Glomerular Territories in the Olfactory Bulb from the Larval Stage of the Sea Lamprey Petromyzon marinus

Transcription

1 THE JOURNAL OF COMPARATIVE NEUROLOGY 465:27 37 (2003) Glomerular Territories in the Olfactory Bulb from the Larval Stage of the Sea Lamprey Petromyzon marinus ANDREA FRONTINI, 1 ALIYA U. ZAIDI, 1 HONG HUA, 1 TOMASZ P. WOLAK, 1 CHARLES A. GREER, 2 KARL W. KAFITZ, 3 WEIMING LI, 4 AND BARBARA S. ZIELINSKI 1 * 1 Department of Biological Sciences, University of Windsor, Windsor, Ontario N9B 3P4, Canada 2 Sections of Neurosurgery & Neurobiology, Yale University School of Medicine, New Haven, Connecticut Pysiologisches Institut, Zellulaere Physiologie, Ludwig-Maximilians-Universitaet Muenchen, Muenchen, Germany 4 Department of Fisheries and Wildlife, Michigan State University, East Lansing, Michigan ABSTRACT 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 campdependent 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. J. Comp. Neurol. 465:27 37, Wiley-Liss, Inc. Indexing terms: Agnatha; olfactory sensory neurons; serotonin; G olf Grant sponsor: Natural Sciences and Engineering Research Council of Canada; Grant number: (B.Z.): Grant number: PG-S1 (H.H.); Grant sponsor: Ontario Council on Graduate Studies (A.Z.); Grant sponsor: the Great Lakes Fishery Commission (W.L., B.Z.); Grant sponsor: National Institutes of Health; Grant number: DC002210; Grant number: NS10174 (C.A.G.); Grant sponsor: the University of Windsor Faculty of Graduate Studies (A.F., A.Z., H.H.). *Correspondence to: Barbara S. Zielinski, Department of Biological Sciences, University of Windsor, Windsor, Ontario N9B 3P4, Canada. zielin1@uwindsor.ca Received 25 July 2002; Revised 14 February 2003; Accepted 7 April 2003 DOI /cne Published online the week of August 18, 2003 in Wiley InterScience ( WILEY-LISS, INC.

2 28 A. FRONTINI ET AL. In all vertebrates, from agnathans to primates, axons of olfactory sensory neurons (OSNs) project from soma in the olfactory epithelium to olfactory bulb glomeruli (Iwahori et al., 1987; Halasz and Greer, 1993; Klenoff and Greer, 1998; Lin and Ngai, 1999). Although functional differences in OSNs that terminate in specific glomerular territories have been observed, glomerular organizing principles are not fully understood (Hildebrand and Shepherd, 1997; Xu et al., 2000). The arrangement of OSN axon terminals into glomerular units may be affected by transduction mechanisms fundamental to OSN function and by modulatory influences from nonolfactory neuronal fibers adjacent to OSN axons. The camp-dependent olfactory GTP-binding protein linked to olfactory receptors (Jones and Reed, 1989), G olf, is a constituent of olfactory sensory transduction and a requisite for olfactory responses in mammals (Belluscio et al., 1998). Because OSN subpopulations in amphibians (Mezler et al., 2001) and teleosts (Hansen et al., 2001) express G olf, this G s subunit may have appeared early in vertebrate evolution. Alternate G-proteins are found in vomeronasal sensory neurons (Jia and Halpern, 1996) and in OSN subpopulations of amphibians (Mezler et al., 2001) and teleosts (Hansen et al., 2001). If G olf is a vital component for vertebrate OSN function, its localization in the olfactory organ of the sea lamprey, an ancestral jawless fish, is expected. If transduction by alternate G-proteins is also a fundamental character or if it evolved in parallel (Eisthen, 2002), clearly defined subpopulations of OSNs that do not express G olf should be present in the lamprey. Previous studies have provided evidence that OSN terminals are distributed according to functional parameters. In mammals, axons of OSNs expressing putative odorant receptors within an epithelial zone converge to spatially conserved glomeruli (Mombaerts, 1996). In addition, OSNs containing elements of a cgmp signal transduction pathway project to specific glomeruli (Juilfs et al., 1997) and identified odorants stimulate synaptic activity in specific glomeruli (e.g., Wachowiak and Cohen, 2001). In zebrafish (Teleostei, Cypriniformes), olfactory glomeruli are arranged in a stereotyped configuration (Baier and Korsching, 1994) and glomerular responsiveness to odor stimulation follows spatial patterning (Friedrich and Korsching, 1998). In catfish (Teleostei, Siluriformes), two OSN subpopulations project axons to specific regions of the olfactory bulb (Morita and Finger, 1998), and in the lamprey, Lampetra fluviatilis, calretinin-immunoreactive OSNs extend to particular glomerular locations (Pombal et al., 2002). In view of these examples of spatially and functionally distinct glomerular arrangements, we propose that subpopulations of OSNs expressing the G-protein, G olf, extend axons into spatially distinct glomeruli in the lamprey. Earlier reports have shown a ring-like arrangement of olfactory glomeruli in Lampetra fluviatilis and L. planeri (Schober, 1964), Ichthyomyzon unicuspis (Northcutt and Puzdrowski, 1988), Petromyzon marinus (Tobet et al., 1996), and Lampetra japonica (Iwahori et al., 1997). However, details of the arrangement of the glomerular territories in lampreys are lacking. The olfactory receptor family is relatively small in lampreys (Berghard and Dryer, 1998; Freitag et al., 1999) and, not surprisingly, few odorants stimulate olfactory activity (Li and Sorensen, 1995). These findings suggest that the larval lamprey has considerably fewer olfactory glomeruli than the 1,800 2,400 glomeruli located in the mammalian olfactory bulb (Royet Fig. 1. Western immunoblot of G olf in a preparation from the olfactory epithelium of an adult sea lamprey. Molecular weight markers are shown on the left. Lane A: 20 g deciliated lamprey olfactory epithelium. Lane B: 10 g deciliated lamprey olfactory epithelium. Lane C: 20 g ciliated lamprey prep. Lane D: 