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Home , Olfactory epithelium, Vomeronasal receptor

UMEÅ UNIVERSITY MEDICAL DISSERTATIONS NEW SERIES NO 911 ISSN 0346-6612 ISBN 91-7305-706-1

ZONAL ORGANIZATION OF THE MOUSE OLFACTORY SYSTEMS

Fredrik Gussing

Department of Molecular Biology Umeå University Umeå 2004

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Cover picture: A coronal section of the mouse with zone-specific expression of the NQO1 gene (white signal) in the olfactory is shown.

Copyright © 2004 by Fredrik Gussing ISBN 91-7305-706-1

Printed in Sweden by Solfjädern Offset AB, Umeå 2004

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HOW DO I SMELL?

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TABEL OF CONTENTS

ABSTRACT 6

PAPERS IN THIS THESIS 7

ABBREVIATIONS 8

INTRODUCTION 9 The main 10 Anatomy 10 The main olfactory epithelium 10 Regenerative capacity 11 The main 12 The odorant receptors 13 Prereceptor events 13 Identification of the odorant receptors 13 Characteristics of odorant receptors 14 Spatial odorant expression patterns 14 in olfactory sensory neurons 15 Downstream of the G-protein coupled receptor 15 Genomic characterization of odorant receptors 17 Functions of the odorant receptors 17 Odorant binding 17 Odorant receptor expression 18 The glomerular maps 19 Axonal convergence and neuronal specificity 19 Zonal organization of olfactory sensory neuronal projections 20 Medial and lateral maps 21 , ephs and ephrins 21 Olfactory bulb projections 21 The septal organ 22 The accessory olfactory system 23 Anatomy 23 The 23 Axonal projections 24 The accessory olfactory bulb 25 The vomeronasal receptors 25 Gene regulation of vomeronasal receptors 26 Receptor signaling in the vomeronasal neurons 27 Vomeronasal receptor - ligand interaction 28 Vomeronasal mediated behaviors 29 Aggression 29 Reproduction related behaviors 30 Genetic modifications in the VSNs 30

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AIMS 32

RESULTS AND DISCUSSION 33 Overview 33 Patterns of gene expression in the primary olfactory neurons: correlation to receptor expression zones (Paper I and II) 33 Genes potentially involved in cell specification in the olfactory epithelium and in axonal guidance 33 Formation of odorant receptor zones by expression gradients 35 Identification of a new zonally restricted gene 35 Differences between the dorsomedial and ventrolateral zones in the olfactory epithelium 37 The zonal dichotomy of the vomeronasal epithelium (Paper III and IV) 37 Gαi2 protein in survival and function of apical vomeronasal neurons 38 Involvement of in the maintenance of basal vomeronasal neurons 39 Two mouse models to study zonal vomeronasal influences on behavior 41 Proposed function of the apical and basal zones 41

CONCLUDING REMARKS 44

ACKNOWLEDGEMENTS 45

REFERENCES 46

5 ABSTRACT

Animals survey their environment for relevant odorous chemical compounds by means of the olfactory system. This system is in most divided into a main and accessory olfactory system with two specialized neuroepithelia, the olfactory and the vomeronasal epithelium, respectively. The sensory neurons reside in these epithelia and together the neurons have an extraordinary sensitivity and are capable of detecting a vast number of different chemical molecules. After processing the chemical information, behavior may be altered. The information about a chemicals structure is deconstructed into a format that the may process. This is facilitated by organizing sensory neurons into a map and that the individual neuron responds only to one chemical feature. The sensory maps appear to have zones with different neuronal subpopulations. This thesis is addressing the fact that establishment, maintenance and function of these zones are unknown. We identify a gene (NQO1) to be selectively expressed in defined zone of the olfactory and the vomeronasal epithelia, respectively. NQO1-positive and negative segregate within the olfactory and maintain a zonal organization when reaching olfactory bulb target neurons. These results indicate that one zone of both the accessory and the main olfactory projection maps is composed of sensory neurons specialized in reducing environmental and/or endogenously produced quinones via an NQO1-dependent mechanism. In addition, we have identified genes expressed in a graded manner that correlates with the dorsomedial-ventrolateral zonal organization of the olfactory epithelia. Considering the known functions of identified genes in establishment of cell specificity and precise axonal targeting, we suggest that zonal division of the primary olfactory systems is maintained, during continuous , as a consequence of topographic counter gradients of positional information. The vomeronasal sensory neurons (VSN) are organized into an apical and a basal zone. The zones differ in expression of e.g. chemosensory receptor families and Gα protein subunits (Gαi2 and Gαo). We have analyzed transgenic mice (OMP- dnRAR) in which the VSNs are unresponsive to the function of one of the genes identified herein (RALDH2). The phenotype observed suggests that endogenous produced retinoic acid is selectively required for postnatal survival of neurons in the Gαo-positive zone. Analyses of another mouse line target deleted in the Gαi2 gene (Gαi2 mutant) reveal a cellular phenotype that is opposite to that of OMP- dnRAR mice. Consequently in these mice, the apical Gαi2-positive zone is reduced whereas VSNs in the basal zone are not affected. Several social and reproductive behaviors are under the influence of the vomeronasal organ. We have analyzed some behavioral consequences of having deficient neurons that corresponds to either of the two zones. We propose that cues important for aggressive behavior are detected by apical vomeronasal zone, while cues detected by both apical and basal VSNs influence gender preference behavior.

6 PAPERS IN THIS THESIS

This thesis is based upon the following articles and manuscript, which will be referred to in the text by their Roman numerals (I-IV).

I Evidence for gradients of gene expression correlating with zonal topography of the olfactory sensory map. Norlin EM, Alenius M, Gussing F, Hägglund M, Vedin V, Bohm S. Mol Cell Neurosci. Sep;18(3):283-95. (2001)

II NQO1 activity in the main and the accessory olfactory systems correlates with the zonal topography of projection maps. Gussing F, and Bohm S. Eur J Neurosci. 2004 May;19(9):2511-8. (2004)

III Vomeronasal phenotype and behavioral alterations in Gαi2 mutant mice. Norlin EM, Gussing F, Berghard A. Curr Biol. Jul 15;13(14):1214-9. (2003)

IV Inhibition of retinoid signaling in mature vomeronasal sensory neurons: postnatal degradation of a population of neurons and behavioral characterization. Gussing F, Hägglund M, Berghard A, Bohm S. Manuscript. (2004)

Articles reprinted with permission from the publisher.

7 ABBREVIATIONS

AC adenylyl cyclase AOB accessory olfactory bulb cAMP cyclic adenosine 3’,5’-monophosphate CNG cyclic nucleotide gated CNS DAG diacylglycerol dnRAR dominant negative retinoic acid receptor IP3 inositol 1,4,5-triphosphate IRES internal ribosomal entry site MHC major histocompatibility complex MUP major urinary protein NADPH reduced nicotinamide adenine dinucleotide phosphate NQO1 NADPH:quinone oxidoreductase 1 O-MACS olfactory specific medium-chain acyl-CoA synthetase OB olfactory bulb OBP odorant binding protein OE olfactory epithelium OEC olfactory ensheathing cell OMP olfactory marker protein OR odorant receptor OSN olfactory sensory neuron P postnatal day PCR polymerase chain reaction PLC C RA retinoic acid RALDH2 retinaldehyde dehyrogenase 2 RAR retinoic acid receptor RGS regulator of G-protein signaling RNCAM Rb8 neural cell adhesion molecule TRP2 transient receptor potential channel 2 VN vomeronasal VNX surgical removal of the vomeronsal organ VR vomeronasal receptor VSN vomeronasal sensory neuron Z1-4 odorant receptor expression zones 1-4

8 INTRODUCTION

My Fifth Wonder is the cell, located in the epithelial tissue high in the nose, the air for clues to the environment, the fragrance of friends, the smell of leaf smoke, breakfast, nighttime and bedtime, and a rose, even, it is said, the of sanctity.… If and when we reach an understanding of these cells and their functions, including the moods and whims under their governance, we will know a lot more about the mind than we do now, a world away. - Lewis Thomas (1983)

In this thesis I give an introduction to the olfactory system, and the research progress in this area during the last decades. This covers both the main and the accessory olfactory system with focus on one of the model organisms, the mouse. The aims and questions that initiated the experimental part of this thesis work are presented before the results are summarized and discussed in relation to other findings in the field.

Figure 1. Localization of the olfactory systems in mouse. A schematic representation of the mouse nasal cavity and brain. Three primary olfactory systems are illustrated, the accessory, the septal and the main olfactory system. Airflow (white arrows) passes the VN organ in the anterior part of the nose before reaching the SO and the OE. The nasal cavity and surface with turbinates that enlarge the receptive area, is lined with olfactory neuroepithelium. Axons of olfactory and VN sensory neurons project to their corresponding regions in the OB and AOB, respectively.

9 The main olfactory system

Anatomy The main olfactory system detects airborne chemical molecules in our environment. When the air is inhaled through the nose of an animal it passes the nasal cavity. The surface of this cavity is enlarged by a series of cartilaginous lamellae, called turbinates, and these are lined with a sensory neuroepithelium. Olfactory sensory neurons (OSN) in this olfactory epithelium (OE) send their axons and form synapses with target neurons in the olfactory bulb (OB) of the brain (Figure 1).

The main olfactory epithelium The main olfactory system is unusual among sensory systems in several respects. Firstly, the OSNs, embedded in the OE, are in direct contact with the environment. This characteristic makes OSNs vulnerable to inhaled toxins, infectious agents and mechanical trauma. Secondly, the OSNs with their axonal projections to target neurons in the brain constitute an exception to the general rule that the central nervous system (CNS) repairs itself poorly after injury. The regenerative capacity of the OSNs helps to maintain sensory function throughout the animals lifetime, despite the exposed position (Graziadei and Graziadei, 1979).

Figure 2. Cell types of the OE. A graphic representation of the parts and cell types of OE, from the covering mucus layer at the apical surface to the OEC surrounding axons fascicles in the lamina propria. Closest to the nasal cavity are the cell bodies of sustentacular cells. Underneath are OSNs, with ciliated in the mucus layer and axons that exit through the basal lamina and project towards the OB. Horizontal basal cells and globose basal cells are located in the most basal part of the OE. The thin layer of mucus is produced in the Bowman’s glands and secreted through the OE spanning ducts.

