the Laurentian Great Lakes. Males may attract females into their nests for spawning by releasing reproductive pheromones, and it has been previously shown that reproductive males synthesize and release the 5ß-reduced and 3α-hydroxyl steroids 3α-hydroxy-5ß-androstane-11,17-dione (11-oxo-etiocholanolone; 11-O-ETIO) and 3α-hydroxy-5ß-androstane-11,17-dione 3-sulfate (11-oxo-etiocholanolone-3-sulfate; 11-O-ETIO-3-s) and 3α,17ß-dihydroxy-5ß-androstan-11-one 17-sulfate. In this study, we investigated properties of these released steroids by recording field potential responses from the olfactory epithelium (electro-olfactogram, EOG). The steroid 3α,17ß-dihydroxy-5ß-androstan-11-one 17-sulfate did not elicit olfactory responses while both 11-O-ETIO and 11-O-ETIO-3-s stimulated olfactory field potentials in the round goby, but not in the goldfish. Cross-adaptation analysis demonstrated that round gobies discriminated between11-O-ETIO and 11-O-ETIO-3-s (as well as etiocholanolone, ETIO) at the sensory level. Second messenger cascades depending on both cAMP and IP(3) were inferred for steroids from pharmacological inhibition studies, while the canonical teleost odors taurocholic acid (a bile acid) and L: -alanine (an amino acid) used only cAMP and IP(3), respectively. The round goby presents itself as an excellent species for the study of olfactory function of fish in the wild, given its possible use of these released steroids as pheromones.

The effect of elevated steroids released by reproductive male round gobies, Neogobius melanostomus, on olfactory responses in females.

The round goby, Neogobius melanostomus, is a highly successful invasive species in the Laurentian Great Lakes. Previous behavioral studies implied that females are attracted by pheromones to the nests of reproductive males, and that males release putative steroidal pheromones--unconjugated as well as conjugated forms of 3α-hydroxy-5ß-androstane-11,17-dione (11-O-ETIO)-following stimulation of the hypothalamic--gonadal axis with salmon gonadotropin releasing hormone analog (sGnRHa). In this study, we tested the olfactory system of females in response to extracts containing these released steroids. We compared electrical field potential responses from the olfactory epithelium (electro-olfactogram, EOG) of non-reproductive females to methanol extracts of water that previously held males, collected before and after injection of the males with sGnRHa or saline. The females showed increased EOG responses to the post-injection extracts when males were treated with sGnRHa but not saline. This finding provides further evidence for interactions between male and female N. melanostomus via steroidal reproductive pheromones.

Sensory cutaneous papillae in the sea lamprey (Petromyzon marinus L.): II. Ontogeny and immunocytochemical characterization of solitary chemosensory cells.

Solitary chemosensory cells (SCCs) and their innervating fibers are located in the respiratory system of many vertebrates, including papillae on lamprey gill pores. In order to gain stronger insight for the role of these chemosensory cells, we examined immunocytochemical and innervation characteristics, as well as abundance at the different stages of the lamprey life cycle. The SCCs were distinguished from the surrounding epithelial cells by calretinin and phospholipase C140 immunoreactivity. Nerve fibers extended into the gill pore papillae, as far as the SCCs and serotonergic fibers extended from the underlying dermis into the papillar base. Gill pore papillae were absent and SCCs were sparse during the larval stage and in newly transformed lamprey. Few SCCs were located on small nub-like papillae during the parasitic juvenile stage, but SCCs were abundant on prominent papillae in migrating and in spawning adults. These findings show similarities between the SCCs in lampreys and other vertebrates and suggest that gill SCC function may be important during the feeding juvenile and the adult stages of the lamprey life cycle.

Multi-glomerular projection of single olfactory receptor neurons is conserved among amphibians.

Individual receptor neurons in the peripheral olfactory organ extend long axons into the olfactory bulb forming synapses with projection neurons in spherical neuropil regions, called glomeruli. Generally, odor map formation and odor processing in all vertebrates is based on the assumption that receptor neuron axons exclusively connect to a single glomerulus without any axonal branching. We comparatively tested this hypothesis in multiple fish and amphibian species (both sexes) by applying sparse cell electroporation to trace single olfactory receptor neuron axons. Sea lamprey (jawless fish) and zebrafish (bony fish) support the unbranched axon concept, with 94% of axons terminating in single glomeruli. Contrastingly, axonal projections of the axolotl (salamander) branch extensively before entering up to six distinct glomeruli. Receptor neuron axons labeled in frog species (Pipidae, Bufonidae, Hylidae, and Dendrobatidae) predominantly bifurcate before entering a glomerulus and 59 and 50% connect to multiple glomeruli in larval and postmetamorphotic animals, respectively. Independent of developmental stage, lifestyle, and adaptations to specific habitats, it seems to be a common feature of amphibian olfactory receptor neuron axons to frequently bifurcate and connect to multiple glomeruli. Our study challenges the unbranched axon concept as a universal vertebrate feature and it is conceivable that also later diverging vertebrates deviate from it. We propose that this unusual wiring logic evolved around the divergence of the terrestrial tetrapod lineage from its aquatic ancestors and could be the basis of an alternative way of odor processing.