10 g ciliated lamprey prep. The G olf antibody concentration was tested at a 1:500 dilution. et al., 1988; Meisami and Sendera, 1993) and that mapping of glomerular territories is feasible in serially sectioned preparations of the lamprey olfactory bulb. In sea lamprey, nonolfactory serotonergic nerve fibers are present along the primary olfactory pathway and enter the olfactory bulb (Zielinski et al., 2000). These fibers may be associated with particular olfactory sensory input and may innervate specific glomerular regions. Therefore, the goal of this study was to probe the olfactory bulb of the developing lamprey for spatially conserved glomerular territories and for the distribution of two substances that may be relevant to OSN activity: the protein G olf and the biogenic amine neurotransmitter serotonin. In this study we observed six glomerular territories, G olf -immunoreactivity at specific glomerular sites, and distinct glomerular subsets with serotonergic nonolfactory fibers. These results imply differences in transduction mechanisms for some OSNs and modulation of specific OSNs by biogenic amines. MATERIALS AND METHODS Experimental animals Year two and three class larval sea lampreys were obtained from naturally occurring populations collected from creeks in Michigan, then housed at the Lake Huron Biological Station, Millersburg, Michigan, or from Oshawa Creek and Bronte Creek, Ontario. All larval lampreys were maintained at 10 C at the Department of Biological Sciences, University of Windsor. Approximately 90 larvae were used for this study (total length mm, weight g). Adult sea lampreys were trapped or collected by hand from tributaries to Lakes Huron and Michigan, transported to the U.S. Geological Survey Lake Huron Biological Station, Millersburg, Michigan. These adults were held in flow-through tanks (1,000 L) with Lake Huron water (7 20 C) before being euthanized for collection of olfactory organs. Prior to experimentation, all lampreys were deeply anesthetized with tricaine methane sulfonate (MS-222) and killed by decapitation. All experimental protocols reported in this study were in compliance with guidelines established by the Canadian Council of Animal Care. Western immunoblot The cilia were dissociated from OSNs by calcium shock (Schandar et al., 1998). In brief, sea lamprey olfactory

3 OLFACTORY BULB IN LARVAL SEA LAMPREY 29 epithelia were dissected out and agitated in a high calcium buffer (10 mm CaCl, 20 mm ACES, 0.3 M sucrose, 10 g/ml leupeptid, 76.8 nm aprotinin, 0.7 M pepstatin, 0.83 mm benzamide, 0.23 mm PMSF, 1 mm iodoacetamide) in an end-over-shaker at 4 C for 20 min. The solution was spun for 15 min at 6,000g and the supernatant was collected. The pellet was resuspended in the same buffer and spun down again for collection of the supernatant. The combined supernatant was spun for 15 min at 18,000g to isolate the cilia. The ciliary pellet was washed with TME buffer (10 mm Tris, 3 mm MgCl, 2 mm EGTA, ph 8.2) and resuspended. Aliquots were stored at 80 C until use. The deciliated olfactory mucosa was prepared from the pellet of the first centrifugation according to DellaCorte et al. (1996). The deciliated tissue was homogenized using a mortar and pestle and centrifuged at 30,000g for 90 min at 0 C. The supernatant was removed and stored at 80 C. The protein concentration was determined by DCA protein analysis kit (Pierce Biotechnology, Rockford, IL). Ten and 20 g protein of cilia and deciliated mucosa were loaded onto a 10% SDS PAGE gel and 5% stacking gel which were subjected to 150 V for 1 hour in a standard running buffer. The protein bands in the gel were transferred to a PVDF membrane. The membrane was then blocked with a blocking solution containing 5% nonfat dry milk in buffer, incubated with the primary antibody (anti-g olf 1:200 or 1:500) in the blocking solution, washed three times with buffer, and incubated with HRP-conjugated secondary antibody (1:5,000) in the blocking solution. Finally, the membrane was washed three times and incubated in chemiluminescence substrate for minutes and film was exposed and developed. Tissue fixation The larval heads were immersed in Zamboni s fixative for 4 20 hours (2% paraformaldehyde, 1.5% picric acid, 0.1 M phosphate buffer, ph 7.4) at 4 C, then cryoprotected by passage through a sucrose gradient ( % in PB). Horizontal sections (20 30 m) were cut on a cryostat (Microm). Sections were air-dried for at least 1 hour, postfixed in acetone at 20 C for 10 minutes, and rehydrated with 0.1 M phosphate buffered saline (PBS) (ph 7.4) for 10 minutes at room temperature. GS1B 4 lectin histochemistry The staining of larval sea lamprey olfactory glomeruli by Griffonia simplicifolia-1 (GS-1 isolectin B 4 ) was previously described (Tobet et al., 1996; Zielinski et al., 2000). Slides were incubated with biotin-conjugated lectin, G. simplicifolia-1 (GS-1 isolectin B 4, Vector, Burlingame, CA; Fig. 2. Localization of G olf -IR in the olfactory epithelium. A and B show a double labeled preparation. A: Acetylated tubulin (AT)- immunolabeling in the larval nasal cavity shows intense labeling of the ciliary layer lining the luminal surface and of OSN soma in the olfactory epithelium (horizontal plane). Nonsensory epithelium is located on the right side of the micrograph. B: The luminal surface of the olfactory epithelium is G olf -IR, and the surface of the nonsensory epithelium is unlabeled (indicated by asterisks). OSN cilia, dendrites, cell bodies, and axons are G olf -IR (arrow). C: Obliquely sectioned OSNs show intense G olf -immunoreactivity in olfactory cilia. D: An OSN in a metamorphosing sea lamprey is intensely G olf -IR (Z-series of seven sections, taken at 0.2 m steps. A, B, and D are the same magnification. Scale bar in B 50 m; 25 m inc.