10 The OE is a pseudostratified columnar epithelium. From the nasal cavity and the apical surface, to the basal lamina, the major cell types are: sustentacular cells, OSNs, and basal cells. The underlying lamina propria contains connective tissue, Bowman’s glands, blood vessels and the OSN axons surrounded by olfactory ensheathing cells. The sustentacular cells are supporting cells with microvilli that span the whole epithelium but with their cell soma lining the nasal cavity (Figure 2). These cells have been shown to participate together with macrophages in the phagocytosis of dead neurons (Suzuki et al., 1995). Due to continuous cell turnover OE contains both immature and mature OSNs (Figure 2). Mature OSNs are bipolar in shape. An apical ends in a knob at the epithelial surface that has 12 or more cilia extending into the covering mucus layer. The odorant receptor proteins that transduce the sensory stimuli are located in the cilia (Barnea et al., 2004; Farbman, 2000). A thin, unmyelinated exits the epithelium basally and fasciculates towards the olfactory bulb (Schwob, 2002). Expression of the olfactory marker protein (OMP) has become a general marker for the mature OSNs, that are located in the upper 2/3 of the OE (Keller and Margolis, 1976). Below the mature neurons are the immature OSNs that have not yet extended cilia and express growth associated protein 43 (Verhaagen et al., 1989). There are basal cells of two types: globose basal cells and horizontal basal cells (Graziadei and Graziadei, 1979) (Figure 2). The globose basal cells lie on top of the horizontal basal cells. Both populations have been postulated to contain the progenitor cells that divide and give rise to OSNs and sustentacular cells (Caggiano et al., 1994; Carter et al., 2004; Huard et al., 1998; Schwartz Levey et al., 1991). The Bowman’s glands (named after Sir William Bowman 1816-1892) reside in the lamina propria and the ducts of the glands extend through the epithelium to the surface (Figure 2). These glands produce the thin protective layer of mucus that covers the OE.

Regenerative capacity The regenerative capacity of OSNs is evident after exposure of OE to either toxic substances that kill cells, e.g. zinc sulphate, methyl bromide and dichlobenil, or by surgical transection of the [reviewed in (Schwob, 2002)]. The lifespan of a neuron in the OE is on average 90 days (Gogos et al., 2000). The constant regeneration of OSNs makes the primary olfactory system suitable for studies of neuronal differentiation and specification, not only during embryonic stages but also in adult life. The thin, unmyelinated axons of OSNs are surrounded by a special type of cells named olfactory ensheathing cells (OEC) (Figure 2). The OECs have gotten a lot of attention lately due to the discovery that transplanted OEC promoted the recovery of injured spinal cord axons (Ramon-Cueto and Nieto-Sampedro, 1994) [reviewed in (Barnett and Chang, 2004)].

11 The main olfactory bulb OSN axons project through the cribiform plate and make synaptic contact with second-order neurons in the OB (Figure 1). Structural features of the OB are common to most mammals [reviewed in (Kosaka and Kosaka, 2004)]. At the surface of the OB is the nerve layer where the incoming axons are sorted before entering the glomerular layer (Figure 3). It has been suggested that sorting is in part aided by the OECs (Astic et al., 1998; Au et al., 2002). The glomeruli are neuropil structures in which the OSNs synapse with dendrites of the second-order neurons. In mice the average number of glomeruli is as many as 1800 per bulb (Royet et al., 1988). Underneath the glomerular layer are the external plexiform-, -, internal plexiform and granule cell-layers (Figure 3). A regenerative capacity is also evident in the OB. Granule cells of the inner- most cell layer as well as periglomerular cells are replaced throughout adulthood (Figure 3). The replacing cells originate in the subventricular zone and migrate in the so called rostral migratory stream towards the OB (Fasolo et al., 2002). The second-order neurons are mitral and tufted cells, and besides histological differences, these are suggested to differently convey the signal from to higher brain areas [(Nagayama et al., 2004) and reference therein] (Figure 3).

Figure 3. Cell layers of the OB. Simplified cellular circuit diagram summarize the organization of the OB (to the left). OSN axons sort out in the nerve layer and innervate their specific glomeruli in the glomerular layer where the axons make synapses with mitral cell dendrites. The mitral cells are the main OB projecting neurons, sending axons to olfactory cortical areas. Inhibitory interneurons in the granule cell layer and periglomerular cells surrounding the glomeruli (black dots; left) modulate the mitral cells. Layers are indicated by arrows, the innermost layer is on top in the left drawing. Medial and lateral glomeruli are depicted as filled circles (middle panel of the left OB). A photomicrograph of a coronal section is showing the right OB. Ventral down.

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An intrabulbar network of cells in the external plexiform layer connect medially and laterally located glomeruli within each bulb (Belluscio et al., 2002; Liu and Shipley, 1994; Lodovichi et al., 2003; Schoenfeld et al., 1985) (Figure 3). Surrounding each glomerulus are the periglomerular interneurons and the short axon cells. Periglomerular cells are thought to release dopamine and GABA (McLean and Shipley, 1988; Smith and Jahr, 2002). The short axon cells have excitatory synapses on inhibitory periglomerular neurons, creating a centre- surround inhibitory network by connecting glomeruli up to 20-30 glomeruli apart (Aungst et al., 2003).

The odorant receptors

Prereceptor events When the small, often hydrophobic, odorant molecules enter the nasal cavity these have to be transported across the protective mucus layer to reach the odorant receptors. One suggestion is that odorant presentation to the receptor is aided by extracellular odorant binding proteins (OBP) that belong to the family of lipocalins [reviewed in (Flower, 1996)]. Different subtypes of OBPs occur simultaneously in a species, all reversibly binding a unique profile of odorants (Lobel et al., 2002; Nespoulous et al., 2004). Based on structural and binding data it has also been suggested that OBP functions as deactivator of odorant binding and as scavenger for toxic or highly concentrated odorants. It is important to assure rapid clearance of odorant molecules and enable the system to perceive a new sensation with the next sniff. In addition to OBP, a set of biotransformation and detoxification enzymes have been reported to be present in the OE and these may fulfill such a function (Brittebo, 1997). The reactions catalyzed by phase I detoxification enzymes (e.g. cytochrome P450s) introduce chemical modifications that are followed up by phase II enzymes (e.g. glutathione- S-transferase or UDP-glucuronosyl transferase) (Lazard et al., 1991; Whitby-Logan et al., 2004). Phase II enzymes have preferentially been found in sustentacular cells of the OE (Banger et al., 1994; Miyawaki et al., 1996; Reed et al., 2003; Whitby- Logan et al., 2004). Together these enzymes may function in metabolism of odorants as well as providing a metabolic barrier contributing to xenobiotic detoxification [reviewed in (Breer, 2003)].

Identification of the odorant receptors Studies of the olfactory system and smell in general have been undertaken for many centuries. In the mid eighteenth century the famous Swedish biologist Linnaeus published his “Odores medicamentorum” where he stated that all odorants are a combination of seven major classes, ranging from pleasant to unpleasant (Watson, 2001). The view on odorant detection changed dramatically after the discovery of the genes encoding odorant receptors (OR) and a new era began (Buck and Axel, 1991).

13 Behind the successful identification of the ORs were three assumptions. Firstly, the finding was based upon the demonstration that cyclic adenosine 3’,5’- monophosphate (cAMP) was the olfactory second messenger (Nakamura and Gold, 1987; Pace et al., 1985). This implied that the receptors should have a seven- transmembrane domain structure and belong to the G-protein-coupled receptors [reviewed in (Kristiansen, 2004)]. Secondly, expression of the receptors should be OSN-specific and finally, the receptors should be very divergent in their sequence since very many structurally different odorant molecules can be detected by the system (Buck, 2004). Using degenerate polymerase chain reaction (PCR), Buck and Axel identified and estimated that there is several hundred different ORs. ORs belong to the super family of G-protein-coupled receptors (Buck and Axel, 1991).

Characteristics of odorant receptors Such a vast number of ORs was unexpected. Estimations of their family sizes range from a 100 in fish to over 1000 ORs in mice, making them by far the largest subfamily of G-protein-coupled receptors (Godfrey et al., 2004; Ngai et al., 1993; Young et al., 2002; Zhang and Firestein, 2002). Sequence analysis of the receptors groups them to the class A G-protein-coupled receptors, which also includes e.g. and catecholamine receptors [reviewed in (Kristiansen, 2004)]. The majority of OR genes has a ~1 kbp coding region in a single exon without introns (Zhang et al., 2004).

Spatial odorant receptor expression patterns The onset of OR gene expression in the OE was determined by in situ hybridization to be around embryonic day 11 of the mouse (Sullivan et al., 1995). However, a recent finding has localized prenatal receptor expression in the cribiform prior to the onset of OR expression in the OE, but the relevance is not known (Conzelmann et al., 2002; Schwarzenbacher et al., 2004). Initial in situ hybridization analyses of OR expression indicated that they were expressed in a restricted zonal manner in the OE (Nef et al., 1992; Ressler et al., 1993; Strotmann et al., 1994; Strotmann et al., 1992; Sullivan et al., 1996; Vassar et al., 1993) (Figure 4). Four OR expression zones organizing the epithelium according to a dorsal-medial to ventral-lateral manner have been defined. Recent studies have reinvestigated the zonal restriction of OR expression and have found that some zones may not be as well defined as originally suggested (Iwema et al., 2004; Norlin et al., 2001; Strotmann et al., 1992). However, spatially circumscribed receptor distribution seems to be a characteristic feature found in many species (Clyne et al., 1999; Gao and Chess, 1999; Marchand et al., 2004; Weth et al., 1996; Vosshall et al., 1999). The neurons expressing a given OR are stochastically scattered within one of these defined zones (Ressler et al., 1993; Strotmann et al., 1994; Vassar et al., 1993) (Figure 4). The reason for the scattered appearance of OSNs expressing a specific OR is described below (see section “Odorant receptor expression”). Beside the zonal and scattered expression a laminar segregation of OR gene expression in the OE has been reported (Reed, 2004; Strotmann et al., 1996).

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Figure 4. OR expression zones in the mouse OE. In situ hybridizations with Z1 and Z4 ORs on OE tissue sections are shown in the left panel. Positive signal is shown in white and nuclear counter stain visualizes the extent of the OE in gray. Localization of Z1 OR gene expression to dorsomedial OE can be seen whereas Z4 ORs are expressed in the most ventrolateral part. Right panel shows a schematic drawing of a mouse OE as seen from inside the nasal cavity. The spatial extent of the OR expression zones are outlined.

Signal transduction in olfactory sensory neurons The initial transduction of the molecular signal into an electrical signal that subsequently is conveyed to the glomeruli, takes place in the cilia of the OSNs in response to activation of the ORs. The pattern of activated glomeruli contains information about the odorant that the brain can process.

Downstream of the G-protein coupled receptor When an odorant binds the OR a conformational change occurs, activating the heterotrimeric G-protein which then initiates a signal transduction cascade that produces a second-messenger that activates an ion channel which finally leads to OSN depolarization (Figure 5). G-proteins are GTPases functioning as molecular switches, flipping between an active GTP-bound and inactive GDP-bound state. This switch may be modulated by a family of proteins named regulators of G- protein signaling (RGS) and a number of these have been identified to be expressed in the OE (Norlin and Berghard, 2001) (Figure 5).

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Figure 5. Signal transduction in OSNs. Schematic drawing of the olfactory sensory transduction, illustrating the downstream effects upon odorant binding to ORs. GTP bound Gαolf activates ACIII to produce cAMP. The elevated levels of cAMP open CNG channels leading to increase of intracellular Ca2+, which in turn activates Cl- channels and further enhances depolarization. Ca2+ can also autoregulate the signaling by inhibiting the CNG channel and stimulate Ca2+-calmodulin kinase II (CAM-KII) and phosphodiesterase (PDE) that lowers cAMP levels.