Organization of glomerular territories in the olfactory bulb of post-embryonic wild chinook salmon Oncorhynchus tshawytscha.

The post-embryonic odor imprinting paradigm suggests Chinook salmon (Oncorhynchus tshawytscha) acquire memory to stream-specific amino acid olfactory odors prior to emergence as fry. Because effects of olfactory experience on development can be examined by mapping olfactory sensory neurons extending into distinct territories of glomerular neuropil in the olfactory bulb, glomerular patterning from early yolk-sac larva to fry was documented in wild salmonids, a temporal scale not yet thoroughly explored. Labeling olfactory sensory neurons with anti-keyhole limpet hemocyanin (anti-KLH) revealed seven spatially conserved glomerular territories visible at hatch and well established by the late yolk-sac larva developmental stage. Because of the responsiveness of microvillous olfactory sensory neurons to amino acids, corresponding glomeruli in the lateral bulbar region were mapped using anti-calretinin. The dorsolateral territory, distinct glomeruli of the lateral glomerular territory and the ventromedial glomeruli were immunoreactive to both KLH and calretinin. This study offers a morphological description of glomerular patterning in post-embryonic stages in wild Chinook salmon, a temporal window previously shown to be significant for olfactory imprinting. J. Morphol. 278:464-474, 2017. © 2017 Wiley Periodicals, Inc.

Sensory Activation of Command Cells for Locomotion and Modulatory Mechanisms: Lessons from Lampreys.

Sensorimotor transformation is one of the most fundamental and ubiquitous functions of the central nervous system (CNS). Although the general organization of the locomotor neural circuitry is relatively well understood, less is known about its activation by sensory inputs and its modulation. Utilizing the lamprey model, a detailed understanding of sensorimotor integration in vertebrates is emerging. In this article, we explore how the vertebrate CNS integrates sensory signals to generate motor behavior by examining the pathways and neural mechanisms involved in the transformation of cutaneous and olfactory inputs into motor output in the lamprey. We then review how 5-hydroxytryptamine (5-HT) acts on these systems by modulating both sensory inputs and motor output. A comprehensive review of this fundamental topic should provide a useful framework in the fields of motor control, sensorimotor integration and neuromodulation.

Glomerular territories in the olfactory bulb from the larval stage of the sea lamprey Petromyzon marinus.

The goal of this study was to investigate the spatial organization of olfactory glomeruli and of substances relevant to olfactory sensory neuron activity in the developing agnathan, the sea lamprey Petromyzon marinus. A 45-kD protein immunoreactive to G(olf), a cAMP-dependent olfactory G protein, was present in the ciliary fraction of sea lamprey olfactory epithelium and in olfactory sensory neurons of larval and adult sea lampreys. This result implies that G(olf) expression was present during early vertebrate evolution or evolved in parallel in gnathostome and agnathostome vertebrates. Serial sectioning of the olfactory bulb revealed a consistent pattern of olfactory glomeruli stained by GS1B(4) lectin and by anterograde labeling with fluorescent dextran. These glomerular territories included the dorsal cluster, dorsal ring, anterior plexus, lateral chain, medial glomeruli, ventral ring, and ventral cluster. The dorsal, anterior, lateral, and ventral glomeruli contained olfactory sensory axon terminals that were G(olf)-immunoreactive. However, a specific subset, the medial glomeruli, did not display this immunoreactivity. Olfactory glomeruli in the dorsal hemisphere of the olfactory bulb, the dorsal cluster, dorsal ring, anterior plexus, lateral chain, and medial glomeruli, were seen adjacent to 5HT-immunoreactive fibers. However, glomeruli in the ventral hemisphere, the ventral ring, and ventral cluster did not display this association. The presence of specific glomerular territories and discrete glomerular subsets with substances relevant to olfactory sensory neuron activity suggest a spatial organization of information flow in the lamprey olfactory pathway.

Morphology and histochemistry of the peripheral olfactory organ in the round goby, Neogobius melanostomus (Teleostei: Gobiidae).