4 30 A. FRONTINI ET AL. Figure 3

5 OLFACTORY BULB IN LARVAL SEA LAMPREY 10 g/ml in 0.1 M PBS, ph 7.5) for 3 hours. These were rinsed in PBS, then incubated in DCS avidin-fluorescein (1:100 in 0.1 M bicarbonate buffer, ph 8.5) for 1 hour, washed, then mounted with Vectashield. Immunoctyochemistry Slides with tissue sections were incubated in diluted normal horse serum for 20 minutes, then in primary antiserum raised in rabbits against G olf (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA) or serotonin (5-HT, 1:3,000; DiaSorin, Stillwater MN) in 0.1 M PBS, ph 7.4, containing 0.1% Triton X-100 overnight at 4 C. The sections were rinsed in PBS, incubated in Alexa 568 goat antirabbit IgG (Molecular Probes, Eugene, OR; 1:100 in PBS, ph 7.4) for 1 hour and rinsed in PBS. Negative controls, with primary antibody omitted from the staining procedure, were included with each immunocytochemical preparation. Specificity of the 5HT antibody was tested by omitting this primary antibody from the staining protocol and by a preadsorption control experiment. Preadsorption control preparations for 5-HT were prepared with diluted antiserum that had been preadsorbed with 10 mm or 100 mm 5HT (Sigma, St. Louis, MO) for 24 hours at 4 C. Application of calcium green dextran The glomerular units containing OSN projections in the olfactory bulb were anterogradely labeled following the application of calcium green dextran into the nasal cavity. Prior to experimentation all lampreys were individually anesthetized by immersion in a 0.05% solution of MS222. After the animal was sedated it was wrapped in wet tissue paper. A solution of 2.5% calcium green 1-dextran (potassium salt 3,000 MW anionic, Molecular Probes), 0.2% TritonX-100, and 1 mm NaCl, 0.1 M Na bicarbonate in water was injected into the nasal cavities using a Hamilton syringe with a 23 gauge needle. Following this treatment the lamprey recovered and another injection was applied the following day. On the fifth day following this injection the lamprey was immersed in a solution of MS222, deeply anesthetized, and killed by decapitation. Fig. 3. The organization of olfactory glomeruli from a 125-mm larval sea lamprey. The horizontal sections were labeled with GS1B 4 lectin and visualized by confocal microscopy. The arrows in A show the orientation of the images. R is rostral, M is medial. The series shows sections taken at intervals of 100 m; however, sections with prominent medial glomeruli are shown at 50- m intervals. A: 100 m. Dorsal cluster. A dorsal group of six glomeruli is seen at the anterior and lateral edges of the olfactory bulb. B: 200 m. Dorsal ring. Glomeruli are arranged in a circular configuration. The olfactory nerve layer is prominent at the anterior/lateral edge. C: 300 m. Anterior plexus and lateral chain. The anterior plexus (AP) and lateral chain (LC) are present at the dorsal edge of the junction of the olfactory nerve with the olfactory bulb. D: 350 m and E: 400 m. Anterior plexus, lateral chain and medial glomeruli. Three medial glomeruli seen at a depth of 350 m: anterior radial (ra), radial posterior (RP), and the glomerulus adjacent to the anterior commissure (AC). At a depth of 400 m the medial glomerulus adjacent to the anterior commissure is present and individually labeled fibers are seen in the neuropil lateral to this glomerulus. F: 500 m and G: 550 m. Ventral ring and medial glomeruli. Ventral to the olfactory nerve the anterior plexus and the anterior medial glomeruli form a circular arrangement. A medial glomerulus extends toward the anterior commissure (500 m) and a posterior medial glomerulus (PM) is prominent (550 m). H: 600 m. Ventral ring. I: 700 m. Ventral cluster. An aggregate of glomeruli is located at the ventral edge of the olfactory bulb. Scale bar 100 m ina. The larval heads were immersed into 4% paraformaldehyde fixative overnight at 4 C, then cryoprotected by passage through a sucrose gradient ( % in PB). Cryosections (20 m) were cut in horizontal planes on a cryostat (Microm). Coverslips were mounted with Vectashield (Vector). The sections were viewed by fluorescence microscopy or confocal microscopy (BioRad 1024, Hercules, CA). Morphometry Larval olfactory bulbs prepared for GS1B4 lectin histochemistry, fluorescent dextran, G olf, and 5HT were serially sectioned and stained (n 25). These sections were viewed on an Axioskop (Zeiss) and prepared as a 3D movie using Empix Eclipse software (Mississauga, Canada). This computer program was used to measure the glomerular diameters and the total glomerular volume. The number of olfactory glomeruli olfactory bulbs was estimated by applying the dissector technique (Gundersen et al., 1988) to the 3D reconstruction of the serially sectioned olfactory bulbs. Production of photomicrographs The confocal images were acquired in BioRad PIC format and converted to TIFF format with Confocal Assistant. The fluorescence microscopy images were acquired using Empix Eclipse software and saved in TIFF format. The photomicroscope images were assembled into figures and labeled with Adobe PhotoShop (Mountain View, CA). RESULTS Western blotting of the ciliary fraction from adult lamprey olfactory epithelium demonstrated the specificity of the G olf antibody. The molecular weight of G olf in the cilia of lamprey OSNs was 45 kda, with a slightly higher molecular weight component likely from the phosphorylated form of this G-protein (Fig. 1). The strong G olf - immunoreactive (IR) band in the ciliary fraction is supported through immunolocalization. Ciliary localization was seen in double-labeled preparations with acetylated tubulin and G olf antibodies (Fig. 2A,B). Aceylated