OSNs use both cAMP and inositol-1,4,5-triphosphate (IP3) pathways although the dominating pathway seems to be that of cAMP (Firestein, 2001; Kaur et al., 2001; Schild and Restrepo, 1998; Vogl et al., 2000). Three olfactory enriched signaling components have been identified. Firstly, the G-protein subunit Gαolf, secondly the adenylyl cyclase III (ACIII) that when activated converts ATP into cAMP and finally the cyclic nucleotide gated (CNG) ion channel (with stoichiometry of two CNGA2 to one CNGA4 to one CNGB1b subunit in OSNs) (Bakalyar and Reed, 1990; Dhallan et al., 1990; Jones and Reed, 1989; Zheng and Zagotta, 2004) (Figure 5). Genetically altered mice with deletions in these genes have been generated. All three mutant mouse strains are anosmic with no odorant evoked electro-olfactogram responses (Belluscio et al., 1998; Brunet et al., 1996; Trinh and Storm, 2003; Wong et al., 2000; Zhao and Reed, 2001). Mice were until recently thought to be anosmic if the cAMP activated CNGA2 ion channel subunit is deleted, when another study showed that glomerular responses can be obtained in such mice (Lin et al., 2004). Influx of Ca2+ through the CNG channel activates an ion channel permeable to chlorides, and its somewhat unusual outward current, further depolarize the membrane potential (Kurahashi and Yau, 1993) (Figure 5). An important aspect of olfactory signaling is the ability of the OSN to adapt its sensitivity to prolonged odorant exposure (Adelman and Herson, 2004). This has been shown to partly depend on a feedback modulation of CNG channels mediated by calmodulin (Bradley et al., 2004). Ca2+ and calmodulin also have a role in

16 regulating the levels of cAMP by either inhibiting the ACIII through Ca2+- calmodulin kinase II or activating a phosphodiesterase (Frings, 2001) (Figure 5). OR activity and signaling via cAMP have also been suggested to increase expression of genes that prolong the survival of OSNs (Watt et al., 2004).

Genomic characterization of odorant receptors A minor revolution in “molecular genetics” has occurred since the first identification of ORs 13 years ago. With the help of genome sequencing and software tools, more accurate numbers of OR genes (including pseudogenes) are now known to be e.g. in the mouse (~1403), dog (~971) and human (~636) (Malnic et al., 2004; Olender et al., 2004; Zhang et al., 2004). These numbers suggest that the OR genes contribute to approximately 2% of genes in the entire human genome and hence is the largest gene family by far. The OR genes are organized into clusters, consisting of one to several hundred genes, that are spread on all mouse chromosomes except 5, 12, 18 and Y (Godfrey et al., 2004). In addition, full or partial OR sequences have been cloned from genomic DNA or OE cDNA from various species across different phyla (Dryer, 2000; Mombaerts, 1999). Analyses show a variable percentage of pseudogenes among the OR genes, making the number of potentially functional ORs considerably less than the total in some species (Glusman et al., 2001; Young et al., 2002; Zhang and Firestein, 2002; Zozulya et al., 2001). The mouse and dog genomes seem to have the same ratio of pseudogenes, i.e. about 20% are pseudogenes. Why there is such a high number (47%) of pseudogenes in humans is not known (Malnic et al., 2004). However, a recent correlation between the loss of OR genes and development of trichromatic vision has been suggested (Gilad et al., 2004). This is a result that could be explained by the shift from olfaction as dominating to vision in primates. The OR genes can be divided into 2 classes based upon amino acid sequence similarity (≥40%) and further subdivided into subfamilies (≥60%). In the mouse genome there is one cluster of ~150 class I genes on chromosome 7, and no class II genes are located to this cluster (Young et al., 2002; Zhang and Firestein, 2002; Zhang et al., 2004).

Functions of the odorant receptors Surprisingly the function of ORs in the OSNs is not restricted to mediating odorant induced signaling. Two other roles, the regulation of OR gene expression and the axonal identity of OSNs, are processes in which there is reason to belive that ORs have a major part.

Odorant binding Despite considerable efforts almost every OR is still an . This slow progress of matching ligands with ORs is mainly due to problems with a versatile functional heterologous expression system (McClintock et al., 1997). In transfected cell lines, ORs are often retained in the endoplasmatic reticulum and hence not correctly transported to the surface of the membrane. Different

17 approaches to aid ligand-receptor matching have been developed including making chimeric receptors, analysis of ORs in OSNs responding to odorant by single cell RT-PCR, or homologous expression using adenoviral vectors (Araneda et al., 2000; Gaillard et al., 2002; Kajiya et al., 2001; Krautwurst et al., 1998; Malnic et al., 1999; Touhara et al., 1999; Wellerdieck et al., 1997; Wetzel et al., 1999; Zhao et al., 1998). Of the mentioned techniques, the first demonstration of an olfactory receptor interaction with its cognate ligand, was performed in vivo using adenovirus-mediated gene transfer of the cloned rat OR-I7 (Zhao et al., 1998). Together all these data support a combinatory model in which one OR is activated due to the presence of a specific molecular feature, named an odotope, which may be present on several different odorant molecules. Thus one given odorant molecule can be detected by several different ORs. Moreover, the concentration of the odorant seems to determine the set of responsive ORs, which in turn may generate different sensations in response to the same compound (Malnic et al., 1999). Using the data obtained by Malnic et al., predictions of 3D structures and function of OR have strengthened the odotope theory further (Floriano et al., 2000; Floriano et al., 2004).

Odorant receptor expression Today one general model for OR gene expression prevails which states that one OSN expresses a single OR gene. Evidence for the one receptor gene - one neuron hypothesis has come from single-cell RT-PCR analyses (Kajiya et al., 2001; Malnic et al., 1999; Touhara et al., 1999). Alleles of the same OR gene can be polymorphic and may thus encode receptors with different specificities. This fact was utilized to determine that OR genes are further regulated by monoallelic inactivation, which means that a given OSN will express only one OR gene from either the maternal or paternal chromosome (Chess et al., 1994; Ishii et al., 2001; Strotmann et al., 2000). After the discovery of the zonal OR expression pattern, genome analysis was carried out to investigate if there were clusters of OR genes expressed in the same zone. No such zonal organization could be found and hence the presence of a single zonal enhancer per cluster was ruled out (Sullivan et al., 1996). Later, researchers found by using transgenic mice that express a reporter gene under the control of a 5’-flanking region of one OR gene, directs OR gene expression in a correct zone-specific pattern (Qasba and Reed, 1998; Vassalli et al., 2002). Such gene regulatory regions contain sequence motifs that may direct gene transcription in OSNs, such as binding sites for O/E (a helix-loop-helix family) and homeodomain proteins (Wang et al., 1997). In the immune system, the selection of a single immunoglobulin (in B cells) or T-cell antigen receptor (in T cells) gene for expression is made by DNA recombination. Use of such a mechanism has also been an attractive hypothesis in the regulation of OR genes (Kratz et al., 2002). To rule out the role of DNA rearrangement, two separate groups have cloned mice from single post-mitotic OSNs (Eggan et al., 2004; Li et al., 2004). Both studies came to the same conclusion: that the stochastic choice of an OR gene is not due to rearrangements in DNA.

18 Several studies have recently shown that a functional OR protein exerts negative feedback on the expression of other OR genes including the other allele of the same OR (Lewcock and Reed, 2004; Serizawa et al., 2003; Shykind et al., 2004). Besides the question on how the OR is mediating this fascinating regulatory mechanism, these findings give further support for the one OR - one OSN rule as well as for monoallelic expression of OR genes. However, co-expression of two ORs has been reported, a finding suggesting that the one OR - one OSN model does not always hold true (Mombaerts, 2004; Rawson et al., 2000).

The glomerular maps The brain identifies an odorant by interpreting the pattern of glomeruli that the odorant activates in the OB. The topographic map of glomeruli is a result of the convergence onto individual glomeruli by OSNs expressing the same OR.

Axonal convergence and neuronal specificity The third function of the OR is its involvement axonal identity and guidance. By in situ hybridization analyses it was shown that the OR mRNA are present in the glomeruli (Ressler et al., 1994; Vassar et al., 1994). This finding was groundbreaking. The intriguing results suggested that the scattered population of OSNs that express the same OR gene performed the remarkable axonal convergence to one (or a few) glomeruli on the medial and lateral halves of the OB (Ressler et al., 1994; Vassar et al., 1994) (Figure 6). Recently, antibodies against two ORs was used to show OR protein localization to cilia on the dendritic knob, perinuclear compartments, and the glomeruli (Barnea et al., 2004). These results indicate the possibility of a direct role of ORs in the specific convergence into one (or a few) glomeruli. In 1996, Mombaerts et al. performed a molecular genetic experiment where both the expressed OR gene and a co-expressed marker could be visualized (Mombaerts et al., 1996). This was accomplished by creating a bicistronic construct, placing the internal ribosomal entry site (IRES) and the lacZ gene behind an OR gene, followed by homologous recombination of the construct into the genome of mouse embryonic stem cells. This led to specific labeleling of the cell bodies and the axons of OSNs expressing the modified OR locus. A number studies have used similar genetic methods to replace the endogenous OR gene, in so called “receptor swap” experiments. The results from such experiments all point to that changing an OR gene results in a new convergence location, that is neither the original nor the glomerulus representing the OR used as substitute (Belluscio et al., 2002; Bozza et al., 2002; Feinstein et al., 2004; Mombaerts et al., 1996; Wang et al., 1998). A recent paper proposed a “contextual” model, in which the ORs guide and sort the axons to the right glomeruli by homotypic interactions between related axons (Feinstein and Mombaerts, 2004). The result confirms what previously has been suggested e.g. by Tsuboi and coworkers, that used OR genes with high sequence similarity from one genomic cluster which all were shown to converge their axons to proximal but distinct glomeruli, relative to each other (Tsuboi et al., 1999).

19 Interestingly, the axonal OR identity can be substituted by another G-protein coupled receptor, the β2 , when expressed from an OR locus (Feinstein et al., 2004). Such data suggests that the mechanism is not specific for ORs. The fact that the β2 adrenergic receptor mediates axonal convergence suggests that neurons other than OSNs may use a similar mechanism.

Zonal organization of olfactory sensory neuronal projections Retrograde tracing studies together with studies of convergence of OSNs expressing ORs limited to different zones has led to a zone-to-zone projection hypotheses (Astic and Saucier, 1986; Astic et al., 1987; Ressler et al., 1994; Saucier and Astic, 1986; Vassar et al., 1994) (Figure 6). Supporting results came from analyses subsequent to the cloning of Rb8 neural cell adhesion molecule (RNCAM/OCAM) (Alenius and Bohm, 1997; Yoshihara et al., 1997). Expression of RNCAM is restricted to zone 2 (Z2), Z3 and Z4 which together constitute the ventrolateral part of the OE. In the glomerular layer the RNCAM protein is localized to OSN axons of all OB regions except the dorsomedial, which thus correlates to the RNCAM negative Z1 in OE (Alenius and Bohm, 2003; Yoshihara et al., 1997). In addition, a marker for oxido-reductive enzymes that requires NADPH as co-factor, NADPH-diaphorase, labels the OSNs in the dorsomedial OE and the axons as they project to the RNCAM-negative glomeruli (Alenius and Bohm, 2003; Dellacorte et al., 1995; Schoenfeld and Knott, 2002).