This first comprehensive study of the peripheral olfactory organ from a representative of the large and economically important order of teleost fishes, the Perciformes, shows a compact structure with olfactory sensory neurons distributed widely throughout the olfactory chamber. The spatial organization of the nasal cavity in the bottom-dwelling round goby (Gobiidae, Neogobius melanostomus) was examined using impression material injection, immunocytochemistry, and transmission electron microscopy. The olfactory chamber contains a single olfactory lamella; prominent dorsocaudal lachrymal and ethmoidal accessory nasal sacs are situated ventrocaudal to the chamber. The location of the olfactory mucosa within the olfactory chamber is novel for teleost fish, as it extends beyond the ventral surface to the lateral and dorsal regions. Microvillar olfactory sensory neurons and ciliated olfactory sensory neurons were identified by transmission electron microscopy and the spatial distribution of these two cell types was assessed through immunocytochemistry against olfactory receptor coupled G-proteins. Both G(alphaolf)-immunoreactive ciliated olfactory sensory neurons and the G(alphao)-immunoreactive microvillar form were located throughout the olfactory epithelium. Ciliated crypt cells were G(alphao) immunoreactive and were found throughout the olfactory epithelium of some specimens. The widespread occurrence of olfactory sensory neurons in the olfactory chamber supports the idea that olfactory signaling is important to the survival of the round goby. The prominence of the lachrymal and ethmoidal accessory nasal sacs indicates the capacity to regulate the flow of odorant molecules over the sensory surface of the olfactory sensory neurons, possibly through a pump-like mechanism driven by opercular activity associated with gill ventilation.

The neuroanatomical organization of projection neurons associated with different olfactory bulb pathways in the sea lamprey, Petromyzon marinus.

Although there is abundant evidence for segregated processing in the olfactory system across vertebrate taxa, the spatial relationship between the second order projection neurons (PNs) of olfactory subsystems connecting sensory input to higher brain structures is less clear. In the sea lamprey, there is tight coupling between olfaction and locomotion via PNs extending to the posterior tuberculum from the medial region of the olfactory bulb. This medial region receives peripheral input predominantly from the accessory olfactory organ. However, the axons from olfactory sensory neurons residing in the main olfactory epithelium extend to non-medial regions of the olfactory bulb, and the non-medial bulbar PNs extend their axons to the lateral pallium. It is not known if the receptive fields of the PNs in the two output pathways overlap; nor has the morphology of these PNs been investigated. In this study, retrograde labelling was utilized to investigate the PNs belonging to medial and non-medial projections. The dendrites and somata of the medial PNs were confined to medial glomerular neuropil, and dendrites of non-medial PNs did not enter this territory. The cell bodies and dendrites of the non-medial PNs were predominantly located below the glomeruli (frequently deeper in the olfactory bulb). While PNs in both locations contained single or multiple primary dendrites, the somal size was greater for medial than for non-medial PNs. When considered with the evidence-to-date, this study shows different neuroanatomical organization for medial olfactory bulb PNs extending to locomotor control centers and non-medial PNs extending to the lateral pallium in this vertebrate.

Chemosensory-induced motor behaviors in fish.

Chemical sensory signals play a crucial role in eliciting motor behaviors. We now review the different motor behaviors induced by chemosensory stimuli in fish as well as their neural substrate. A great deal of research has focused on migratory, reproductive, foraging, and escape behaviors but it is only recently that the molecules mediating these chemotactic responses have become well-characterized. Chemotactic responses are mediated by three sensory systems: olfactory, gustatory, and diffuse chemosensory. The olfactory sensory neuron responses to chemicals are now better understood. In addition, the olfactory projections to the central nervous system were recently shown to display an odotopic organization in the forebrain. Moreover, a specific downward projection underlying motor responses to olfactory inputs was recently described.

Evidence for behavioral preference toward environmental concentrations of urban-use herbicides in a model adult fish.

Fish live in waters of contaminant flux. In three urban, fish-bearing waterways of British Columbia, Canada, we found the active ingredients of WeedEx, KillEx, and Roundup herbicide formulations (2,4-D, dicamba, glyphosate, and mecoprop) at low to high ng/L concentrations (0.26 to 309 ng/L) in routine conditions, i.e., no rain for at least one week. Following rain, these concentrations increased by an average of eightfold, suggesting runoff as a major route of herbicide introduction in these waterways. To determine whether fish might be able to limit point-source exposures through sensory-driven behaviors, we introduced pulses of representative herbicide mixtures to individual adult zebrafish (a model species) in flow-through tanks. Fish did the opposite of limit exposure; they chose to spend more time in pulses of herbicide mixtures representative of those that may occur with rain events. This attraction response was not altered by a previous 4-d exposure to lower concentrations of the mixtures, suggesting fish will not learn from previous exposures. However, previous exposures did alter an attraction response to an amino acid prevalent in food (L-alanine). The present study demonstrates that fish living within urban waterways may elect to place themselves in herbicide-contaminated environments and that these exposures may alter their behavioral responses to cues necessary for survival.