tubulin immunoreactivity indicated the presence of cilia (Fig. 2A). In larvae, the boundary between the olfactory epithelium and non-g olf -expressing nasal epithelium was sharp and clear (Fig. 2B). High-power views confirmed strong G olf -IR in cilia of OSNs (Fig. 2C). G olf expression in cilia, dendrites, cell bodies, and axons persisted after metamorphosis (Fig. 2D), when OSNs were considerably larger than during larval development (Vandenbossche et al., 1995, 1997). OSN subcellular components located in the olfactory epithelium, dendrites, soma, and axons contained G olf -IR (Fig. 2D). The presence of G olf -immunoreactivity in the OSN axons led us to examine spatial distribution of G olf -axons in the olfactory bulb. Glomerular territories The detailed glomerular organization in the lamprey olfactory bulb has not been documented. Therefore, we mapped the olfactory bulbar neuropil innervated by OSNs before charting G olf -IR glomeruli. Examination of the olfactory bulb from serial sections in the horizontal plane, stained with GS1B 4 lectin (Fig. 3), and by anterograde labeling with fluorescent dextran, revealed a consistent pattern of glomerular organization. All glomeruli that were identified were labeled by both techniques. The spa- 31

6 Figure 4

7 OLFACTORY BULB IN LARVAL SEA LAMPREY tial distribution of the glomerular groupings was consistent in the larvae examined ( mm long). The thickness of the olfactory bulb was 500 m in the smaller lampreys. Spatial analysis is presented in a series of evenly spaced sections through the olfactory bulb of larvae with a length of cm, where the thickness of the olfactory bulb was m. The olfactory nerve entered the olfactory bulb at a depth of m. The diameter of olfactory glomeruli ranged from m. Tallies of glomerular modules in serially sectioned larval olfactory bulbs indicated that the total number of glomeruli ranged from (n 10). In addition to forming olfactory glomeruli, OSN fibers were seen entering the olfactory bulb from the olfactory nerve and proceeding medially beyond the olfactory bulb to the ventral diencephalon, as previously reported (Tobet et al., 1996). We consistently observed the following glomerular territories in larval sea lampreys. Dorsal cluster, m. A cluster of 6 10 olfactory glomeruli was apparent close to the dorsal edge of the olfactory bulb (Fig. 3A). Dorsal ring, m. Approximately 7 10 closely spaced glomerular modules were arranged in a circular pattern along the medial, anterior, and lateral edges and the olfactory nerve layer was prominent at the anterior and lateral edge (Fig. 3B). Anterior plexus, m. The anterior plexus consisted of a cohesive mesh of OSN axon terminals (Fig. 3C E). Intense GS1B 4 labeling at the medial edge indicated the location of olfactory nerve fibers entering the olfactory bulb. Fig. 4. G olf -IR in olfactory glomeruli of the larval and adult lamprey. Scale bar in A 100 m. The depth of each section is indicated. A, B, F, and G are single-labeled preparations (G olf -immunocytochemistry). C and D are double-labeled (G olf -immunocytochemistry and fluorescent dextran). E is double-labeled (G olf -immunocytochemistry and GS1B 4 lectin). A: 200 m. Dorsal ring. B: 300 m. Anterior plexus and lateral chain. C: 350 m and D: 400 m. Anterior plexus, lateral chain, and medial glomeruli. In C the medial edge of the olfactory bulb is located on the right edge of the micrograph. In D the center of the micrograph shows the medial regions of adjacent olfactory bulbs. The anterior and lateral glomeruli show double-labeling for G olf and anterogradely applied fluorescent dextran. These labels do not show complete colocalization, as the green (dextran) is within the cytoplasm of the OSN axons and the red G olf is localized on the membranous surface. OSN axons in the medial glomerulus facing the anterior commissure (AC) labeled with the dextran, but not with G olf immunocytochemistry. E: 500 m. Ventral ring and medial glomeruli (Z series of eight sections taken at 1- m intervals). The glomeruli of the ventral ring show double-labeling for G olf and for GS1B 4 lectin. Both stains are localized on the cell membrane. The medial glomeruli, including posterior medial glomeruli (PM), are not G olf -IR. F: 600 m. Ventral ring. G: 700 m. Ventral cluster. H to J show a horizontal view of the olfactory bulb from an adult sea lamprey, double-labeled, GS1B 4 lectin histochemistry, and G olf immunocytochemistry. H and I are fluorescence micrographs shown at the same magnification. Scale bar in H 500 m. H: GS1B 4 lectin stains olfactory glomeruli in the anterior, lateral, and medial regions. I: Anterior and lateral olfactory glomeruli are G olf -IR. The area enclosed by a green box is shown at high power in J. J: A high-power confocal micrograph of the medial olfactory glomerular region. Green is GS1B 4 lectin histochemistry, red is G olf - immunolabeling, and yellow is colocalized G olf -immunolabeling and GS1B 4 lectin staining. The red and green channels were imaged in series, to avoid fluorescein bleed-through on the red channel. Scale bar 25 m. Lateral chain, m. A space about 50 m wide separated the anterior plexus from the lateral chain, a relatively narrow group of glomerular modules extending along the lateral edge of the olfactory bulb (Fig. 3C E). The anterior plexus and lateral chain were previously observed in the zebrafish olfactory bulb (Baier and Korsching, 1994). Medial glomeruli, m. At bulbar depths of m, medial glomeruli were positioned posterior to the junction of the olfactory nerve and olfactory bulb (Fig. 3D,E). At 350 m, a small anterior-radial glomerulus was positioned posterior to the medial edge