Figure 6. Projections of OSNs. OSNs expressing a specific OR gene are scattered within a zone among OSNs expressing other OR genes (dots in the OE). Passing the cribiform plate, axons expressing a specific OR gene sort out in the nerve layer and converge into one or a few glomeruli (black, light gray or dark gray circles). Each mitral cell (MC) innervate only one glomerulus. The OSN axons project to the olfactory bulb in a zone – to – zone fashion. Notice that the sharp border between the dorsomedial (Z1) and ventrolateral (Z2-Z4) in the OE is maintained in the OB. The MCs send their axons to the olfactory cortex.

20 Medial and lateral maps Axons from OSNs expressing a specific OR gene are guided to one or two glomeruli located on each side of the bulb (Mombaerts et al., 1996; Ressler et al., 1994; Vassar et al., 1994) (Figure 3). The parts of OE that correspond to the medial-lateral subdivision of the OB has been visualized by injecting different retrograde tracers into one OR specific glomerulus on each side of the bulb (Levai et al., 2003). This experiment revealed that within the OR’s zone, the scattered OSNs are separated into different areas which correspond to targeting of the medial or lateral glomerulus, respectively. The medial and lateral glomeruli are connected through an intrabulbar network of connections (Schoenfeld et al., 1985). This network is made by the external tufted cells and their precision in axonal targeting is high since the connected medial and lateral glomeruli are innervated by OSNs expressing the same OR (Belluscio et al., 2002). As a result it has been argued that the glomerular maps, one lateral and one medial, should be considered as one instead of two (Lodovichi et al., 2003).

Neuropilins, ephs and ephrins was originally identified to segregate the OSN axons in the Xenopus frog (Satoda et al., 1995). Moreover, the role of neuropilin-1 in OSN axonal guidance was suggested by an experiment in chick where axons expressing a dominant-negative neuropilin-1 overshoot the bulbar target (Renzi et al., 2000). In mice, the semaphorin-3A ligand for neuropilin-1 is the most studied, with several reports proposing a function for these guidance molecules in the correct glomerular targeting of OSNs (de Castro et al., 1999; Schwarting et al., 2000; Schwarting et al., 2004; Taniguchi et al., 2003; Walz et al., 2002). OR genes that are closely linked on the chromosome and have high sequence similarity were shown to project to adjacent glomeruli that can be described as “glomerular domains” (Tsuboi et al., 1999). In addition, manipulation of the expression of the eph – ephrin axonal guidance family results in an anterior – posterior shift of location in these smaller domains of glomeruli with similar OR identity (Cutforth et al., 2003). Such results suggest that a functional domain organization of the glomerular sheet exists. Progress in this area has been made through data collected by in vivo optical imaging methods of OB after odorant stimulation [(Mori, 2003) and reference therein]. The results indicate that OSNs with a similar molecular receptive range (i.e. the range of carbon-chain length that a given OSN respond to) innervate glomeruli within the same domain (Bozza et al., 2002; Bozza et al., 2004; Takahashi et al., 2004). Interestingly, these domains are arranged at predictable positions in relation to the zonal organization of the OB (Takahashi et al., 2004).

Olfactory bulb projections Despite great gain in our understanding of the olfactory sense during the last decade we are so far only at first or perhaps second base. Organization of the projections carrying olfactory information to higher brain centres from the OB is

21 still elusive. The mitral cells project their axons to the olfactory cortex and limbic system (Christensen and White, 2000). In an experiment by Zou et al., a genetic tracing approach was used to mark the projections of OSN expressing two zonally different ORs with barley lectin. Barley lectin has previously been shown to cross multiple synapses (Horowitz et al., 1999; Zou et al., 2001). This approach revealed a sensory map in the olfactory cortex where information originating from one OR is send to specific clusters of neurons in multiple olfactory cortical areas. Moreover, the information from specific glomeruli in the OB seems to be integrated in the cortex as mitral axons, which correspond to different OR- specificities, innervated different neuronal clusters that sometimes overlap.

The septal organ

I will just briefly mention an organ that sometimes is thought of as the third olfactory system. Located on the nasal septum between the OE and the vomeronasal organ, near the entrance of the nasopharynx is the septal organ (SO, also termed “organ of Masera”) (Figure 1). Due to its location at the entrance of the nasal cavity it has been proposed that it has an alerting function. However experimental evidence for this theory has not been presented (Giannetti et al., 1995). Recently the ORs expressed by sensory neurons of the septal organ were identified (Kaluza et al., 2004). The septal receptors were found to be expressed also in the OE but only in the ventrolateral zones (Z2-4). No evidence for a zonal organization in the septal organ was found (Kaluza et al., 2004).

22 The accessory olfactory system

Anatomy In mouse olfaction, there is a collaborating or accessory system which peripheral sensory organ is located in the vomer bone at the base of the nasal septum, hence the name vomeronasal (VN) organ (Figure 1). Danish anatomist Ludvig Jacobson identified this organ in 1813, nearly 200 years ago, and later also gave this structure its eponym, the Jacobson’s organ (Jacobson et al., 1998). Sensory neurons in this VN organ project their axons to the accessory olfactory bulb (AOB) located dorsal/posterior in the olfactory bulb (Halpern and Martinez-Marcos, 2003) (Figure 1). The accessory olfactory system can be found in most vertebrate tetrapods (amphibians, reptiles and mammals) and has been designated to influence various behaviors including social, aggressive, sexual and reproductive behaviors (Doving and Trotier, 1998). However, in humans the VN axons degenerate during late gestation and a discernable AOB has not been identified in the adult brain. Taken together, it is not likely that humans have a functional accessory olfactory system [reviewed in (Meredith, 2001)]. During the last decade a dichotomy of the primary accessory olfactory system has been studied. There is a duality of the VN organ due to the precense of two subpopulations being apical and basal VN sensory neurons (VSN). The apical and basal VSNs differ with respect to a number of parameters, e.g. gene expression (Gα-protein expression, NADPH–diaphorase staining, lectin staining and other markers), types of VN receptors, target areas for their axonal connections in the AOB and responses to stimulating substances [reviewes in (Brennan and Keverne, 2004; Halpern and Martinez-Marcos, 2003)]. I here give an introduction to these findings with a focus on the accessory olfactory system of the mouse.

The vomeronasal organ The VN organ is a bilateral symmetrical tubular structure located in the lower anterior part of the nasal septum (Figure 1). A single VN in the nasal cavity is the only opening to the lumen of the VN organ of the mouse. The large blood vessel that runs along the anterior-posterior extent of the organ, opposite the crest shaped sensory epithelium, is responsible for the pumping action that draws substances into the lumen (Meredith and O'Connell, 1979) [reviewed in (Doving and Trotier, 1998)] (Figure 7). The VN organ contains a pseudostratified epithelium with bipolar sensory neurons and supporting cells, much like the main OE, but with some cellular characteristics. Both apical and basal VSN have microvilli on their dendritic knobs instead of cilia. The basal lamina is undulating due to the presence of blood vessels protruding from the lamina propria. As a result the boundary between the apical and basal VSNs is irregular (Figure 7). There are no Bowman’s glands in the VN organ.

23 Like the OE, the VN epithelium has a population of neuronal progenitors (Wilson and Raisman, 1980). The turnover of VSNs has for long been thought to take place at the boundary between the sensory epithelium and the ciliated (Barber and Raisman, 1978a). New studies have questioned this with results pointing to an additional area of neurogenesis in the central basal layer of the epithelium (Cappello et al., 1999; Giacobini et al., 2000; Matsuoka et al., 2002). VSN replacement after sectioning the VN nerve has also been observed. Interestingly, regeneration after cutting the VN nerve appears not to be complete with regard to axonal outgrowth. During normal cell development however new axonal connections appear to be formed (Barber, 1981a; Barber, 1981b; Barber and Raisman, 1978b).

Axonal projections The VN nerve consists of 3-4 fascicles that cross the cribiform plate, enter the CNS, and run along the medial surface of the OB on the way its AOB part. The dichotomy of the VN epithelium by its division into an apical and a basal VN zone is maintained in the AOB glomerular layer as the VN subpopulations target the anterior and posterior target AOB, respectively (Figure 7). This was first visualized by analysis for specific Gα proteins that labels the two zones separately (Berghard and Buck, 1996; Jia and Halpern, 1996).

Figure 7. Projection of VSNs. The apical VSNs (light gray) send their axons to the anterior part of the AOB. The posterior glomerular layer in the AOB receives innervation from basal VSNs (dark gray). VSNs expressing the same VR project to several glomeruli. Mitral/tufted cells send dendrites to multiple glomeruli that receive input from VSNs that express the same VR. Thus it is likely that the mitral/ receive input from a single VR. BV, blood vessel; L, lumen; M/T, mitral/tufted cells.

A few years after the cloning of the OR genes, researchers identified two large and unrelated families of VN receptors (VR), termed V1R and V2R (Dulac and Axel, 1995; Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997). In situ hybridizations with receptor probes for V1Rs and V2Rs show that the VSNs segregate into two zones after birth. The apical VSNs express V1Rs whereas the V2Rs are expressed by VSNs located in the basal part (Figure 7). The segregation of neurons in the VN epithelium resembles the zonal organization of the OE.

24 By virtue of being selectively expressed in apical VSNs, RNCAM mRNA and protein demarcate this VN zone and its VSN axonal projections (Alenius and Bohm, 1997; von Campenhausen et al., 1997; Yoshihara et al., 1997). Another gene that is co-expressed with Gαi2, V1Rs and RNCAM in apical VSNs is neuropilin-2 (Cloutier et al., 2002; Walz et al., 2002). Results from mice with a targeted null mutation of the neuropilin-2 gene show that the mice exhibit defasciculated VSN axons with some innervating the main OB. In addition, apical VSNs project incorrectly to the posterior AOB in neuropilin-2 mutant mice (Cloutier et al., 2002).

The accessory olfactory bulb Mixed VSN axons are sorted in the VN nerve right before reaching the AOB and their correct targets in the anterior and posterior glomerular layer (Figure 7). Synapses are made in glomeruli with dendrites on the projection mitral/tufted neurons which convey the signal further [reviewed in (Meisami and Bhatnagar, 1998)]. As in the main OB, the AOB periglomerular and granule interneurons are replaced during adult life of the mice [(Halpern and Martinez-Marcos, 2003) and references therein]. Mitral/tufted cells situated in the anterior or posterior AOB send their dendrites into anterior or posterior glomeruli, respectively, keeping the segregation of incoming information (Figure 7). Underneath the mitral/tufted cell layer are the lateral and a granular cell layer. The mitral/tufted cells axons target the bed nucleus of the accessory olfactory tract, medial amygdaloid nucleus, posteromedial cortical amygdaloid nucleus and bed nucleus of the [(von Campenhausen and Mori, 2000) and references therein]. Interestingly, in these structures there are no differences in target areas for the axonal projections from the anterior and posterior AOB (Salazar and Brennan, 2001; von Campenhausen and Mori, 2000). Thus, the integration of VN information, conveyed separately by apical and basal VSNs is likely to first take place in these nuclei.