Differential behavioral responses by reproductive and non-reproductive male round gobies (Neogobius melanostomus) to the putative pheromone estrone.

Previous studies have shown that the frequency of gill ventilation during exposure to estrone and gonadal extracts in the round goby (Neogobius melanostomus) is linked to olfactory sensory input. Control over gill ventilation may be a regulatory mechanism used for odorant sampling during reproductive periods. In this study, we examined changes in gill ventilation in osmic and anosmic (nasal occluded), reproductive and non-reproductive male round gobies to a putative steroidal pheromone estrone (1,3,5(10)-estratrien-3-ol-17-one). We tested 5 different concentrations of estrone (10(-12) to 10(-8) M) and showed that the response threshold for estrone varied with the male's reproductive status; it was 10(-11) M in reproductive males, and rose to 10(-9) M in non-reproductive males. However, anosmic reproductive and non-reproductive males did not respond to estrone. These findings suggest that olfactory responses to putative pheromones may change depending on the reproductive status of the fish.

Olfactory sensory input increases gill ventilation in male round gobies (Neogobius melanostomus) during exposure to steroids.

In teleostean fish, ventilation increases have been observed in response to low dissolved oxygen levels, visual stimuli, and gustatory cues. However, olfactory sensory input may also stimulate gill ventilation rate. We investigated whether olfactory sensory input mediates gill ventilation responses, as suggested by the observation that steroidal compounds detected by the olfactory system elicited increases in opercular activity in the perciform teleost, the round goby (Neogobius melanostomus). Close parallels between gill ventilation and olfactory responses, led us to conduct an empirical study that used two different olfactory sensory deprivation techniques to seek a causal relationship between olfactory epithelial activity and hyperventilation. Chemical lesion of olfactory sensory neurons or mechanical occlusion of the nasal cavities inhibited gill ventilation responses of reproductive male round gobies to estrone (1,3,5(10)-estratrien-3-ol-17-one) and to ovarian extracts. This direct evidence demonstrates the role of olfactory sensory input for the gill ventilation response to putative reproductive pheromones and may represent an important regulatory mechanism for odorant sampling during pheromone communication.

Diversity in the olfactory epithelium of bony fishes: development, lamellar arrangement, sensory neuron cell types and transduction components.

In this study we use a taxon-based approach to examine previous, as well as new findings on several topics pertaining to the peripheral olfactory components in teleost fishes. These topics comprise (1) the gross anatomy of the peripheral olfactory organ, including olfactory sensory neuron subtypes and their functional parameters, (2) the ultrastructure of the olfactory epithelium, and (3) recent findings regarding the development of the nasal cavity and the olfactory epithelium. The teleosts are living ray-finned fish, and include descendants of early-diverging orders (e.g., salmon), specialized descendants (e.g., goldfish and zebrafish), as well as the Acanthopterygii, numerous species with sharp bony rays, including perch, stickleback, bass and tuna. Our survey reveals that the olfactory epithelium lines a multi-lamellar olfactory rosette in many teleosts. In Acanthopterygii, there are also examples of flat, single, double or triple folded olfactory epithelia. Diverse species ventilate the olfactory chamber with a single accessory nasal sac, whereas the presence of two sacs is confined to species within the Acanthopterygii. Recent studies in salmonids and cyprinids have shown that both ciliated olfactory sensory neurons (OSNs) and microvillous OSNs respond to amino acid odorants. Bile acids stimulate ciliated OSNs, and nucleotides activate microvillous OSNs. G-protein coupled odorant receptor molecules (OR-, V1R-, and V2R-types) have been identified in several teleost species. Ciliated OSNs express the G-protein subunit G(alphaolf/s), which activates cyclic AMP during transduction. Localization of G protein subunits G(alpha0) and G(alphaq/11) to microvillous or crypt OSNs, varies among different species. All teleost species appear to have microvillous and ciliated OSNs. The recently discovered crypt OSN is likewise found broadly. There is surprising diversity during ontogeny. In some species, OSNs and supporting cells derive from placodal cells; in others, supporting cells develop from epithelial (skin) cells. In some, epithelial cells covering the developing olfactory epithelium degenerate, in others, these retract. Likewise, there are different mechanisms for nostril formation. We conclude that there is considerable diversity in gross anatomy and development of the peripheral olfactory organ in teleosts, yet conservation of olfactory sensory neuron morphology. There is not sufficient information to draw conclusions regarding the diversity of teleost olfactory receptors or transduction cascades.

Morphological and electrophysiological examination of olfactory sensory neurons during the early developmental prolarval stage of the sea lamprey Petromyzon marinus L.

This study examined olfactory sensory neuron morphology and physiological responsiveness in newly hatched sea lamprey, Petromyzon marinus L. These prolarvae hatch shortly after neural tube formation, and stay within nests for approximately 18 days, before moving downstream to silty areas where they burrow, feed and pass to the larval stage. To explore the possibility that the olfactory system is functioning during this prolarval stage, morphological and physiological development of olfactory sensory neurons was examined. The nasal cavity contained an olfactory epithelium with ciliated olfactory sensory neurons. Axons formed aggregates in the basal portion of the olfactory epithelium and spanned the narrow distance between the olfactory epithelium and the brain. The presence of asymmetric synapses with agranular vesicles within fibers in the brain, adjacent to the olfactory epithelium suggests that there was synaptic connectivity between olfactory sensory axons and the brain. Neural recordings from the surface of the olfactory epithelium showed responses following the application of L-arginine, taurocholic acid, petromyzonol sulfate (a lamprey migratory pheromone), and water conditioned by conspecifics. These results suggest that lampreys may respond to olfactory sensory input during the prolarval stage.

In vitro biosynthesis of novel 5beta-reduced steroids by the testis of the round goby, Neogobius melanostomus.

Previous studies indicate that, in the round goby Neogobius melanostomus, the reproductively mature male releases a pheromone that attracts ripe females. Furthermore, studies suggest that the pheromone may be a steroid (more specifically a 5beta-reduced androgen) produced by specialized glandular tissue in the testes. In the present study, it is shown that the testis of the male round goby contains such specialized glandular tissue. In vitro, the testes convert [3H]androstenedione into 3alpha-hydroxy-5beta-androstane-11,17-dione (i.e., 11-oxo-etiocholanolone, 11-oxo-ETIO); 11-oxo-ETIO sulfate (11-oxo-ETIO-s); 11-oxo-testosterone (i.e., 11-ketotestosterone), 3alpha-hydroxy-5beta-androstan-17-one (etiocholanolone, ETIO); 11beta-hydroxy-androstenedione; ETIO sulfate and testosterone. Glucuronidated steroids were not identified. Neither 11-oxo-ETIO nor 11-oxo-ETIO-s has previously been identified in teleost gonads. Both these steroids are formed in the round goby testis even when [3H]17-hydroxyprogesterone is used as a precursor. The fact that, for both steroids, the carbon A ring has a 5beta-configuration (already linked with olfactory sensitivity and behavior induction in two other species of gobies) makes them likely candidate pheromones in the round goby. However, their in vivo production and pheromonal activity remain to be proved.

Metamorphosis of the olfactory organ of the sea lamprey (Petromyzon marinus L.): Morphological changes and morphometric analysis.

In larval sea lampreys (Petromyzon marinus), a small, relatively inconspicuous olfactory organ sac contains small, densely packed olfactory receptor neurons and sustentacular cells. During metamorphosis, the larval organ transforms into a prominent lamellar structure with large distinct olfactory epithelial cells that is characteristic of the adult lamprey. In the present study, scanning electron microscopy and light microscopy are used to examine changes during the seven stages (1-7) of metamorphosis. The magnitude of growth over the course of metamorphosis is evident from the doubling of the relative weight of the nasal sac. During early metamorphosis (stages 1 and 2), the larval olfactory organ enlarges, and by stage 3 specific adult structures begin to form, namely a nasal valve between the nasal tube and the organ, lamellar folds, and diverticuli of the accessory olfactory organ. Subsequent development involves widening of the cells lining the lamellar folds to the form characteristic of postmetamorphic lampreys. Although the cells in the troughs initially retain numerical density values that are significantly higher than those on the lamellar surfaces, by stage 7 values decline both in troughs and along lamellar surfaces to those observed in adults. These results show that although expansion of the olfactory organ is ongoing throughout metamorphosis, remodeling occurs early (by stage 3). This timing provides space for extensive olfactory receptor neuron neurogenesis and differentiation and correlates with the transformation of some organs that were previously examined. This is the first report in any species of olfactory receptor neuron zonation based on morphometric characteristics. J. Morphol. 231:41-52, 1997. © 1997 Wiley-Liss, Inc.