of the anterior plexus; a second medial glomerulus, the posterior-radial glomerulus, was positioned radially (Fig. 3D), and a third glomerulus faced the anterior commissure (Fig. 3D,E). Medial glomeruli were located ventral to the olfactory nerve ( m): a posterior glomerulus and a glomerulus adjacent to the anterior commissure (Fig. 3F,G). Ventral ring, m. A circular arrangement of glomerular units were present in sections in the olfactory bulb taken ventral to the olfactory nerve at depths of m (Fig. 3F H). At 500 m, GS1B 4 - positive fibers were seen extending from anterior glomeruli of this ring into the granular layer. Ventral cluster, m. The bottom portion of the olfactory bulb contained a cluster of 7 10 olfactory glomeruli (Fig. 3I). Glomerular G olf -immunolabeling. Glomerular territories in the dorsal, anterior, lateral, and ventral regions of the olfactory bulb contained G olf -IR. However, the medial glomeruli were not G olf -IR (Fig. 4). The G olf - immunolabeling was also intense in the olfactory nerve and the olfactory nerve layer of the olfactory bulb. Dorsal cluster and dorsal ring. The glomerular units in this dorsal region of the olfactory bulb contained G olf -immunoreactivity (Fig. 4A). Anterior plexus and lateral chain. These glomeruli contained G olf -immunoreactivity (Fig. 4B). Medial glomeruli. G olf -immunoreactivity was absent from the region occupied by medial glomeruli. In preparations double-labeled with G olf immunocytochemistry and anterogradely applied fluorescent dextran, medial glomeruli were devoid of G olf -immunoreactivity (Fig. 4C E). These included the posterior radial glomerulus, the glomerulus facing the anterior commissure, the posterior medial glomerulus, and individual fibers extending into the neuropil. The anterograde labeling shows directly that OSN axons extend to the medial glomeruli of the olfactory bulb, and that these axons do not stain by G olf - immunocytochemistry. The anterograde labeling was limited to the OSN axons that took up the dextran when it was applied to the nasal cavity in vivo, and labeled OSN axons were less populous than with GS1B 4 lectin labeling (Fig. 4E). The lectin, which reacts with carbohydrate residues on extracellular axonal surface, stained more robustly than the dextran, which was confined to the cytoplasm within the narrow intracellular axonal space. Ventral ring and ventral cluster. Glomeruli in this region were G olf -IR (Fig. 4F,G). Glomerular subsets with differing G olf expression persisted in the adult stage of the sea lamprey. Medial glomeruli maintained an absence of G olf -IR compared to the remaining glomerular units (Fig. 4H I). Spatial relationship between 5HT-IR fibers and olfactory glomeruli The dorsal region of the olfactory bulb contained more robust 5HT-IR than the ventral region (Fig. 5). Double- 33

8 34 A. FRONTINI ET AL. Fig. 5. Double-labeled preparations of the olfactory bulb from the larval lamprey. GS1B 4 lectin is green and 5HT immunocytochemistry is red. Yellow indicates regions of overlap between the axons of OSNs and 5HT-IR fibers. Glomerular regions with 5HT-IR fibers adjacent to OSN axons are circled in white. A, B, and C are the same magnification. The distance from the dorsal surface of the olfactory bulb of each section is indicated for each micrograph. A: 100 m. Dorsal cluster. Five regions with close apposition between OSN and 5HT-IR fibers are present. The neuropil proximal to the glomeruli is populated by 5HT-IR fibers. B: 300 m. Anterior plexus and lateral chain. 5HT-IR fibers are located in glomerular units adjacent to the olfactory nerve and in the space between the anterior plexus and lateral chain (arrow). C: 400 m and D: 420 m. Medial glomeruli. High-power views of medial glomerular neuropil show seven regions with 5HT-IR fibers adjacent to OSN axons. E: 500 m. Ventral ring and medial glomerulus. In the ventral region 5HT-IR fibers are weakly stained and confined to the granular cell layer of the olfactory bulb. Scale bars 100 m ina;50 m inc. labeling experiments from eight larvae showed a constant pattern of 5HT innervation of glomerular territories. Dorsal cluster and dorsal ring. Serotonergic fibers entered the glomerular units and were located between the closely spaced glomeruli (Fig. 5A). Anterior plexus and lateral chain. Serotonergic fibers were present in the space between the anterior plexus and the lateral chain and surrounding the periphery of the anterior plexus and the lateral chain (Fig. 6B). As previously observed, 5HT-IR fibers extended along the

9 OLFACTORY BULB IN LARVAL SEA LAMPREY 35 TABLE 1. Summary of the Distribution of Relevant Molecules in Glomerular-Territories in the Olfactory Bulb from cm Long Larval Sea Lampreys Glomerular territory Depth ( m) G olf -IR glomeruli 5HT-IR fibers Dorsal cluster Labeled Within glomeruli Dorsal ring Labeled Within glomeruli Anterior plexus Labeled Medial edge of anterior plexus* Lateral chain Labeled Outer edge of the lateral chain* Medial glomeruli Unlabeled Through medial glomeruli Ventral ring Labeled Absent Ventral cluster Labeled Absent The glomerular territories were labeled by GS1B 4 lectin and by anterograde application of fluorescent dextran. *The region between anterior plexus and lateral chain contains 5HT-IR fibers. olfactory nerve into the olfactory bulb (Zielinski et al., 2000). In the lateral portion of this region, 5HT-IR fibers extended into the olfactory bulb neuropil through the anterior plexus. Medial glomeruli. Serotonergic fibers extended into the medial posterior glomerulus from the medial region of the olfactory nerve (Fig. 5C,D). Two medial glomeruli, the radiomedial glomeruli and