The vomeronasal receptors In the mouse, the number of V1Rs has been estimated to 332 genes of which 164 potentially are intact (Zhang et al., 2004). The V1R genes can be divided into 12 subfamilies clustered on at least 8 chromosomes and are not intermingled with OR genes (Zhang et al., 2004). No full genome analysis of the V2Rs has been undertaken, leaving only a rough prediction that there are 100-150 different genes (Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997). Like the OR genes, the VN receptors are G-protein-coupled receptors. However, V1R and V2R family members exhibit no sequence similarity between each other or the ORs. The two VR families also differ from each other in that V2Rs has a large extracellular N-terminal domain that is believed to be the ligand binding domain due to a high level of sequence variability (Herrada and Dulac, 1997). Antibodies against V2Rs have been made that localize V2R protein to the cell body and microvilli, supporting the role of V2Rs as true VN receptors (Martini et

25 al., 2001). The interesting finding that an antibody against one specific V2R (V2R2) stain the majority of basal neurons, has been interpreted to suggest that V2Rs form receptor dimers in analogy to other G-protein coupled receptors of the same class (Martini et al., 2001; Matsunami and Amrein, 2003). In the search for other genes expressed only in the VN organ and not in the OE, two groups found the unusual major histocompatibility complex (MHC) class 1b genes to be expressed along with their accessory protein, β2-microglobulin. MHC class 1b is co-expressed with the V2Rs in basal VSNs (Ishii et al., 2003; Loconto et al., 2003). Data indicates that one function of the MHC class 1b containing multimolecular complexes is to locate and stabilize the V2R at the cell surface (Loconto et al., 2003). Importantly, the expression of these non-classical MHC molecules is not random, instead certain combinations of gene(s) are found with specific V2R genes (Ishii et al., 2003). The function of this specific co-expression is not known today. Several studies have been undertaken to answer if humans have functional VRs (Giorgi et al., 2000; Kouros-Mehr et al., 2001; Lane et al., 2002; Rodriguez et al., 2000). The results show that all receptors are pseudogenes except five V1Rs, of which one has been shown to be expressed in OE (Pantages and Dulac, 2000; Rodriguez et al., 2000) The genetic labeling approach that allows for visualization of OSNs that express a given OR gene has also been used to study VN projections (Belluscio et al., 1999; Del Punta et al., 2002b; Rodriguez et al., 1999). The observed convergence of VSN axons onto the glomerular target is not at all as distinct as for OSN axons. Instead of targeting one or a few glomeruli, the axons expressing either a tagged V1R or V2R, synapse on 6-30 glomeruli located in broadly defined regions of the anterior or posterior part of the AOB, respectively (Belluscio et al., 1999; Del Punta et al., 2002b; Rodriguez et al., 1999) (Figure 7). However, another genetic experiment demonstrated that the mitral/tufted cells of the AOB send their dendrites to multiple glomeruli, which all were innervated by axons from VSNs expressing the same VR (Del Punta et al., 2002b). So, the convergence of sensory information from a defined VR is as a result achieved in the mitral/tufted cells in the AOB (Figure 7). In addition, VRs appear to be required for axonal convergence and VSN survival (Belluscio et al., 1999; Rodriguez et al., 1999).

Gene regulation of vomeronasal receptors Compared to the OR genes, little is known about the regulation of V1Rs and V2Rs. Monoallelic regulation has been shown for V1Rs by the use of different genetic markers for both alleles of a given V1R (Rodriguez et al., 1999). The isolation of only one V1R or V2R gene from single-cell cDNA libraries and the scattered expression pattern of VRs which is similar to that of ORs imply that the one receptor - one neuron rule seems to apply also for VR genes (Dulac and Axel, 1995; Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ressler et al., 1993; Vassar et al., 1993).

26 Receptor signaling in the vomeronasal neurons VSNs react to sensory stimuli with a receptor mediated response, leading to increased intracellular free Ca2+ levels and increased action potential firing [(Halpern and Martinez-Marcos, 2003) and references therein] (Figure 8). Many of the major signal transducing proteins in the OSNs are not expressed in the VN organ, and the signal mechanisms subsequent to the activation of the VN receptors are still not well understood (Berghard et al., 1996). It is believed that the VSNs preferentially use the phospholipase C (PLC) pathway with IP3 and diacylglycerol (DAG) as second messengers in the signaling cascade (Spehr et al., 2002; Zufall et al., 2002). The discovery that the transient receptor potential channel 2 (TRP2) is exclusively expressed in all VSNs and the subsequent analyses of TRP2β null mice, have shown that the TRP2 channel is essential for VN signaling (Hofmann et al., 2000; Leypold et al., 2002; Liman et al., 1999; Menco et al., 2001; Stowers et al., 2002). The co-immunoprecipitation of TRP2 and the type-III IP3 receptor from VSNs implies a role for IP3 in elevating 2+ Ca levels by protein-protein interactions between the type-III IP3 receptor and the TRP2 channel (Brann et al., 2002). In addition, a direct role for DAG in gating this channel has recently been reported (Lucas et al., 2003). (Figure 8) Organization of VSNs into an apical, Gαi2 positive and a basal, Gαo expressing subpopulation was subsequently supported by the interesting co-expression of these G-protein subunits with the different identified VR families, V1R and V2R, respectively (Berghard and Buck, 1996; Dulac and Axel, 1995; Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997) (Figure 8).

Figure 8. Signal in apical and basal VSNs. Ligand binding of VRs leads to activation and dissociation of the associated G-proteins. In the apical population (left) the V1Rs couples to Gαi2β2γ2 while the basal V2Rs expressing zone couples to Gαoβ2γ8. The major second messengers in VSNs are likely to be IP3 and DAG that are produced by phospholipase C when stimulated by Gβγ dimers. Influx of Ca2+ through TRP2 channels initiates a depolarization.

27 Upon ligand binding, the heterotrimeric G-proteins transduce and modulate the signal by eliciting diverse intracellular responses [reviewed in (Hamm, 1998)]. This type of G-protein is made up of α, β, and γ subunits that can be distinguished into four main classes based on their Gα subunits: Gs/olf – that activates adenylyl cyclase, Gi/o – that inhibits adenylyl cyclase, Gq/11 – that activates PLC, and lastly G12/13 [see review (Cabrera-Vera et al., 2003)]. Beside the Gα subunit there are several different Gβ and Gγ subunits that act together as dimers, which seem to occure in tissue specific combinations. When GTP-bound Gα dissociates from Gβγ after receptor activation, both parts are free to modify the activity of downstream targets. In addition, a certain combination of Gα and Gβγ subunits are likely to be needed for connecting a particular receptor to a specific signaling pathway. The high expression of Gαi2 and Gαo subunits will upon activation of VRs conceivably lead to high levels of free Gβγ dimers that in turn can activate PLC. Localization of the two Gα subunits to the VN sensory microvilli and co- localization of TRP2 argues for a functional importance (Berghard and Buck, 1996; Liman et al., 1999; Menco et al., 2001). Further exploring the function of Gαo in VSNs by analyzing Gαo knock out mice, confirmed its significance for survival of the basal zone neurons (Tanaka et al., 1999). A role for the Gβγ dimer in activating PLC in VSNs has been shown (Runnenburger et al., 2002). It was shown that scavengers for Gβγ dimers and antibodies against Gγ2 and Gγ8 blocked IP3 induction in VSN membrane preparations in response to urine and α2u-globulin stimulation. The Gγ8 subunit is preferentially expressed by basal VSNs, Gγ2 is expressed in apical VSNs, whereas the Gβ2 subunit is expressed in both populations (Runnenburger et al., 2002). (Figure 8) The switch between a GTP-active and GDP-inactive state of the Gα-protein is accelerated by the regulators of G-protein signaling (RGS) family of GTPase- activating proteins. A thorough investigation of the different RGS expressed in the VN organ demonstrated that RGS9 and RGSZ1 are present equally apical and basal VSNs while RGS3 is co-expressed with V1R and Gαi2 in apical VSNs (Norlin and Berghard, 2001). The cAMP producing enzyme ACII is expressed by Gαi2 and Gαo neurons (Berghard and Buck, 1996). The enzymes degrading cAMP, phosphodiesterase-4A (PDE4A) and PDE4D are located to apical and basal VSNs, respectively (Cherry and Pho, 2002; Lau and Cherry, 2000). The significance of these different expression patterns is presently unknown. That VR signaling appears important for survival of VSNs has been noticed in -/- -/- -/- studies of several knockout mice, e.g. TRP2β , Gαo , β2-microglobulin and mice targeted deletions of a large number of V1Rs (Del Punta et al., 2002a; Leypold et al., 2002; Loconto et al., 2003; Stowers et al., 2002; Tanaka et al., 1999).

Vomeronasal receptor - ligand interaction Most animals use for communication between members of the same species. The original definition by Karlson and Lüscher (1959) of a reads ‘‘Pheromones are defined as substances which are secreted to the outside by an individual and received by a second individual of the same species, in which

28 they release a specific reaction, for example, a definite behavior or a developmental process’’ (Karlson and Luscher, 1959). This definition is a little problematic to use when it comes to mammals since specific behaviors can be hard to define (Brennan and Keverne, 2004). Moreover, in the last two-three years the concept of dividing the olfactory system into two functionally separate systems turns out not to be that simple [reviewed in (Brennan and Keverne, 2004) (Rodriguez, 2003) (Restrepo et al.)]. Detection of pheromones by OE of mammals has been demonstrated in the following behaviors: the nipple search in rabbit pups, maternal behavior in ewes, and attraction and mating stance in sows (Hudson and Distel, 1986) (Dorries et al., 1997) (Levy et al., 1995). Vice versa, odorant molecules other than pheromones can be stimulate the VSNs (Sam et al., 2001) (Trinh and Storm, 2003). Thus it is important with an awareness and not to think of the VN organ as the only pheromone sensor. Urine contains both volatile chemicals and non-volatile major urinary proteins (MUP) belonging to the lipocalin family [MUP are reviewed in (Beynon and Hurst, 2003)]. These stimuli may activate different VN zones in mice. A biochemical response to small volatile compounds can be inhibited by antibodies against the apically expressed Gαi2 and Gγ2, whereas antibodies against basally expressed Gαo and Gγ8 reduce the response to α2u-globulin (a MUP) stimuli (Krieger et al., 1999; Runnenburger et al., 2002) . An appropriate working hypothesis may thus be that there are different types of ligands for the apical V1Rs and basal V2Rs. High sensitivity of the VSNs to stimuli has been shown both in vitro and in vivo (Holy et al., 2000; Leinders-Zufall et al., 2000). Male pheromones activate VSNs of the female mice in a dose dependent way, as shown by increased influx of Ca2+, without recruitment of additional VSNs as the concentration increases. Assuming the one VR - one VSN rule leads to the hypothesis that the VRs are narrowly tuned, i.e. that a given VR respond to a specific ligand.

Vomeronasal mediated behaviors What types of behavior may be influenced by signals from the VN organ? Most of our knowledge about VN function comes from results obtained on animals after surgical removal of the VN organ, known as VNX, in different rodent species [reviewed in (Halpern and Martinez-Marcos, 2003; Wysocki and Lepri, 1991)]. Recently an article demonstrated that AOB of freely moving mice, was activated differently depending on the strain and sex of stimulus animals (Luo et al., 2003). Some of the behavioral phenotypes and physiological responses influenced by VNX of mice include: male and maternal aggression, male sexual preference, puberty/estrus regulation, and block of pregnancy [(Halpern and Martinez-Marcos, 2003) and references therein].