the glomerulus adjacent to the anterior commissure, were devoid of 5HT-IR. Ventral ring and ventral cluster. Glomerular modules in this region of the olfactory bulb did not contain 5HT-IR fibers (Fig. 5E). In summary, 5HT-IR fibers and OSN axons were in close proximity in the dorsal cluster and the medial posterior glomerulus, and 5HT-immunoreactive fibers were least populous in the ventral olfactory bulb. DISCUSSION Examination of the distribution of substances that may be relevant to OSN function in the sea lamprey revealed spatially conserved glomerular associations. The olfactory receptor linked protein G olf was expressed by sea lamprey OSNs projecting to dorsal, anterior, lateral, and ventral glomerular subsets. Innervation by nonolfactory 5HT-IR fibers was concentrated in the dorsal cluster and the medial posterior glomerulus. The principle differences between the glomerular groups is summarized in Table 1 and in Figure 6. Although these lampreys were from naturally occurring populations that represented diverse gene pools, there was remarkable consistency in these morphological, biochemical, and spatial characteristics. Bulbar G olf -IR and OSN subtypes The molecular weight of the lamprey G olf, 45 kda, is identical to that of G olf that regulates adenlyl cyclase activity in mammals (Jones and Reed, 1989) and teleosts (Abogadie et al., 1995; Dellacorte et al., 1996). The ciliary localization of lamprey G olf, shown both through Western immunoblotting and immunocytochemically, supports the involvement of G olf in olfactory sensory transduction. Therefore, this G-protein may have been present in vertebrate ancestors over 400 million years ago, or it may have evolved in parallel during agnathan evolution. G olf - labeling was prominent in the glomerular units of the dorsal ring, anterior plexus, lateral chain, and ventral cluster. This is somewhat in contrast with prior reports in the rodent where G olf was not detected in the olfactory bulb, although other molecules, including olfactory marker protein (Danciger et al., 1989) and mrna for the Fig. 6. A schematic summary of the position of glomerular territories with G olf -IR and 5HT-IR labeling in the olfactory bulb from larval lampreys cm long. The diagram represents an anterior view of the olfactory bulb. The depth of the olfactory bulb is shown on the left ( m). The glomerular territories are shown as rectangles and the regions with 5HT-IR fibers are shown as cylinders. Glomeruli with G olf -IR are dark gray; the medial glomeruli, which are not G olf -IR, are shown by a patterned rectangle. olfactory marker protein (Wensley et al., 1995) and odor receptor are present in OSN axons (Mombaerts, 1996). This difference may underscore a species-specific phenotype but nevertheless emphasizes that subsets of lamprey OSNs may use G olf transduction cascades. Detection in the axons may reflect further on the importance of G olf in not only odor transduction, but also modulation of growth cone behavior through cascades that involve G olf and cyclic nucleotide-gated channels, as suggested by Kafitz et al. (2000). The absence of G olf -IR in the lamprey medial glomeruli points to a differing signal transduction mechanism for these compared to the other glomerular groupings. In various teleosts the medial olfactory path is associated with bile acid and steroid perception (e.g., Thommesen, 1978; Hara and Zhang, 1998), and in the zebrafish, medial glomeruli respond to chemostimulation by these compounds (Friedrich and Korsching, 1998). The OSN medial glomeruli in the lamprey may also constitute a subtype of OSN specialist expression. It is not surprising to find a subpopulation of lamprey OSNs without G olf -IR. The expression of various G-proteins in OSN subtypes appears to be a principle of the vertebrate olfactory system (e.g., Shinohara et al., 1992; Berghard and Buck, 1996; Jia and Halpern, 1996; Wekesa and Anholt, 1999; Hansen et al., 2001; Mezler et al., 2001). The medial location of the glomeruli that do not express G olf implies that the OSNs projecting to these glomeruli use an alternate G-protein during olfactory sensory transduction. In tetrapods, chemoreceptive neurons of the vomerosal system contain the G-proteins G i 2 and G 0 (Halpern et al., 1995; Berghard and Buck, 1996; Jia and Halpern, 1996), and in the mam-

10 36 A. FRONTINI ET AL. malian olfactory system, necklace olfactory glomeruli adjacent to the accessory olfactory bulb contain cgmpstimulated phosphodiesterase and guanylyl cyclase-d (Juilfs et al., 1997) and heterogenous immunoreactivity for neural proteins (Ring et al., 1997). Therefore, the use of G-protein subtypes may have preceded the gnathostome radiation, or have evolved in parallel in gnathostome and agnathostome vertebrates. Nonolfactory fibers adjacent to dorsal and medial glomeruli In the lamprey olfactory bulb 5HT-IR fibers were located between glomerular groupings, particularly in lateral and medial locations of the dorsal hemisphere. In mammals, periglomerular serotonergic fibers originate from raphe nuclei (McLean and Shipley, 1987; Philpot et al., 1994). However, in the lamprey the nonolfactory 5HT-IR fibers from the primary olfactory pathway enter the olfactory bulb (Zielinski et al., 2000). The present study extends previous results by investigating the spatial relationship between these fibers and olfactory glomeruli. The most prominent glomerular sites with 5HT innervation were the dorsal cluster and the medio-posterior glomerulus. This anterior dorsal/posterior medial ventral distribution is reminiscent of the dorsomedial pathway of the terminal nerve in the African lungfish (Von Bartheld and Mayer, 