Aggression Removal of the VN organ severely reduces aggressive intermale behavior in mice, as is urine marking by the male (Bean, 1982a; Clancy et al., 1984; Maruniak et al., 1986). Female mice rarely show any aggressive behavior as compared to males [reviewed in (Miczek et al., 2001)]. However, lactating females defend their pups

29 against unfamiliar male intruders (maternal aggression), a response that can be virtually abolished by VNX surgery (Bean and Wysocki, 1989).

Reproduction related behaviors Male mice ultravocalize (~70 kHz) when encountering a female and such gender recognition behavior is dependent on an intact VN organ (Bean, 1982b; Wysocki et al., 1982). Moreover, VNX males display significantly reduced copulatory behavior (Clancy et al., 1984). The role of the VN organ in sexual development is evident by a block in puberty acceleration when the young females are VNX (Drickamer and Assmann, 1981; Kaneko et al., 1980; Lepri et al., 1985; Lomas and Keverne, 1982; Vandenbergh, 1973). Moreover, the urine from adult VNXed females does not delay onset of puberty in females (Lepri et al., 1985). The estrus cycle in mice is normally 4 days. A feature seen in control mice, but not in adult VNX females, is the ability to suppress the estrus cycle when group housed, (Archunan and Dominic, 1991; Lepri et al., 1985; Reynolds and Keverne, 1979). This suggests a role for the VN organ in sensing the number of reproductive females in the population. A return to estrus occurs when newly mated female mice are exposed to strange males before embryo implantation, an effect termed the Bruce effect [reviewed in (Halpern and Martinez-Marcos, 2003)]. The implant failure is blocked by VNX thus allowing the pregnancy to continue (Bellringer et al., 1980; Lloyd-Thomas and Keverne, 1982). Recently different fractions of male urine were tested separately for their ability to mediate the pregnancy block effect (Peele et al., 2003). Only the low molecular fraction could convey information about the mating male identity and thus elicit the Bruce effect. In some VNX experiments, different effects were seen depending on age and prior experience of the mouse. However, with genetic tools it has now become possible to avoid the pre-experience problem by studying mice that never had a functional VN organ due to effects of targeted deletions of genes, with a critical function in VSN signal transduction (Leypold et al., 2002; Stowers et al., 2002).

Genetic modifications in the VSNs A mouse mutant strain that resembles the VNX mice in the sense that no stimulus can activate the VSNs is the TRP2β knock out mice. These mice show, in similarity to VNX mice, no intermale aggression or maternal aggression, however with regard to male sexual behavior the TRP2β -/- mice behave differently compared to control mice (Leypold et al., 2002; Stowers et al., 2002). Strikingly, the TRP2β -/- males display sexual behavior towards both females and males. There is no effect of the TRP2β -/- mutation for male courtship behaviors and sexual performance towards females, which is in contrast to the reduced sexual behavior seen for VNX males (Clancy et al., 1984; Leypold et al., 2002; Stowers et al., 2002). Furthermore, TRP2β mutant males vocalize both to male and female cues (Stowers et al., 2002).

30 The first analysis addressing the function of one defined VSNs subpopulation was done by Del Punta et al., who generated mice that were targeted deleted in a V1R gene cluster (Del Punta et al., 2002a). In these mice ~12% of the functional V1R genes are deleted. Behavioral phenotypes of V1R mutated mice were reported to include a modest reduction in maternal aggression, reduced male-male mounts in first aggression trail and a slight reduction in male sexual performance towards females (Del Punta et al., 2002a). Basal VSNs co-express β2-microglobulin and V2Rs. In β2-microglobulin -/- mice, basal VSNs are presumably deficient in V2R signaling since the localization of V2Rs to dendrites is severely compromised. Results of behavioral analysis of these mice were interpreted to suggest that basal VSNs were required for intermale aggression in the resident-intruder assay (Loconto et al., 2003). Together the results described above have led to the conclusion that certain VN mediated behaviors require that one VN subpopulation is intact. However, it is still not clear to what extent the two VN subpopulations are redundant with regard to detection of different cues that influence the same behavior.

31 AIMS

This thesis deals with one of our – the (olfaction). The aim of this thesis has been to elucidate molecular and behavior regulatory differences between defined zones of the primary neurons of main and accessory olfactory systems in mouse. In order to further our understanding of the initial information processing in these systems.

The specific aims were to:

Identify genes that are spatially expressed in a manner that correlate with zonal organization of the olfactory sensory map.

Investigate vomeronasal phenotype and behavioral differences in mice with altered gene expression affecting defined zones of the vomeronasal sensory map.

32 RESULTS AND DISCUSSION

This section gives a summary and discussion of the main results from the papers included in the thesis. Details about methodology and results are found in the papers. Overview

The regulation and function of the olfactory and VN sensory epithelia’s zonal organization is still elusive. In the first two papers (I and II), identification of genes expressed in a manner that correlates with the zonal expression patterns of different OR and VR genes are presented. The possible role of identified genes in establishment of cell specificity, precise axonal guidance and zone-specific neuronal function is discussed. Paper III and IV address the zonal dichotomy of the VN epithelium and the AOB by using genetic modifications that affect the apical and basal zone, respectively. Proposed functions of the two zones in regulating behaviors influenced by the VN organ are discussed. Patterns of gene expression in the primary olfactory neurons: correlation to receptor expression zones (Paper I and II)

Genes potentially involved in cell specification in the olfactory epithelium and in axonal guidance The fact that OSNs regenerate and that a zonal organization re-establishes after injury, even without target cells, raises interesting questions how the specificity of new OSNs is regulated. Signals could be intrinsic to the progenitor cells, such that basal cells are pre-programmed to generate neurons with a specific zonal cell fate. Alternatively, signals from the local OE environment could give the progenitors positional cues that instruct them in the choice of a zonal gene program. Results obtained from transgenic mice suggest that zone-specific expression of ORs is regulated in part by DNA regions upstream of OR genes (Qasba and Reed, 1998; Serizawa et al., 2000). We performed a RT-PCR-based screen for the expression of known transcription factors in the adult OE. With the criterium that identified genes should be spatially expressed in the OE, the obtained PCR clones were used to screen an olfactory cDNA library to obtain probes that were subsequently used in in situ hybridization analyses. By this approach we identified the Msx-1 to be expressed, in what appears as a scattered basal cell population, in a zonal manner (Figure 9). The Msx-1 mRNA levels are high in zone 4 (Z4) and progressively lower in Z3 to Z2 (Paper I). No Msx-1 signal above background can be detected in the basal cells of Z1.

33 Retinoic acid (RA) is a well known and involved in development and differentiation of many parts of the nervous system by regulating gene expression [reviewed in (Maden, 2002)]. Studies have indicated that retinoic acid- synthesizing retinaldehyde dehyrogenase 2 (RALDH2) expression is important for development of the lateral structures of the nasal cavity (LaMantia et al., 2000). Intriguingly, we find that a rate limiting enzyme in RA biosynthesis, RALDH2, shows graded expression in cells of the lamina propria (Figure 9). In other systems RA has been shown to regulate the expression of Msx-1 and in the OE the RALDH2 expression gradient correlates to that of Msx-1 (Z4>Z1) (Paper I). In addition, given that Msx-1 protein is a downstream transcription factor of BMP- signaling we have searched for regional differences in expression of members within the BMP/Activin gene family and their receptors. We find that Alk-6, a BMP type I receptor, is expressed in sustentacular cells in a gradient opposing to that of Msx-1 (Z1>Z4) (Figure 9 and Paper I). BMP may therefore regulate spatial gene expression in sustentacular cells. The graded expression of Alk6 in sustentacular cells does however not exclude an indirect mechanism that establishes topographic constrains important for spatial gene regulation in juxtaposed OSNs. In a search for zonally expressed axonal guidance molecules we found that neuropilin-2 was expressed in OSNs in a manner that correlated with Msx-1 and RALDH2 (Figure 9 and Paper I). Together, this suggests that the identified counter gradients of gene expression may be involved in the specification of OSN progenitor cells and for OSNs (see Paper I for references).

Figure 9. Gene expression gradients correlating with OR gene expression zones. We identified four genes expressed a gradient manner in four different cell types of the OE. Alk6 is the only one gene that has a high expression level in Z1 declining towards Z4 (sustentacular cells). RALDH2 is in the lamina propria (Z4>Z1) and Msx-1 is expressed in basal cells (Z4>Z2) and both match the OSN step gradient of neuropilin-2 (Z4>Z2).

These results show that in adult OE tissue some genes are indeed expressed in patterns correlating to the OR zones (Paper I). Moreover, these genes are expressed in different cell types of the OE (Paper I). (Figure 9)

34 Formation of odorant receptor zones by expression gradients A solution for creating borders in a cell layer is to set up two opposing gene expression gradients. The zonal organization of OR gene expression could be a result of two or more counter gradients where one gene product is abundant in Z1 and low in Z4 whereas the other shows decreasing expression from Z4 to Z1 . We investigated the boundaries between the four zones and found that only Z1 and Z2 have a sharp boundary (Paper I). Interestingly, due to considerable distribution of neurons expressing Z3 receptors within both Z2 and Z4 the Z2-3 and Z3-4 borders are not as defined as the Z1-Z2 border (Paper I). Zonal organization may thus not be as well defined as first believed, rather zonal OR expression can in some cases overlap (Iwema et al., 2004) (Paper I). However, there is still an OSN population in the center of the Z3 that exclusively expresses Z3 receptors, a finding that argues for the existence of three zones in the ventrolateral OE (Paper I). (Figure 9) Gradients play an important role in axonal guidance. The axonal growth cone encounters along its path various attractive or repulsive cues and the gradients of these may steer it along the right track and to the final target [(Osterfield et al., 2003) and references herein]. Gradients in the OB have been proposed to have a role in the guidance of OSNs axons to the specific glomerulus (Gierer, 1998). Indeed we find that the axonal guidance receptor neuropilin-2 has an expression pattern in the OSNs that match such a hypothesis (Figure 9 and Paper I). The Z4 to Z1 stepwise higher gradient of neuropilin-2 seems to follow the same topography as RALDH2 indicating a function of RA in the regulation of neuropilin-2. To strengthen the indication of neuropilin-2 involvement in OSN axon guidance, two semaphorins specifically acting as ligands to neuropilin-2 are expressed by the projecting target neurons and/or interneurons in the OB (Paper I). The studies on the neuropilin-2 null mice show some axon targeting defects in the OB but the major phenotype of these mice is in the VSN projection (Cloutier et al., 2002; Walz et al., 2002). Redundancy due to overlapping functions by other guidance molecules may underlie the absence of a major phenotype in OSNs of neuropilin-2 null mice.

Identification of a new zonally restricted gene Of the genes we identified in paper I none shows expression restricted to a specific zone. Previous results have shown that cell adhesion molecule RNCAM is expressed evenly within the Z2-4 OSNs whereas Z1 OSNs show no expression (Alenius and Bohm, 1997). OSNs located in Z1, i.e. that are complementary to RNCAM-positive OSNs, have been shown to have enzymatic activity that catalyzes the reduction of nitroblue tetrazolium to insoluble blue formazan in the presence of the electron donor NADPH (Paper II). In addition to Z1 the apical zone of the VN organ has the same enzymatic activity. In paper II we identify one enzyme responsible for this activity as NADPH:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2) (Figure 10). NQO1 is a flavoprotein that catalyses the two-electron reduction of quinones e.g. generated by a cytochrome P450 (Ross et al., 2000). NQO1 is the first protein identified that selectively mark axons of Z1

35 neurons (Paper II). Specifically, NQO1 protein can be detected in dendritic knobs, soma and the entire length of the axons from OE to glomeruli.

Figure 10. Gene expression restricted to either dorsomedial or ventrolateral OE zone. NQO1 is expressed by OSNs in the RNCAM negative Z1. Consecutive coronal OE sections analyzed by in situ hybridization revealed a sharp border between Z1 and Z2. Dorsal is up and medial left.

Interestingly, we find that NQO1 positive (Z1) and RNCAM-positive (Z2-4) axons segregate in the olfactory nerve (Paper II). Since the OSN axons are non- myelinated this selective fasciculation allows for interactions between neighboring axons that could e.g. alter the frequency of action potentials (Bokil et al., 2001). Another possibility is that these zone-specific fascicles facilitate path finding for axons of the newly generated OSNs as they grow towards the OB. The finding that NQO1 is restricted to the Z1 OSNs and the apical VN zone raises interesting questions on the function of this phase II enzyme in olfaction. Zonal expression of detoxifying enzymes in OE has been reported previously but not in OSNs (Miyawaki et al., 1996). Intraperitoneal injections of olfactory toxins such as dichlobenil can result in a preferential Z1 neuronal degeneration (Brandt et al., 1990; Vedin et al., 2004). The co-localization of the toxic cell damage and NQO1 expression is interesting. However, a role in the detoxification of volatile xenobiotic chemicals or quinone containing odorants is perhaps not that likely considering the zonally restricted NQO1 expression and that such chemicals are likely to spread over the entire OE sheet. Nevertheless, a recent paper showed that to a scarab beetle pheromone can be caused by the inhibition of a pheromone-degrading enzyme (Maibeche-Coisne et al., 2004). The fact that this enzyme is a cytochrome P450 and the pheromone is a quinone is intriguing. The quick enzymatic removal of a pheromone could also be important in the mammalian VN organ. The anatomical constrains of a blind ended VN tube may possibly benefit of an enzymatic removal of pheromones. Endogenous quinones may be abundant rest products of auto-oxidized dopamine in the OB. The function of NQO1 in the dorsal glomeruli may thus be to protect the axons from such neurotoxic compounds. However, no direct evidence for topographic variations in dopamine or its metabolites in OB or AOB is present.

36 Differences between the dorsomedial and ventrolateral zones in the olfactory epithelium Together these genes are expressed in different patterns: graded or evenly expressed, zone restricted or not (Paper I and II). Two notable features that emerge are the sharp border between the Z1 and the Z2-4 and the few genes identified to be exclusively expressed in Z1 OSNs and the basal progenitor cell layer of Z1. Besides NQO1, only two genes have so far been reported to be selectively expressed in Z1 OSNs. The signal regulating protein RGS9 is localized to the dendrites of mature Z1 OSNs. However, RGS9 is also expressed in the progenitor cell layer throughout the epithelium (i.e. in all zones; ref. (Norlin and Berghard, 2001). Olfactory specific medium-chain acyl-CoA synthetase (O-MACS) is a gene expressed in Z1 OSNs as well as Z1 sustentacular cells and progenitor cells (Oka et al., 2003). O-MACS is zonally expressed as early as embryonic day 11.5 in the rat. O-MACS participates in fatty acid activation and intriguingly, disturbances in fatty acid metabolism is indicated from studies of NQO1 knock-out mice (Gaikwad et al., 2001; Oka et al., 2003). So far no gene, beside the ORs has been found to be solely expressed in either Z2, Z3 or Z4. In an attempt to identify the transcription factors responsible for the Z1 specific regulation of NQO1 we analyzed for two transcription factors, known to bind to the aromatic response element in promoter region of NQO1, but these did not show zone restricted expression (Paper II). An interesting experiment is to use a transgenic approach to analyze if the gene regulatory regions of NQO1 direct heterologous gene expression specifically to Z1 OSNs. An interesting possibility is that Z1 OSNs has a longer life span due to a neuroprotective function of NQO1 since OE in Z1 is thicker. One way to address this possibility is to study if NQO1 knockout mice have more apoptosis in Z1 OSNs. The zonal dichotomy of the vomeronasal epithelium (Paper III and IV)

Many mammals are born with their eyes and ears closed and thus rely primarily on their sense of olfaction. Of the two olfactory systems in mice, the main system is thought to be the one primarily used during pre-pubertal ages, while the accessory system adds olfactory information around puberty and there after. Surgical removal of the VN organ and recently genetic disruption of VSN function have provided information about role of the accessory olfactory system in the detection of cues that influence sexual, social and aggressive behaviors as well as physiological changes in e.g. hormone levels (for references, see introduction). Previous experiments have suggested that there may be different ligands for the two VR families. Two recent reports addressing the behavioral roles of the apical V1R positive and the basal V2R positive zone have begun the intriguing dissection of the significance of these zones (Del Punta et al., 2002a; Loconto et al., 2003). However, this research is still in an early phase.

37 We have started to dissect the molecular and behavior regulatory differences between the apical and basal zones of the VN organ by analyzing genetically modified mouse lines (Paper III and IV).

Gαi2 protein in survival and function of apical vomeronasal neurons One of the genes that differ in the expression between the two VSN zones is the heterotrimeric Gαi2 subunit (Berghard and Buck, 1996; Jia and Halpern, 1996). To examine what happens if the apical VSNs are lacking the major G-protein and hence presumably are not able to respond to pheromones, we analyzed the Gαi2 null mutant mice (Rudolph et al., 1995) (Paper III). We found that the apical VSN zone specifically decreased to approximately 50% in Gαi2 mutant as compared to control mice (Paper III) (Figure 11). In addition, the basal zone increased slightly in size. Apical VSN axons target the anterior glomerular layer of the AOB. In accordance with the reduction of the apical zone, analyses of the glomerular layer in the AOB revealed that the anterior part was significantly reduced in volume. Genetic targeting of VRs and other signaling components acting downstream of VRs, have been made (Belluscio et al., 1999; Rodriguez et al., 1999; Tanaka et al., 1999). Interestingly, mice that lack the olfactory specific CNG channel 1 have a reduced number of OSNs and OB volume (Baker et al., 1999). Taken together, these results indicate that both OSNs and VSNs die if OR and VR signaling is inhibited.

Figure 11. Reduction of apical or basal VSNs in two mutant mouse models. Loss of signaling through the Gαi2 protein leads to an obvious phenotype in the apical VSN population (top panel). The opposite was found in the OMP-dnRAR transgenic mice that virtually lack basal VSNs (lower panel). Grayscale images of PDE4A immuno- histochemistry with a light gray color indicating the apical PDE4A positive VSN population. In the top panel a double headed arrow demarcates the difference in apical thickness between control and Gαi2 mutant. Asterisks indicate the VN lumen. OMP- dnRAR transgenes have almost no basal VSNs compared to control (thick dashed line). The basal lamina of the VN organ is marked with a dashed line.

38 A useful marker for neuronal activity of VSNs is the induced expression of the c-fos immediate early gene in AOB target neurons (Brennan et al., 1992). To test the functionality of the VSNs remaining in the Gαi2 mutant we performed a soiled bedding assay on mutant and control males. The mutant males that were allowed to investigate female soiled bedding did not show any c-fos induction in the anterior glomerular layer that harbors the target neurons for apical VSNs (Paper III). This result indicates that apical VSNs in Gαi2 mutant mice are not activated in response to V1R ligands produced by female mice. Although unlikely it is possible that the Gαi2 participate in signaling from receptors other than V1Rs that are important for cell survival. Additional experiments need to be done to identify the course of events leading to the cellular Gαi2 phenotype. Since the apical VSNs selectively express Gβ2γ2, it would be interesting to study the levels of this dimer in the Gαi2 mutant, with a prediction that it will be reduced. In addition, the duct that allows passage of ligands to the VN organ does not open until postnatal day 5 (P5) (Coppola et al., 1993). One interesting experiment is to study if Gαi2 function is required for cell survival prior to P5. The corresponding experiment to the Gαi2 mutant, on the basal VSNs has been made by targeted deletion of the Gαo gene (Gαo mutant) (Tanaka et al., 1999). Interestingly, the morphological phenotype of the basal VSNs population in Gαo mutant mice is analogous to that described above for the apical VSN population in mice lacking a functional copy of the Gαi2 gene. The signaling deficient Gαi2 and Gαo mice also phenocopy each other in that the anterior and posterior glomerular target areas, respectively, are severely reduced in volume.

Involvement of retinoic acid in the maintenance of basal vomeronasal neurons In paper I, we identified a graded expression of RALDH2 in the lamina propria of the OE. We also show expression of this RA producing enzyme underneath the basal lamina of the VN organ. This expression pattern suggests that RA is produced close to the VSN axons (Paper IV). A transgenic approach was applied to study the effect of RA in mature VSNs. This was accomplished by expressing a dominant negative RAR (dnRAR) under the control of the OMP promoter that block all RA receptor (RAR) isoforms in their ability to initiate transcription (Hägglund et al., manuscript) (Figure 12).

39

Figure 12. RA synthesis and a transgenic construct to inhibit RA induced signaling. RALDH2 is a rate limiting step in the synthesis of RA from retinol. When the dominant negative RARα403 is expressed in the primary sensory neurons using the OMP promoter, this blocks RAR-mediated gene transcription.

Unexpectedly, we found that these OMP-dnRAR transgenic mice had a reduced basal Gαo-positive subpopulation of VSNs (Figure 11 and Paper IV). Measuring the volume of the target for Gαo VSNs in the posterior AOB showed that at 7 month of age it was reduced to 25% of that in control mice (data not shown). Comparing the total glomerular area at 1 week and 4 weeks of age demonstrated that the expansion of posterior AOB seemed to stop at early postnatal development in the OMP-dnRAR mice (Paper IV). To determine if the reduction in the basal VSN zone was due to increased cell death or reduced proliferation rate we analyzed for activated caspase-3 and BrdU incorporation in embryonic and postnatal pups. Only after birth did we observe a significant increase in activated caspase-3 positive cells in the VN epithelial layer of OMP-dnRAR mice compared to controls. Results of the proliferation assay showed similar numbers of BrdU positive postnatal VSNs in transgenic and control mice. Retrograde activation of caspase-3/-9 has been reported to drive apoptosis of mature OSNs in response to target deprivation (Cowan et al., 2001). whether Gαo- positive and Gαi2-positive VSNs are specified into being destined for the apical or basal zone is not evident at birth. Thus, to determine if the increase of activated caspase-3 positive VSNs at early postnatal dates corresponded to apical or basal neurons we analyzed the glomerular layer of the AOB. A robust increase in activated caspase-3 was observed in the posterior AOB of dnRAR transgenic mice at P4 and P7 (Paper IV). The results thus suggest that inhibition of RAR-mediated transcription results in selective death of basal VSNs in postnatal mice. This is the first in vivo study that indicates a role for RA mediated signaling in the maintenance of axonal projections.