1988; Schober et al., 1994) and may indicate that this 5HT-immunoreactive pathway is a derivative of the nasal terminal nerve. Spatial pattern of OSN projections The pattern of glomerular organization in the larval lamprey was similar yet considerably reduced from the pattern formed by 18 glomerular groups in the olfactory bulb of the teleost Danio rerio (Baier and Korsching, 1994). In both, glomeruli were clustered in dorsal and ventral regions, the anterior plexus stretched from the dorsal to the ventral olfactory bulb, and the lateral group appeared as a chain of glomeruli. The overall simplicity of the lamprey s glomerular arrangement can be seen from the ring of glomeruli encircling the dorsal and ventral regions. Images from previous studies of the adult silver lamprey Ichthyomyzon unicuspis, (Northcutt and Puzdrowski, 1988) and larval Petromyzon marinus (Tobet et al., 1996) have shown this ring-like pattern, as well as the medially located glomeruli. The four medial glomeruli (radial anterior, radial posterior, adjacent to the anterior commissure, and posterior medial) that we observed in the larval lamprey are fewer than the several medial glomeruli located in the olfactory bulb of the zebrafish (Baier and Korsching, 1994). The scarcity of glomeruli in the lamprey s dorsomedial region of olfactory bulb may be due to the fact that this neuropil is occupied by contralateral secondary olfactory projections (Northcutt and Puzdrowski, 1988). The larval olfactory glomeruli were smaller than the 140 m (average) diameter reported for glomeruli in adult Lampetra japonica (Iwahori et al., 1987), and within the range observed in adult zebrafish, (25 and 140 m; Baier and Korsching, 1994) and rats ( m; Pinching and Powell, 1971). It is not surprising that the glomerular size is larger in adult lampreys than in larvae, as the olfactory epithelial surface area and the diameter of the olfactory nerve fascicles also increase during metamorphosis (Vandenbossche et al., 1997). In conclusion, there are spatially conserved glomerular territories and substances that may be relevant to OSN activity which are distributed in a consistent manner. Serotonin-IR fibers are adjacent to specific glomerular modules and the G-protein G olf is expressed by lamprey OSNs; however, a subpopulation of OSNs do not express G olf. These data are the first to show that expression of G olf, the GTP-binding protein linked to olfactory receptors, is present at the base of gnathostome radiation. ACKNOWLEDGMENTS The authors thank the staff at Department of Fisheries and Oceans, Sault Ste Marie, Ontario, Canada; Hammond Bay Biological Station, Millersburg, MI, and the U.S. Fish and Wildlife Service, Marquette Biological Station, Marquette, MI. for animal collection; and assistance from Dr. S.S. Yun (Michigan State University, Dept. of Fisheries and Wildlife), A. Belanger, C. Dakhil, and D.W. Dingler (University of Windsor, Ontario, Canada). LITERATURE CITED Abogadie FC, Bruch RC, Farbman AI G-protein subunits expressed in catfish olfactory receptor neurons. Chem Senses 20: Baier H, Korsching S Olfactory glomeruli in the zebrafish form an invariant pattern and are identifiable across animals. J Neurosci 14: Belluscio L, Gold GH, Nemes A, Axel R Mice deficient in G(olf) are anosmic. Neuron 20: Berghard A, Buck LB Sensory transduction in vomeronasal neurons: evidence for G alpha o, G alpha i2, and adenylyl cyclase II as major components of a pheromone signaling cascade. J Neurosci 16: Berghard A, Dryer L A novel family of ancient vertebrate odorant receptors. J Neurobiol 37: Chiba A, Sohn YC, Honma Y Immunohistochemical and ultrastructural characterization of the terminal nerve ganglion cells of the Ayu, Plecoglossus altivelis (Salmoniformes, Teleostei). Anat Rec 246: Danciger E, Mettling C, Vidal M, Morris R, Margolis F Olfactory marker protein gene: its structure and olfactory neuron-specific expression in transgenic mice. Proc Natl Acad Sci U S A 86: DellaCorte C, Restrepo D, Menco BP, Andreini I, Kalinoski DL G alpha 9/G alpha 11: immunolocalization in the olfactory epithelium of the rat (Rattus rattus) and the channel catfish (Ictalurus punctatus). Neuroscience 74: Eisthen HL Why are olfactory systems of different animals so similar? Br Behav Evol 59: Freitag J, Beck A, Ludwig G, von Buchholtz, Breer H On the origin of the olfactory receptor family: receptor genes of the jawless fish (Lampetra fluviatilis). Gene 226: Friedrich RW, Korsching SI Chemotopic, combinatorial, and noncombinatorial odorant representations in the olfactory bulb revealed using a voltage-sensitive axon tracer. J Neurosci 18: Greer CA Structural organization of the olfactory system. In: Getchell TV, editor. Smell and taste in health and disease. New York: Raven Press. p Gundersen HJG, Bagger P, Bendtesen TF, Evans SM, Korbo L, Marcussen N, Moller A, Nielsen K, Nyengaard JR, Pakkenberg B, Sorense FB, Vesterby A, West MJ The new stereological tools: dissector, fractionator, nucleator and point sampled intercepts and their use in diagnosis and diagnosis. APMIS 96: Halasz N, Greer CA Terminal arborizations of olfactory nerve fibers in the glomeruli of the olfactory bulb. J Comp Neurol 337: Halpern M, Shapiro LS, Jia C Differential localization of G proteins in the opossum vomeronasal system. Brain Res 17: Hansen A, Anderson KT, Finger TE Immunohistochemical and ultrastructural identification of G-proteins in the olfactory epithelium of catfish. Chemical Senses 26:1128. Hara TJ, Zhang C Topographic bulbar projections and dual neural pathways of the primary olfactory neurons in salmonid fishes. Neuroscience 82: Hildebrand JG, Shepherd GM Mechanisms of olfactory discrimina-