40 Two mouse models to study zonal vomeronasal influences on behavior -/- The fact that Gαi2 and OMP-dnRAR mice showed deficiencies in complementary subpopulations of VSNs (apical and basal, respectively) allows for behavior studies that may reveal the specific function of this division of the accessory olfactory system (Rudolph et al., 1995) (Paper III and IV). VN-mediated behaviors that are changed in the Gαi2 mutant strain could thus be ascribed to being dependent on the apical zone while an unaltered behavior tentatively could be assigned to be mediated by the unaffected basal zone. Consequent behavioral testing of the -/- transgenic OMP-dnRAR and Gαi2 mice could facilitate the study of whether the apical and basal zones are working independently or together in influencing behavior in the mouse.

Proposed function of the apical and basal zones TRP2β gene targeted mice lack signaling capacity in all VSNs. Behaviorally these TRP2 mutant mice display abolished maternal aggression, intermale aggression and gender preference in mating (Leypold et al., 2002; Stowers et al., 2002) (Figure 13). To determine whether any of these altered behaviors could be ascribed to one VN zone, we utilized similar behavioral assays for Gαi2 mutants (Paper III) and for the transgenic OMP-dnRAR mice (Paper IV). Although the apical VSN likely is the cell type that expresses the highest levels of Gαi2, the mutant mice lack low level Gαi2 expression in many other tissues. We assured that the adult mutated mice behaved normally in a set of tests. For example, Gαi2 mutant mice behave identical to controls with regard to eye blink, ear, whisker, and postural reflexes (Crawley and Paylor, 1997) (Paper III). All mice reacted normally to an approaching object. Moreover, there was no significant difference between mutants and controls on several VN-independent behavioral tests. Importantly, using the resident-intruder assay to evaluate the isolation induced aggression revealed a significant reduction in several parameters of male aggressive behavior (Paper III) which is also true for VNX males in this type of assay (Bean, 1982a; Clancy et al., 1984). In contrast to the results of the aggression tests, Gαi2 mutant males behaved as controls in the gender preference assay (Paper III). Maternal aggression was investigated by introducing a male to the home cage of a lactating female. During this behavior assay, 90% of control and only 20% of the Gαi2 mutant females displayed any sign of aggression. In addition, the aggressive display was significantly lower in the few aggressive mutant females compared to control females. Thus maternal aggression is to a very large extent lost in Gαi2 mutant mice. However, the lack of maternal aggression did not affect other aspects of normal maternal behavior such as pup retrieval in which the mutant females behaved normally, as are VNX females reported to do (Bean and Wysocki, 1989). To summaries the behavioral phenotype of Gαi2 mutant mice; these mice show normal sexual performance and discrimination between sexes, but in contrast the display of aggressive response towards intruders is severely compromised (Figure

41 13). The results suggest that the apical V1R positive VSNs are involved in the regulation of aggressive behavior. The model is supported by the phenotype of V1R -/- mice which, despite the relatively minor interference with function of the apical zone, display a reduced maternal aggressive behavior (Del Punta et al., 2002a) (Figure 13).

Figure 13. Proposed functions of apical and basal VN zones. In TRP2 mutant mice (second from top), both apical and basal VSNs are non-functional. Behavioral studies show that these mice lack male and female aggression as well as male gender preference. Work from several labs makes it possible to propose a role for the apical VSNs in influencing aggressive behavior. The lack of a few V1Rs (V1R -/-) gives a behavioral phenotype of reduced maternal aggressiveness. The more severe loss of the apical VSN population in Gαi2 mutant mice renders both male and female aggression -/- affected while no effect is seen on gender preference (Gαi2 ). In OMP-dnRAR transgenic mice, with severely reduced basal VSN population but normal apical VSNs, intermale aggressive behavior is normal. Altogether, the behavior experiments point to a function of the apical VSNs in aggressive behavior. ? indicates not determined.

As described above, there is a severe reduction of the basal VSNs in OMP- dnRAR transgenic mice (Paper IV). In contrast to the behavior of Gαi2 mutants, initial studies of OMP-dnRAR males have not revealed any change in known VN- mediated behaviors. Transgenic male mice showed normal sexual preference for

42 females and equally high frequency of mounting attempts as control mice (Paper IV). A slight, but not significant, increase in intermale aggression was observed in the OMP-dnRAR mice compared to controls in the resident-intruder assay (Paper IV) (Figure 13). Such a result is not in accordance with a previously reported result from analysis of β2-microbglobulin null mice (Loconto et al., 2003). However, the data presented for β2-microglobulin null males with regard to intermale aggressive behavior is not comprehensive and differences in genetic background in the control and β2-microglobulin null mice in that study makes proper comparison difficult. It is well known that there are strain differences in male aggression [reviewed in (Miczek et al., 2001)]. Caution should be taken in interpreting the results from studies of the OMP- dnRAR due to the fact that the few remaining basal VSNs could still be functional. At the start of courtship towards females, male mice ultra-vocalize. This behavior has been shown to be VN mediated (Wysocki et al., 1982) (Figure 13). Interestingly, we observed altered female-female ultrasound vocalization in OMP- dnRAR mice. Isolated OMP-dnRAR females ultrasound vocalized significantly more frequently compared to controls when a female intruder was introduced into the home cage. Since very few experiments have been made on the female-female ultrasound vocalization behavior, it remains to be shown that the increase in ultrasound vocalization is due to the lack of basal VSNs only (Figure 13).

43 CONCLUDING REMARKS

The result and discussion section in this thesis can be summarized as follows:

There are counter gradients of gene expression in different OE cell types. Specifically we find that subpopulations of OSNs express the axonal guidance gene neuropilin-2 in a ventrolateral to dorsomedial step gradient that correlates with zonal OR expression. In cell types other than OSNs, we find a ventrolateral to dorsomedial gradient of Msx-1 and RALDH2 expression, whereas Alk6 is expressed in a reverse gradient. The identified genes may play a role in the formation and/or maintenance of a zonal organization of OSNs and their axonal projections. One defined zone in both the OE and the VN epithelium selectively express NQO1. This result indicates that one zone of both the main and accessory axonal projection maps is formed by sensory neurons that are specialized to reduce environmental and/or endogenous quinones. Using NQO1 as marker-protein to study the organization of axons in nerve-bundles suggests that axons of OSNs located in one defined zone form zone-specific fascicles before reaching the OB. The loss of Gαi2 function results in a reduced number of apical VSNs. VR-mediated signaling through the heterotrimeric G-protein thus appears important for cell survival. Inhibiting RA signaling in all mature VSNs severely reduces the basal VN zone. This result indicates that RA signaling is important for zone-specific survival of VSNs and suggests a previously unknown function of RA in maintaining precise neuronal connections. Behavioral studies of Gαi2 and OMP-dnRAR identify differences in aggressive behavior. Gαi2 mutant mice, with a deficient apical VN zone, do not display a complete aggressive response towards intruders whereas they retain gender preference. OMP-dnRAR transgenic mice, with a deficient basal VN zone, display normal male aggression and gender preference. These mice however, display an increased female-female ultrasound vocalization. We propose a model with apical VSNs detecting cues important for aggressive behavior, whereas both the apical and the basal VSNs detect cues that are important for gender preference.

The zonal organization of primary main olfactory system is still unknown in functional significance although identified new markers will increase the success rate of future investigations. However, knowledge about the functional relevance of dichotomy in the primary accessory olfactory system has increased a lot during these thesis years.

44 ACKNOWLEDGEMENTS

Somliga (ja nästan alla jag pratat med) har sagt att ett acknowledgement skrivs med förkärlek de sista skälvande timmarna eller kanske rent av minuterna innan avhandlingen går på tryck. Detta är inte ett undantag. First to my two supervisors, Staffan and Anna. During the last year I’ve realized more and more how really talented and hardworking you are. From the early years of crayfish parties, bowling victories and NJÖG to the last months of struggle with the thesis I have never questioned whether or not I choose the right lab for my PhD. Sometimes questioned the whole carrier, true but never this choice. I’m pretty sure that I’ll be even more amazed over your capacity an enthusiasm when I look back from wherever I end up. Shortly, thanks for all the help and guiding in my life, so far. The people that have come and gone in the lab during my years, thanks to all for keeping me in a good spirit and of course for your good music . Especially, a thought goes to Björn and Lennart. Björn I will always remember your face when you got a postcard from a certain person that didn’t know it was your birthday (I promise). Seriously, the whole lab misses you! And to Lennart, I only need to say “I år vinner Brynäs!” Thank you both. Mattias and Marianne, the original pack! We have had some really fine ordinary days together, the kind of days that just pass by but made the journey worth while. All the best in the future! Maria I really didn’t mean for you to jump out of a plane just because of that little post-it ”det går utför”. I really enjoy working with you, or should I say discuss science with you at 9:an. Which reminds me, we need another challenge now that the “delicato contest” is over… Viktoria, another artist in the lab. I don’t know what to say, more than - missed you when you’ve been away. Marie, as Karen said some time ago, you’re a star to meet in the corridor. Perhaps even sometimes a bokmärksängel! Johan, I really hope you will have some good years in the lab, keep up your curiosity and it can’t go wrong. Big thanks to Julie! We have to work together in the future, we’re such a team! You plan and work and I … well one could say. Big Thank You to the hardworking people at GDL and Media, and of course the secretary queens. Many people have passed by during my years in Umeå and if you’re not mentioned in here read the first lines again… Pekka&Maria och Stefan&Stina, det känns som en evighet sen vi var nere i Värmland och härjade runt. Ganska länge sen vi var i Sundsvall… men vi kommer fortsätta göra sådana små turer hoppas jag. Om di gamle orkar! Till Fredrik, Andreas och Annica – jag är snart där snart snart.. får jag vara med er då? Har saknat middagarna med er och framtids- planeringarna… tänk nu är vi snart där i framtiden (ja ni har ju redan varit där ett tag men jag var ju tvungen att vänta ;-). Joakim som gjorde en blixtvisit uppe i Umé på dryga året.. Kul att veta att vännerna från förr alltid finns där! Det gäller ju även dig Philip. Nu till lista på människor som jag har saknat så de sista månaderna. Johan, Gunnarn, David, Henrik, Niklas, Peter, Sven, Sofia, Sara, Petra, Petra, Kicki, Martina, Christina, Maria, Linda, Charlotte, Jeanette. Vilka glömda? Erik och Magnus nä knappast! Är det inte dags för en liten semesterresa nu? Och ja, jag ska kolla lab i London! Och nä du är knappast glömd ANNA jansson. Finns inget bättre gäng. Till sist går tankarna hem till Skåne som så många gånger under dessa år i norrlands mörka och kalla skogar ;-) Har tyvärr blivit längre mellan gångerna som man kommer hem till er men desto oftare i tanken… Mamma, Pappa, Richard, Annette, Niklas och resten av släkten. Vad kan man säga mer än att jag saknar och älskar er ! ! Slut på tiden…☺

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