11 OLFACTORY BULB IN LARVAL SEA LAMPREY tion: converging evidence for common principles across phyla. Annu Rev Neurosci 20: Iwahori N, Kiyota E, Nakamura KA Golgi study on the olfactory bulb in the lamprey, Lampetra japonica. J Neurosci Res 5: Jia C, Halpern M Subclasses of vomeronasal receptor neurons: differential expression of G proteins (Gi alpha 2 and G(o alpha)) and segregated projections to the accessory olfactory bulb. Brain Res 719: Jones DT, Reed RR Golf: an olfactory neuron specific-g protein involved in odorant signal transduction. Science 244: Juilfs DM, Fulle HJ, Zhao AZ, Houslay MD, Garbers DL, Beavo JA A subset of olfactory neurons that selectively express cgmp-stimulated phosphodiesterase (PDE2) and guanylyl cyclase-d define a unique olfactory signal transduction pathway. Proc Natl Acad Sci U S A 94: Kafitz KW, Lenders-Zufall T, Zufall F, Greer CA Cyclic GMP evoked calcium transients in olfactory receptor cell growth cones. Neuroreport 11: Klenoff JR, Greer CA Postnatal development of olfactory receptor cell axonal arbors. J Comp Neurol 390: Li W, Sorensen PW The olfactory system of migratory adult sea lamprey (Petromyzon marinus) is specifically and acutely sensitive to unique bile acids released by conspecific larvae. J Gen Physiol 105: Lin DM, Ngai J Development of the vertebrate main olfactory system. Curr Opin Neurobiol 9: McLean JH, Shipley MT Serotonergic afferents to the rat olfactory bulb. I. Origins and laminar specificity of serotonergic inputs in the adult rat. J Neurosci 7: Meisami E, Sendera TJ Morphometry of rat olfactory bulbs stained for cytochrome oxidase reveals that the entire population of glomeruli forms early in the neonatal period. Brain Res Dev Brain Res 71: Mezler M, Fleisher J, Conzelmann S, Korchi A, Widmayer P, Breer H, Boekhoff I Identification of a nonmammalian G(olf) subtype: functional role in olfactory signaling of airborne odorants in Xenopus laevis. J Comp Neurology 439: Mombaerts P Visualizing an olfactory sensory map. Cell 87: Mombaerts P Seven-transmembrane proteins as odorant and chemosensory receptors. Science 286: Morita Y, Finger TE Differential projections of ciliated and microvillous olfactory receptor cells in the catfish, Ictalurus punctatus. J Comp Neurol 398: Northcutt RG, Puzdrowski RL Projections of the olfactory bulb and nervus terminalis in the silver lamprey. Brain Behav Evol 32: Philpot BD, Jazaeri AA, Brunjes PC The development of serotonergic projections to the olfactory bulb of Monodelphis domestica (the grey short tailed opossum). Brain Res Dev Brain Res 77: Pinching AJ, Powell TP The neuropil of the glomeruli of the olfactory bulb. J Cell Sci 9: Pombal MA, de Arriba MC, Sampedro C, Alvarez R, Megias M Immunocytochemical localization of calretinin in the olfactory system of the adult lamprey, Lampetra fluviatilis. Brain Res Bull. 57: Ring G, Mezza RC, Schwob JE Immunohistochemical identification of discrete subsets of rat olfactory neurons and the glomeruli that they innervate. J Comp Neurol 388: Royet JP, Souchier C, Jourdan F, Playe H Morphometric study of the glomerular population in the mouse olfactory bulb: numerical density and size distribution along the rostrocaudal axis. J Comp Neurol 270: Schandar M, Laugwitz KL, Boekhoff I, Kroner C, Gudermann T, Shultz G, Breer H Odorants selectively activate distinct G protein subtypes in olfactory cilia. J Biol Chem 273: Schober W Vergleichend-anatomische untersuchungen am gerhirn der larven un adulten tiere von Lampetra fluviatilis (Linne, 1758) und Lampetra planeri (Bloch 1784). J Hirnforschung 7: Schober A, Mayer DL, von Bartheld CS Central projections of the nervus terminalis and the nervus praeopticus in the lungfish brain revealed by nitric oxide synthase. J Comp Neurol 349:1 19. Shinohara H, Kato K, Asano T Differential localization of G proteins, Gi and Go, in the olfactory epithelium and the main olfactory bulb of the rat. Acta Anat (Basel) 144: Thommesen G The spatial distribution of odour induced potentials in the olfactory bulb of char and trout (Salmonidae). Acta Physiol Scand 102: Tobet SA, Chickering TW, Sower SA Relationship of gonadotropinreleasing hormone (GnRH) neurons to the olfactory system in developing lamprey (Petromyzon marinus). J Comp Neurol 376: VanDenbossche J, Seelye JG, Zielinski BS The morphology of the olfactory epithelium in the larval, juvenile and upstream migrant stages of the sea lamprey, Petromyzon marinus. Brain Behav Evol 45: VanDenbossche J, Youson H, Pohlman D, Wong E, Zielinski BS Metamorphosis of the olfactory organ of the sea lamprey (Petromyzon marinus L.): morphological changes and morphometric analysis. J Morphol 231: von Bartheld CS, Mayer DL Central projections of the nervus terminalis in lampreys, lungfishes, and bichirs. Brain Behav Evol 32: Wachowiak M, Cohen LB Representation of odorants by receptor neuron input to the mouse olfactory bulb. Neuron 32: Wekesa KS, Anholt H Differential expression of G proteins in the mouse olfactory system. Brain Res 837: Wensley CH, Stone DM, Baker H, Kauer JS, Margolis FL, Chikaraishi DM Olfactory marker protein mrna is found in axons of olfactory receptor neurons. J Neurosci 15: Wright DE, Demski LS Organization of GnRH and FMRF-amide systems in two primitive bony fishes (Order Polypteriformes). Brain Behav Evol 47: Xu F, Greer CA, Shepherd GM Odor maps in the olfactory bulb. J Comp Neurol 422: Zhang X, Firestein S The olfactory receptor gene superfamily of the mouse. Nat Neurosci 5: Zielinski BS, Moretti N, Hua HN, Zaidi AU, Bisaillon AD Serotonergic nerve fibers in the primary olfactory pathway of the larval sea lamprey, Petromyzon marinus. J Comp Neurol 420: