BUILDING a RISK ASSESSMENT MODEL FOR MANAGEMENT OF PERSISTENT ENDODONTIC LESIONS

Abstract Olfactory studies in zebrafish have provided enormous insights into how olfaction occurs in an aquatic environment, triggering innate and stereotypical responses that allow surv

Copyright by Kee Zhi Ling Michelle

2015

Abstract

Olfactory studies in zebrafish have provided enormous insights into how olfaction

occurs in an aquatic environment, triggering innate and stereotypical responses that

allow survival in the vast habitat One of the odors detected by zebrafish is an alarm

substance from the lesioned skin of conspecifics, known as Schreckstoff A feature of

the alarm substance is increased potency caused by heating, and glycan content To

further characterize Schreckstoff, I have found that hyaluronan (HA), a simple linear

polysaccharide, which is broken to active signaling fragments by heat, evokes activity

in the olfactory system of larval zebrafish Through the use of wide-field

fluorescence microscopy to perform calcium imaging on transgenic zebrafish

expressing the genetically encoded calcium indicator GCaMP, I demonstrated that

HA is able to evoke calcium responses in the olfactory epithelium and bulb of

zebrafish as young as 5 days post fertilization This suggests HA can also function as

an odor, in addition to its diverse size-dependent roles in cell signaling Additionally,

I also describe and demonstrate the use of a microfluidic chip, as a concept” to aid in characterization of the olfactory sensory neurons that are responsible for detecting HA

“proof-of-v

Dedicated in loving memory of Yeye

“It doesn’t matter how long you take, but learn from the experience and enjoy the process.”

Thank you Yeye, I finally did it

Acknowledgement

This thesis would not be possible without the following people, to which my heartfelt

gratitude and appreciation cannot be expressed in mere written words:

To Associate Professor Suresh Jesuthasan – I thank you sincerely for your continuous mentorship, patience, guidance and support since 2007, especially during

the course of my degree and my two pregnancies Research aside, you will always

be the source of my motivation to seek maternal rights even as a PhD student

To my thesis advisory committee members, Assistant Professor Marc Fivaz,

Assistant Professor Adam Claridge-Chang, Associate Professor William F

Burkholder – My sincere thanks and gratitude for your invaluable knowledge, advice and critical suggestions throughout these years

To my colleagues in SJ lab - Thank you all for sharing your wealth of knowledge and

advice, in all aspects of science and life Dr Ajay Mathuru, thank you for being my

“personal critic” Special thanks to my fellow graduate student - Joanne, Lin Qian, Charlie and Mahathi - for keeping me healthy and mentally alive with our daily “4 p.m chocolate and apple time” Thank you all members of SJ lab, for converting me to a caffeine addict, without which I would probably not appreciate the close relationship

between coffee and science

To the members of MSB lab, both past and present – Thank you all for sharing your invaluable knowledge and advice on qPCR, microfluidics and RNA sequencing

vii

Last but not the least, I would like to express my deepest gratitude and appreciation

for my dear family members, especially to my parents for finally supporting me in

neuroscience endeavors; my husband for never failing to listen to my thoughts and

providing me with a loving refuge from science; my lovely sons, for keeping me

grounded, sane and youthful in the lab when the going gets tough and inspiring me

to understand cognitive development, processing and functions in young children

Contents

Title Page i

Abstract Signature ii

Copyright iii

Abstract iv

Dedication v

Acknowledgements vi

Table of Contents viii

List of Tables ix

List of Figures x

Chapter 1 Introduction 1

1.1 Difference between olfaction and gustation 2

1.2 Olfaction 3

1.2.1 How olfaction differs in terrestrial and aquatic animals 4

1.2.2 Olfaction begins in utero 5

1.2.3 Food, foraging and homing odors 7

1.2.4 Olfaction in social context 8

1.2.4.1 Recognizing individuals 8

1.2.4.2 Recognizing kin 9

1.2.4.2.1 Kin recognition and mate choice – role of MHC 10

1.2.4.2.2 Kin recognition and mate choice – role of major urinary proteins 11

1.2.4.3 Recognizing predators 12

1.3 Recognizing danger – smell of threatening situations 13

1.3.1 Source of Schreckstoff 14

1.3.2 Components of Schreckstoff 14

1.4 Hyaluronan 17

1.4.1 Introduction of hyaluronan 17

1.4.2 Role of HA depends on its polymer size 18

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1.4.3 Role of HA depends on its binding proteins 20

1.4.4 HA degradation 21

1.4.4.1 Enzymatic fragmentation of HA 21

1.4.4.2 Thermal fragmentation of HA 22

1.4.4.3 Degradation of HA by free radicals 23

1.5 Introduction to zebrafish olfactory system 24

1.5.1 Advantages of zebrafish as a model system 24

1.5.2 Anatomy of the zebrafish olfactory system 25

1.5.3 Types of OSNs 26

1.5.4 Olfactory bulb 30

1.5.4.1 Odor coding in the olfactory bulb 32

1.5.5 Projection from OB to higher brain centers 33

1.6 Aims for this thesis 36

Chapter 2 Zebrafish detect hyaluronan as an odor 37

2.1 Abstract 37

2.2 Introduction 37

2.3 Material and Methods 40

2.3.1 Animals 40

2.3.2 Skin extract preparations 41

2.3.3 Calcium imaging experiments 41

2.3.4 Odors stimulation 42

2.3.5 Calcium image processing and analysis 43

2.3.6 Dissection of olfactory epithelia 44

2.3.7 Confocal imaging 44

2.4 Results 45

2.4.1 Experimental workflow 45

2.4.2 Et(sqKR15-3A) labels olfactory sensory neurons 47

2.4.3 OSNs respond distinctly when exposed to separate odors 50

2.4.4 A component in the WGA column elution buffer activates calcium

activity in OSNs 56

2.4.5 Different sizes of HA activates the olfactory bulb 57

2.4.6 Distinct glomeruli can be identified with Tg(NBT:GCaMP5) 60

2.4.7 Exposure to a range of HA sizes at similar concentrations displayed distinct responses over time 62

2.4.8 Nanomolar concentration range of HA-L inhibited medial anterior glomeruli of the olfactory bulb 65

2.4.9 Similar concentrations of HA-L and HA-M evoked activity at maG, mdG and vmG clusters 69

2.4.10 Exposure to HA-S evoked excitatory activity specifically in mdG and vmG clusters 71

2.4.11 HA evoked similar glomerular activities across sizes, which were distinct from glomerular activities after L-lysine stimulation 75

2.4.12 Different sizes of HA at similar concentrations activated OSNs 79

2.4.13 The olfactory bulb remains active to HA exposure after two trials 80

2.5 Discussion 83

Chapter 3 A microfluidic device to sort olfactory sensory neurons based on dynamic response to different odors 95

3.1 Abstract 95

3.2 Introduction 96

3.3 Material and Methods 98

3.3.1 Microfluidic device fabrication 98

3.3.2 Isolation and dissociation of olfactory epithelial cells 98

3.3.3 Cell viability assay 99

3.3.4 Calcium indicator dyes 99

3.3.5 Ionophore and odorants stimulation 100

3.3.6 Image acquisition and processing 100

3.3.7 qPCR of single OSN 101

3.4 Results 102

3.4.1 Microfluidic device design 102

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3.4.2 Device operation and preparation 103

3.4.3 Olfactory epithelial (OE) cells viability and sorting efficiency 106

3.4.4 Microfluidic chip allows active monitoring of intracellular calcium changes in response to external stimulus 108

3.4.5 Microfluidic chip allows correct sorting and recovery of subpopulation of cells 110

3.5 Discussion 114

Chapter 4: References 117

Appendix A 135

Biography 144

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List of Figures

Figure 1-1: Schmatic representation of GAGs 19

Figure 1-2: Olfactory system of zebrafish 27

Figure 1-3: Four unique classes of olfactory sensory neurons (OSNs) 29

Figure 1-4: Organization of the olfactory bulb network 31

Figure 1-5: Schematic overview of axon projections from the olfactory bulb to other forebrain regions 35

Figure 2-1: Schematic diagram of experimental workflow 46

Figure 2-2: Double fluorescent labeling of Et(sqKR15-3A)/NBT:GCaMP5 transgenic zebrafish 49

Figure 2-3 (A-F): Calcium activity in Et(sqKR15-3A)/NBT:GCaMP3 olfactory epithelium at dorsal (A-F) planes 53

Figure 2-3 (G-L): Calcium activity in Et(sqKR15-3A)/NBT:GCaMP3 olfactory epithelium at ventral (G-L) planes 54

Figure 2-4: Thunder analyses of Et(sqKR15-3A)/NBT:GCaMP3 olfactory epithelium on exposure to various odors at dorsal and ventral planes 55

Figure 2-5: Changes in calcium fluorescence intensity of L-lysine and different sizes of HA with similar concentrations in olfactory bulb of NBT:GCaMP5 59

Figure 2-6: Distinct glomeruli identified in olfactory bulb of Tg(NBT:GCaMP5) 61

Figure 2-7: Thunder analyses of different sizes of HA with similar concentrations in olfactory bulb of Et(sqKR15-3A)/NBT:GCaMP5 64

Figure 2-8: Thunder analyses of lower concentrations of HA-L and L-lysine in olfactory bulb of Tg(OMP:lyn-mRFP)/NBT:GCaMP5 67

Figure 2-9: Thunder analyses of higher concentrations of HA-L and L-lysine in olfactory bulb of Tg(OMP:lyn-mRFP)/NBT:GCaMP5 68

Figure 2-10: Thunder analyses of HA-L and HA-M at similar concentrations, with L-lysine in olfactory bulb of Tg(OMP:lyn-mRFP)/NBT:GCaMP5 70

Figure 2-11: Thunder analyses of HA-M and HA-S at similar concentrations, with L-lysine in olfactory bulb of Et(sqKR15-3A)/NBT:GCaMP5 73

Figure 2-12: Thunder analyses of HA-M and HA-S at a ten-fold higher concentration, with L-lysine in OB of Et(sqKR15-3A)/NBT:GCaMP5. 74

Figure 2-13: Changes in calcium fluorescence intensity of L-lysine and different sizes of HA with similar concentrations in OSNs of Et(sqKR15-3A)/NBT:GCaMP3 77

Figure 2-14: HA evoked distinct glomerular activities from L-lysine stimulation in various glomeruli clusters 81

Figure 2-15: Et(sqKR15-3A)/NBT:GCaMP5 OB responses to two trials of various stimuli 82

Figure 2-16 Mass spectrometry peak of active fraction of Schreckstoff 89

Figure 2-17: Juvenile zebrafish significantly swam slower and closer to the wall after

250 nM-1µM HA-M delivery than when Schreckstoff was delivered 94 Figure 3-1: Overview of experimental design and diagram of microfluidic device…104 Figure 3-2: Operational characteristics for odorant stimulation and cell recovery 105

Figure 3-3: Ionophore A23187-induced calcium influx in zebrafish olfactory epithelial cells 109

Figure 3-4: Lysine stimulation of a heterogenous cell population from OE of

TrpC2:gapVenus transgenic zebrafish 112

Figure 3-5: qPCR analyses of cells isolated after exposure to various odorants 113

Chapter 1: Introduction

"Smell is a potent wizard that transports us across thousands of miles and all the

years we have lived The odors of fruits waft me to my southern home, to my

childhood frolics in the peach orchard Other odors, instantaneous and fleeting,

cause my heart to dilate joyously or contract with remembered grief Even as I think

of smells, my nose is full of scents that start to awake sweet memories of summers

gone and ripening fields far away."

- Helen Keller

Imagine you are walking down an empty street on a cool early morning, with rays of

sunlight shining through the leaves of tall trees You take a deep breath of fresh crisp

air, enjoying the moment as you hold your tumbler of freshly brewed coffee Without

realizing it, we are surrounded by a treasure of sights, sounds, smells, and textures

that arouse our various sensory modalities, guiding our behavior in ways that we are

not conscious of These modalities are vital for survival; without them, we would not

be able to select our food, communicate and navigate through our environment

These sensory systems may even determine life or death Animals use sensory cues

to explore the environment and retrieve vital information that are integrated and

processed in the brain, consequently allowing animals to respond in an adaptive

manner to enhance survival Of these sensory cues, chemosensory communication

is phylogenetically the oldest form of communication between organisms It is

present in organisms across phyla, from bacteria (Berg, 1975) to nematodes

(O’Halloran et al., 2006), arthropods (Rittschof, 1992) and mammals (Johnston et al., 1993; Meyerhof and Korsching, 2009), discriminating other organisms from their

conspecifics and biological status through the release of specific chemicals into the

local surroundings

1.1 Differences between olfaction and gustation

Chemosensory communication is deeply intriguing, as it functions in all environs (air,

land and sea) and continues to persist in spite of rapid evolutionary change

(Symonds and Elgar, 2008) While chemosensation includes chemethesis (pain,

touch and thermal dermal sensation), olfaction (smell) and gustation (taste) (Finger

et al., 2000), olfaction and gustation are nowadays identified as the most ubiquitous

of all sensory systems (Hildebrand and Shepherd, 1997; Ache and Young, 2005;

Penn, 2006) Furthermore, these two chemosensory systems are the most

perceptually intertwined: in humans, as presumably in other animals, the flavor of a

nutriment is highly regulated by the simultaneous perception of its odor

In vertebrates, olfaction is defined as the detection chemicals at a long-range

distance, either in air or water, by olfactory sensory neurons (OSNs) with axons in

the olfactory nerve (cranial nerve I) (Hildebrand and Shepherd, 1997) Gustation,

however, requires direct contact with chemical compounds dissolved in solution and

is mediated by non-neuronal, polarized epithelial cells known as taste receptor cells

(TRCs) Additionally, TRCs are innervated by the facial (cranial nerve VII),

glossopharyngeal (cranial nerve IX) and vagal (cranial nerve X) nerves that project to

different brain structures and have diverse functions (Atema, 1977; Caprio et al.,

1993)

Studies underlying the molecular biology and neurobiology of sense of taste and

smell have also revealed fundamental differences in stimulus processing by these

two sensory systems Olfactory circuits are optimized for combinatorial detection of a

vast number of odorants (Friedrich and Korsching, 1997; Malnic et al., 1999;

Araneda et al., 2000; Katada et al., 2005; Nakagawa et al., 2005; Hallem and

Carlson, 2006), while the gustatory system categorize taste into unique and

topographically defined modalities – bitter, sour, salty, sweet and umami (the savory taste of monosodium glutatmate) (Nelson et al., 2001; Zhang et al., 2003; Zhao et al.,

2003; Chandrashekar et al., 2006; Huang et al., 2006; Behrens and Meyerhof, 2009;

Chen et al., 2011)

Lastly, olfaction and gustation can be distinguished by their function Gustation is

known to mediate simple, immediate reflexive behaviors, especially toward food

(such as choice and ingestion) Olfaction, on the other hand, tends to mediate more

complex behaviors, such as in mate selection, and sensing for predators (Todd et al.,

1967; Brechbühl et al., 2008; Gerlach et al., 2008; Getz and Page, 2010)

Although both olfaction and gustation are chemosensory systems, olfaction is the

principal chemosensory system used in most animals Previously ranked as one of

the “lower” senses by ancient philosophers such as Plato and Aristotle, the olfactory system remained the most enigmatic of our senses until 1991, when Linda Buck and

Richard Axel published a joint groundbreaking paper that sheds light on olfactory

receptors and how the brain recognizes and remembers a plethora of odors (Buck

and Axel, 1991) They were awarded the 2004 Nobel Prize in Physiology or Medicine

for their pioneering work that permits a deeper understanding of the olfactory system

In this dissertation, the focus will mainly be on olfaction

Olfaction, the sense of smell, has baffled and also thrilled epistemologists since

philosophy began (Ackerman, 1990) It is known for its chemical sensitivity, with only

a few molecules of an odorant needed to cross a large distance before recognition,

awareness and distinction of the odor takes place with great precision Yet, pure

odors are rare in nature; odorants are commonly an array of chemical molecules

Hence, as aptly highlighted by Bargmann, the olfactory system, unlike the visual and

auditory system which detects “immutable physical entities”, is analogous to the immune system, with its “remarkable ability to track constantly changing cues generated by other organisms, and continually generate, test and discard receptor

genes and coding strategies over evolution” (Bargmann, 2006)

1.2.1 How olfaction differs between terrestrial and aquatic animals

To further appreciate the complexity of olfaction, we must first understand and

distinguish how olfaction takes place in various animals and its many roles in

ensuring survival

In the olfactory environment of terrestrial organisms, odors of distant objects are

brought to the nose through diffusion of odorant molecules by wind These molecules

leave their source into the air, which itself contains other air packets of odorant

molecules from diverse sources The airborne, volatile chemical molecules are then

mixed, as they move together with the wind Consequently, odors that reach the

nose are composed of multi-molecular mixtures, often containing hundreds of volatile

components (Knudsen et al., 1993) Added complexity arises as the precise

composition of the odor is progressively mixed with other odors in the environment

over time (Bossert and Wilson, 1963; Wright, 1982; Murlis, 1992) Moreover, the odor

source changes during its lifetime, causing variations to the composition of an odor

due to processes such as oxidation (Maqsood and Benjakul, 2011)

Conversely, odors in aquatic environment are soluble chemicals in water Despite not

encountering airborne volatile odorants, aquatic organisms possess olfactory

systems that are anatomically similar to terrestrial animals (Hildebrand and Shepherd,

1997; Laurent, 2002) Since chemicals cues move more slowly in water than in air,

most aquatic organisms have their OSNs positioned anteriorly, where they will be

exposed to moving water, allowing the organism to have a better chance to smell

and detect chemical changes in the environment These chemical cues also form a

plume, which is well conserved at great distances from the source (Murlis, 1992)

Additionally, the ability to detect chemical cues over great distances is of particular

importance and advantage to aquatic animals due to limitations on vision at depth

and in complex or turbid environments

1.2.2 Olfaction begins in utero

How early in life does olfaction begin? Ontogenetically, the olfactory system is

developed closely with the somesthesic and vestibular modalities (Gottlieb, 1971a) It

is fully functional during gestation, preceding the development of both visual and

auditory systems (Gottlieb, 1971b) Despite this fact, olfaction in utero was not

considered seriously as early researchers assumed the necessity for presence of

airborne chemical molecules and fluid currents flowing through the fetus’ nose to trigger olfaction (Humphrey, 1978) However, later advancements in olfactory system

research changed this notion Teicher and Blass (1977) first provided the initial

evidence that newborn albino rats are attracted to amniotic fluid and that the olfactory

cues contained in it guide the initial sucking episode when auditory and visual

systems are not fully developed Subsequent studies also showed that neonates are

highly responsive and had more specific preference to maternal biological odors,

which they have never been exposed to prior to the post-natal experience These

odor preferences can be acquired within a short time window after birth (Sullivan and

Wilson, 1991; Kindermann et al., 1994) and include their own mothers’ saliva (Teicher and Blass, 1977), breast milk and amniotic fluid in mammals such as

rodents (Smotherman, 1982; Hepper, 1987; Smotherman and Robinson, 1990),

piglets (Parfet and Gonyou, 1991), sheep (Schaal et al., 1995) and humans (Varendi

et al., 1996; Schaal et al., 1998, 2000) From these studies, the most obvious way to

stimulate fetal olfactory receptors is through the amniotic fluid, either through direct

infusion or maternal ingestion In fact, the human fetus inhales more than twice the

volume it swallows (Duenhoelter and Pritchard, 1976), suggesting an intense

movement of fluid through the nose, thus allowing olfaction in utero The chemical

composition in the amniotic fluid at a specific period of fetal development may also

induce a specific pattern of olfactory receptor expression, therefore determining their

odorant specificity and sensitivity (Wang et al., 1993)

Along with the ability to detect, learn and retrieve odor information from the late

gestation period, the fetal chemoreceptor system is also genetically predisposed to

detect particular biological odor cues (Todrank et al., 2005) These are essential for

neonatal survival as the fetus prepares itself for life outside the womb, by locating

food, eating and attaching to its biological mother for protection during development

(Schaal et al., 1998) However, the neonate does not solely determine the

mother-infant interaction In humans, mothers also have the capacity to distinguish between

the smell of their infant and other children (Porter et al., 1983) The importance of

olfaction for establishing maternal behavior is also observed in ewes (Lévy et al.,

1996) Aminotic fluids, generally repulsive, briefly become attractive at parturition

(Lévy et al., 1996) In contrast, the lack of amniotic fluids in neonates, as well as

suppression of olfactory cues is detrimental for maternal acceptance, leading to an

absence of licking, refusal to nurse and aggressive maternal behavior (Lévy et al.,

1996)

1.2.3 Food, foraging and homing odors

Olfaction in utero is also responsible for allowing one to recognize whether food is fit

for ingestion Future food preferences are influenced by odors perceived in the womb

as well Several experimental paradigms have demonstrated neonatal rats and

humans can associate chemosensory stimulation experienced in the womb with

aversive (Smotherman, 1982) or appetitive (Pedersen et al., 1983; Schaal et al.,

1995, 2000) reinforcements that may influence their behavior throughout their lifetime

Reversible changes in food odor preferences can also occur over short periods

because of simple exposure, in a phenomenon known as sensory-specific satiety

(Rolls et al., 1981) In general, responsiveness and attraction towards certain food

odors are modified by hunger and specific feeding history - one that is fed to satiety

on a particular food, may have decreased attraction to it and continue to seek other

foods to satisfy their hunger

In aquatic animals, olfactory information from a distance may also elicit foraging

behavior as with lobsters (Panulirus interruptus), which have a marked specific

preference for abalone and mackerel muscle that are presented from one to two

meters apart (Zimmer-Faust and Case, 1982)

Additionally, one of the classic functions of olfaction in aquatic animals is for homing

and home range recognition, as observed in salmon (Oncorhynchus spp.), which

return to their natal stream to spawn after spending years in the Pacific Ocean based

on learned odor cues (Dittman and Quinn, 1996) Salmon first rendered anosmic by

plugging their nostrils and were recaptured evenly among all streams of the basin,

whereas salmon with unplugged noses had higher correlation of returning to home

and recognize individuals are crucial in understanding social behavior Odors from

the same animal may provide diverse information about the animal, as demonstrated

in mammals Castroreum secreted by beavers is involved in territorial demarcation

and may mediate recognition of family members, whereas anal gland secretion

contains individual, kin and sex information (Sun and Müller-Schwarze, 1999) On

the other hand, odor cues from the same animal may provide redundant information

For example, in golden hamsters, five different odor sources were individually

distinctive, namely flank gland, ear glands, urine, feces and vaginal secretions,

where other sources of odors were not (Johnston et al., 1993) In three-spined

sticklebacks (Gasterosteus aculeatus), the odor of an individual is strongly influenced

by both recent habitat use and diet (Ward, 2004) From wasps to fish, rodents and

primates, this odor information is a consequence of varying proportions of individual

chemical compounds in a complex mixtures of chemicals, producing a distinct odor

gestalt that is easily recognized by individuals of the same species (Penn and Potts,

1998a; Dani et al., 2001; Smith et al., 2001; Leinders-Zufall et al., 2004; Ward, 2004;

Hinz et al., 2013)

1.2.4.2 Recognizing kin

Olfaction is also fundamental in kin recognition as it facilitates cooperation among

relatives, avoiding excessive kin competition or inbreeding (Hamilton, 1964)

Individuals with the advantage of discerning a kin member could have had higher

levels of survival, successful reproduction and consequently higher fitness At least

two mechanisms have been demonstrated to recognize kins through olfaction: by

self-inspection (also known as “self-referent phenotype matching” and the “armpit effect”) or indirect familiarity (“phenotype matching”)

The “armpit effect” is defined as an individual compares its own phenotype (such as odors) to that of other conspecifics The other conspecific would be treated as kin if

there is a high similarity to odor of self Such example has been shown in female

golden hamsters Through cross-fostering studies shortly after birth, estrous females

were more attracted to unfamiliar non-kin than to unfamiliar kin (Mateo and Johnston,

2000)

In phenotype matching, individuals learn, recognize and associate characteristics of

other conspecifics they grow up with instead Tang-Martinez showed that vertebrates

who resemble their own kin later in life are treated as related (Tang-Martinez, 2001)

Aquatic vertebrates such as juvenile zebrafish (Gerlach and Lysiak, 2006) and cichild

fish (Pelvicachromis taeniatus) (Hesse et al., 2012) have demonstrated to use

phenotype matching based on olfactory cues to differentiate between kin and non-kin,

preferring odor of unfamiliar siblings to unfamiliar and unrelated individuals This

leads to an immediate selective advantage as juvenile fishes housed in kin groups

grew significantly faster than those in groups of unrelated individuals (Gerlach et al.,

2007) Wild Atlantic salmon (Salmo salar L.) that have an accelerated growth

frequently correlates with enhanced survival and earlier reproductive age (Garant et

al., 2003)

As summarized by Krause and Krüger (2012), the finding that even zebrafinches

(Taeniopygia guttata), previously known for recognizing relatives through auditory

signals, can rely on olfactory cues for kin recognition, leads to the conclusion that

general mechanism of kin recognition might be based on phenotype matching

through genetically-based markers, such as the major histocompatibility complex

(MHC) A specific set of MHC peptides have been recently found to play a novel role

in olfactory imprinting of kin in a specific line of zebrafish, activating specific

populations of neurons in the olfactory bulb that do not overlap with food odor (Hinz

et al., 2013)

1.2.4.2.1 Kin recognition and mate choice – role of MHC

MHC is one of the two key polymorphic and multigenic complexes found in odors that

contribute to the determination of individual differences Known to play a central role

in immunological self/non-self-recognition (Janeway et al., 2001), MHC genes are

characterized by their high polymorphism, making MHC similarity between

individuals a good indicator for their relatedness House mice have shown preference

to mate with individuals carrying dissimilar MHC genes under laboratory (Eklund,

1997; Carroll et al., 2002) and semi-natural conditions (Potts et al., 1991) Female

mice in estrus show preferences towards odors of males with a dissimilar MHC type

when tested in a Y-maze, whereas those not in estrus did not show such preference

(Egid and Brown, 1989) Similarly, women in the fertile phase of their menstrual cycle

are most attracted to the odor of MHC-dissimilar individuals (Wedekind and Füri,

1997) and have mating preferences towards these individuals (Ober et al., 1997)

Such preferences are reversed when women are taking oral contraceptives – odors

of men with a similar MHC type are then preferred (Roberts et al., 2008)

These selective behaviors reduce inbreeding (Penn and Potts, 1998a) and enhance

MHC variability and individual odor distinctiveness, as mating with a male of

dissimilar MHC type produces offspring that are more heterozygous across all of the

genome, not just at the MHC loci These offspring also respond effectively to a wider

range of pathogens than homozygous pups (Penn and Potts, 1998b), hence

increasing their ability to survive Similarly, females of three-spined sticklebacks

(Gasterosteus aculeatus) used an odor-based selection strategy to achieve optimal

number of MHC alleles in their offspring, equipping them with optimal resistance

toward pathogens and parasites When gravid female sticklebacks are exposed to

sources of water from two males, females appear to compare their own set of MHC

alleles and show preferences for the scent of the male with the optimal complement

of alleles (Reusch et al., 2001; Milinski et al., 2005)

1.2.4.2.2 Kin recognition and mate choice - role of major urinary proteins

In contrast, a recent study using wild mice in large enclosures has shown that MHC

is not a relevant marker that animals use for avoiding inbreeding (Sherborne et al.,

2007) Instead, major urinary proteins (MUPs) are the main responsible markers,

containing individually distinctive odor information (Hurst et al., 2001; Cheetham et

al., 2007) MUPs are mostly produced in the liver and become concentrated in the

urine of mice, though similar lipocalin proteins in scent-producing organs of other

species have been shown in other rodents (Hurst et al., 2001; Beynon and Hurst,

2004) Although they are non-volatile molecules, they bind to smaller volatile

molecules and release them slowly, hence extending the efficacy of volatile

compounds enclosed in these scent marks MUPs also appear to be a better

chemical fingerprint of an individual due to the consistent pattern of MUPs expressed

by an individual, which is unaffected by factors such as diet or social status (Nevison

et al., 2003)

1.2.4.3 Recognizing predators

The recognition of predators through olfaction occurs in a variety of vertebrates, both

in the field (Ward et al., 1997) and in the laboratory environment (McGregor et al.,

2002) These predator odors contain a class of chemosignals known as kairomones,

chemicals that work like a pheromone but only communicate between different

species In the laboratory settings, such kairomones most commonly employed to

induce fear in rodents are lipocalins produced by cats and rats,

2,5-dihydro-2,4,5-trimethylthiazoline (a synthetic compound isolated from fox feces) (Fendt, 2006) and

more recently, 2-phenylethylamine (Ferrero et al., 2011) Prey exposed to these

odors often display behavioral and physiological responses indicative of fear

including freezing and avoidance (Ward et al., 1997; McGregor et al., 2002)

For many prey species, the ability to recognize a predator through olfaction plays an

important role in their survival This is especially so in habitats with poor visibility,

such as in turbid waters or under low-light conditions Detection of predators through

odor cues is critical for detecting cryptic ambush predators as well Hence, an

alternative way to enhance survival by hiding from predators is to smell like its habitat,

as demonstrated recently in coral-feeding filefish (Oxymonacanthus longirostris)

(Brooker et al., 2014). Crabs (Tetralia glaberrima), which inhabit the corals (Acropora

spathulata) consumed by these filefish, prefer the odor of these filefish that fed on

the same coral the crabs lived in Predatory cod (Cephalopholis spp.) are also less

attracted to filefish odor when presented alongside the coral it has been fed on,

suggesting evidence of diet-induced chemical crypsis in vertebrate

In a threatening situation, animals warn their conspecifics of the imminent danger by

emitting an alarm substance (von Frisch, 1942; Kiyokawa et al., 2005; Mitchell et al.,

2012; Brechbühl et al., 2013) These pheromones signal injury, distress or the

presence of predators, eliciting innate and stereotypical behavioral responses such

as avoidance or defensive attack (Verheggen et al., 2010)

In 1938, when Austrian ethologist Karl von Frisch was performing experiments on

sense of auditory in fishes, he marked individual European minnows (Phoxinus

phoxinus) with an incision close to the tail, severing the sympathetic nerve (von

Frisch, 1942) When these marked minnows were released into the water, he noticed

that the other members first approached the injured individual, before appearing

stressed and rapidly swimming away to avoid the latter Upon encountering a

minnow that was accidentally injured, a school of fish was also observed to rapidly

swim away Von Frisch then termed the substance causing these alarm reactions

“schreckstoff” (or “fear substance” in German) In following studies, von Frisch began observing the alarm responses in these fish He noticed an increase in

respiratory rate movement, before the minnows exhibited a sudden fright and rapid

swimming into a hiding place, followed by an immediate emergence and increasingly

erratic movements within the tank Some were observed to dwell to the bottom of the

tank, swimming excitedly with their heads against the bottom of the tank This

behavior is akin to disturbing mud and debris to hide themselves in their natural

habitat

1.3.1 Source of Schreckstoff

What is the source of Schreckstoff? Specialized cells lying in the surface layers of

the epidermis, known as “club cells”, have been proposed to be where Schreckstoff

originate (Pfeiffer, 1977) These cells are not in contact with external water, but are

among the first cells to be damaged after a tissue wound or physical insult, frequently

caused by a predator Hence, it was proposed that club cells release Schreckstoff

into the water only upon physical damage, causing alarm reactions in other

surrounding fishes Club cells were shown to be auto-fluorescent, with spectral

properties similar to that of crude skin extract (Reutter and Pfeiffer, 1973) However,

Lebedeva and colleagues found that the fluorescent fraction of skin extract do not

elicit an alarm response in European minnows (Lebedeva et al., 1975) Therefore,

more molecular and histoimmunological work needs to be done to establish that club

cells are indeed the source of Schreckstoff

1.3.2 Components of Schreckstoff

Many researchers were intrigued by characteristics of the active component of

Schreckstoff, leading to extensive studies on the components of Schreckstoff since

its discovery In the first attempt to isolate and identify its chemical nature, Hüttel

(1941) obtained a colorless and odorless dry powder that was found repulsive to

minnows In a later study with a colleague, Hüttel discovered that the components

resembled a pterin from their UV-spectra, hence naming the alarm substance

ichthyopterin (S7N8O3N4) However, this icthyopterin sample was not potent, as it failed to evoke alarm responses in any trial involving schools of minnows

From the 1970s, researchers found it difficult to consider the alarm substance to be

made out of a single purified compound For over thirty years,

hypoxanthin-3(N)-oxide (H3NO), which consists of a purine skeleton with a nitric hypoxanthin-3(N)-oxide (NO) side

group, was a favorite candidate as the active component in the alarm substance

(Pfeiffer et al., 1985; Brown et al., 2000) However, further findings proved this to be

inconclusive First, synthetic H3NO elicited only a slight increase in darting behavior

and did not appear to induce a bottom dwelling nor a tight shoaling behavior

(Mathuru et al., 2012) Second, through functional imaging, synthetic H3NO did not

activate the same part of the olfactory bulb as Schreckstoff (Mathuru et al., 2012)

Interestingly, both Lebedeva et al., and Kasumyan et al., reported that

carbohydrates were found in the alarm substance isolated from common minnows

(Lebedeva et al., 1975; Kasumyan and Lebedeva, 1977, 1979; Kasumyan and

Ponomarev, 1987) These studies motivated Mathuru and his colleagues (Mathuru et

al., 2012) to seek to characterize the components in Schreckstoff.

Two fractions were obtained after anion-exchange chromatography followed by

high-resolution gel filtration of skin extract from zebrafish The high molecular weight

(HMW) fraction elicited slow swimming and descent to the bottom of the tank,

whereas the low molecular weight (LMW) fraction evoked a darting response A

series of tests was conducted in order to classify the components of Schreckstoff As

pronase and peptidase treatment did not reduce the activity of the extract, proteins

are unlikely to be part of Schreckstoff. Surprisingly, an increase in alarm responses

was observed upon heating Schreckstoff for 2 hours at 95°C This, along with mass

spectrometry data, suggests glycosaminoglycans (GAGs) are present, since heating

can cause the breakdown and release of GAGs, such as hyaluronan and chondroitin

sulfate (CS) from the mucous (Shephard, 1994) GAGs are long unbranched

polysaccharide chains that consist of a repeating disaccharide unit The repeating

unit (except for keratan) consists of an amino sugar (N-acetylglucosamine (GlcNAc)

or N-acetylgalactosamine (GalNAc), along with a uronic sugar glucuronic acid (GlcA)

or iduronic acid (IdoA) or galactose (Gal) (Figure 1-1A) Further tests determined the

GAG involved is CS Functional calcium imaging data from this study also revealed

that the dorsomedial loci of the olfactory bulb are activated by Schreckstoff,

proposing that the crypt olfactory sensory neurons (OSNs) may be involved in

detection of Schreckstoff (Gayoso et al., 2011; Oka et al., 2011)

However, after further analysis of Mathuru’s study, I found a candidate that could fit the profile of a component of Schreckstoff, which was overlooked earlier. First, active

fraction of Schreckstoff was not only sensitive to chondroitinase ABC treatment, the

treated active fraction also displayed an increased alarm behavioral response

(Mathuru et al., 2012) Although Mathuru’s study used this as a confirmation to state that CS is one of the glycans in Schreckstoff, it must be highlighted that

chondroitinase ABC is also able to fragment hyaluronan Second, as one of the

purification steps to classify the component of Schreckstoff, a lectin-based column,

more specifically, wheat-germ agglutinin (WGA) column was used However, CS that

was identified as a component of Schreckstoff in the previous study does not bind to

WGA (Carlsson et al., 1976) These results suggest that Schreckstoff contains the

same glycan that binds to WGA GlcNAc is known to bind tightly to WGA, and the

only GAG that contains GlcNAc and adheres strongly to WGA is hyaluronan

(Monsigny et al., 1980) Furthermore, the mass spectrometry analysis of Schreckstoff

done by Mathuru and his colleagues showed a peak at m/z = 775.2 (Mathuru et al.,

2012) This peak correlates to hyaluronan tetramers as reported by Volpi and

colleagues ((Volpi, 2007) These suggest that hyaluronan may be a component of

Schreckstoff. However, to prove that, it is fundamental to show a priori that

hyaluronan is an odor that is detected by zebrafish

What is hyaluronan? What are its properties and how is it similar to Schreckstoff?

These questions will be addressed in the subsequent section

1.4.1 Introduction of hyaluronan

The ability of the HMW fraction to bind to wheat germ agglutinin (WGA) column was

the first indication that glycans are constituents of the active fraction in Schreckstoff

Additionally, the alarm substance was also found to be heat-sensitive One of the

glycans reported to be sensitive to thermal degradation is hyaluronic acid (HA)

(Bothner et al., 1988; Lowry and Beavers, 1994; Reháková et al., 1994)

HA was first biochemically purified from the vitreous humor of bovine eyes by Karl Meyer in 1934 (Meyer and Palmer, 1934) He first named the substance “hyaluronic acid”, due to its glassy appearance upon soaking in water and the plausible presence

of uronic acid as one of the components It was not until the 1950s, when Meyer and

his colleagues determined that HA was an unbranched, linear polysaccharide chain

composed of repeating β-1,4-linked GlcA and β-1,3-linked GlcNAc disaccharide units (Meyer, 1958) (Figure 1-1B) Moreover, HA behaves differently under various

conditions When Meyer first isolated it, it behaved like a mild acid Conversely,

under physiological conditions, HA exists mostly as a sodium salt, and hence was

given the name “sodium hyaluronate” This name was later revised to “hyaluronan” to encompass all forms of the molecule (Balazs et al., 1986)

1.4.2 Role of HA depends on its polymer size

Although HA consists of a single polysaccharide chain like other GAGs, its molecular

weight can be a diverse range, from 1.6 kDa to the millions Consequently, polymer

size appears to confer specific and diverse functions on HA fragments An integral

component of the extracellular matrix, large HA polymers (from 4x102 kDa) are traditionally considered as solvating, space-filling macromolecules largely

responsible for tissue support and integrity, such as synovial fluid (Balazs and

Denlinger, 1985) In the immunology field, these large polymers are pro-angiogenic

(Feinberg and Beebe, 1983; Deed et al., 1997) and immunosuppressive (Delmage

et al., 1986) High concentration of large HA polymers circulating in fetal circulation

and amniotic fluid also accounted for immuno-suppression in the developing fetus

(Dahl et al., 1983; Decker et al., 1989) Furthermore, HMW-HA plays an important

role in anti-inflammation, protecting cells against injury (Jiang et al., 2005) and

maintaining the integrity of a protective coat in tumor cells from cytotoxic effects

(McBride and Bard, 1979)

19

polysaccharide chains consisting of a disaccharide unit of a hexose sugar (GlcNAc or GalNAc) and uronic sugar molecule (GlcA, IdoA or) or galactose (Modified from Esko, Kimata, & Lindahl, 2009) (B) Chemical structure of hyaluronan Hyaluronan is made from repeating disaccharide units of GlcA and GlcNAc

Legend

N-acetylglucosamine (GlcNAc)

Glucuronic acid (GlcA)

Iduronic acid (IdoA)

N-acetylgalactosamine (GalNAc)

Galactose (Gal)

Glycosaminoglycans consist of repeating disaccharide units

Chondroitin sulfate

Dermatan sulfate

Heparian sulfate / Heparin

Glucuronic acid (GlcA)

Iduronic acid (Ido )

N-acetylgalactosamine (GalNAc)

Galactose (Gal)

Heparin

B

In general, HMW-HA promotes tissue integrity and quiescence, whereas smaller

fragments of HA signal that an injury has occurred LMW-HA accumulates during

inflammatory phase, stimulating fibroplast proliferation and collagen synthesis

(Rooney et al., 1993) It was also observed to facilitate tumor cell migration in a

CD44-dependent manner (Sugahara et al., 2003) Additionally, LMW-HA plays a

pro-angiogenic role (Sattar et al., 1994; Slevin et al., 2002) Although these LMW

polymers appear to function as endogenous “danger signals”, even smaller fragments (~ 0.8 to 4 kDa) can ameliorate these effects by first identifying areas of

wounding through toll-like receptors (TLRs) (Taylor et al., 2004) These short

oligosaccharides of HA then stimulate cytokine production by dendritic cells (Termeer

et al., 2002) Moreover, smaller fragments of HA regulate microglia at site of

ischemic brain damage, enhancing synthesis of nitric oxide synthase (Wang et al.,

2004)

1.4.3 Role of HA depends on its binding proteins

The interplay between various sizes of HA with their corresponding binding protein or

receptor may contribute to their diverse functions as well TLR, pattern-recognition

receptors that regulate immunity, have been shown to interact with HA, specifically

TLR4 and TLR2 (Akira, 2006) Although both reside on the cell surface, TLR4 has

been shown to mediate signals from LMW (~800 Da) to HMW HA (~2.8 x 103 kDa), whereas TLR2 preferentially binds to intermediate-sized HA (~ 2.7 x 102 to 4 kDa) This is in line with the fact that TLR4 recognizes a plethora of structurally unrelated

pathogen-associated molecular patterns, while TLR2 recognizes limited ligands such

as lipids and some glycoproteins (Akira, 2006) In zebrafish, TLR4 has two paralogs,

TLR4a and TLR4b Unlike previous finding of TLR4 (Hoebe et al., 2003), these

paralogs are unresponsive to lipopolysaccharides, suggesting that they have evolved

to provide alternative ligand specificities to the TLR immune defense system in

zebrafish (Sullivan et al., 2009) HA role as an agonist of TLR has also been

inconclusive Termeer et al. first identified HA as an agonist for TLR4 (Termeer et al.,

2002), however, Scheibner et al (2006) disproved TLR4 agonistic activity, describing

HA as an agonist for TLR2 instead

Other cell surface binding proteins of HA have also been studied Besides

participating in inflammatory and immunological responses (Nedvetzki et al., 2005),

receptor for hyaluronan-mediated-motility (RHAMM) is responsible for focal

adhesions and cell motility (Hall et al., 1996), and also cell migration (Masellis-Smith

et al., 1996; Savani et al., 2001) HA receptor for endocytosis (HARE), CD44 and

lymphatic vessel endothelial HA receptor-1 (LYVE1) clear HA and other GAGs from

circulation (Banerji et al., 1999; Zhou et al., 2000; Hirose et al., 2012), taking part in

HA uptake and degradation (Culty et al., 1992), as well as endocytosis (Zhou et al.,

2000; Pandey et al., 2013) It is also noteworthy that LYVE1 was suggested to serve

as “chemosensory factors to sense change interstitial fluid composition” (Dunworth and Caron, 2009)

1.4.4 HA degradation

Since the size of HA polymer is tightly linked to its activity, fragmentation of HA is

thus a critical aspect in HA signaling capacity Previous findings report three major

ways in which HA can be degraded: (1) through enzymatic activity such as

hyaluronidases (Hyals); (2) heat (Bothner et al., 1988; Lowry and Beavers, 1994);

and (3) free radicals or reactive oxygen species (ROS) (McCord, 1974; Greenwald

and Moy, 1980) HA catabolism in cells occurs in a series of synchronized events A

recent proposal by Stern (2004) suggests that HMW-HA is cleaved progressively by

a series of enzymes in which the substrate for a reaction is the product of the

preceding one HA degradation is generally caused by Hyals which are hydrolases

and have recently been identified to include six enzymes in mammals (Hya-1, -2, -3,

-4, -5 and PH20) (Csoka et al., 2001) Above all, Hyal1 and Hyal2 are involved

predominantly in degrading HA in somatic tissues (Csoka et al., 1999) Yet, these

two Hyal are unique Hyal1 is widely expressed and have a sharp optimal enzymatic

activity at very low pH (pH 3.7), suggesting little activity outside lysozymes Hyal1

also produce fragments up to 800 Da (Hofinger et al., 2008) In contrast, Hyal2 is

often found to be glycosylphosphatidylinositol (GPI)-anchored, bound to the

extracellular side of the plasma membrane and was suggested to reside in

lysosomes (Lepperdinger et al., 1998) Unlike Hyal1, Hyal2 is active over a larger

range of pH, with its optimal activity at about pH 6.0 – 7.0 Besides this, Hyal2 degrades HA in larger fragments of up to 20 kDa in size (Harada and Takahashi,

2007) Coincidentally, HA can also be fragmented by chondroitinase ABC, which was

used in the study that determined CS as a component in Schreckstoff (Taylor et al.,

2004; Mathuru et al., 2012)

Other studies have shown thermal degradation of HA Using laser light scattering

techniques, Bothner observed an extensive decline of molecular weight of long

polymers of HA after autoclaving at 128°C (Bothner et al., 1988) Rhelogical studies

show that HA solutions retrograde in water; at increasing temperatures, the viscosity

of solution reduces in time exponentially as a function of temperature (Lowry and

Beavers, 1994) Comparatively, another study also reported degradation of HA after

heating from 60°C to 90°C for one hour, albeit rather moderately (Reháková et al.,

1994) Fortuitously, heated crude skin extract did elicit more intense alarm behavioral

responses (Mathuru et al., 2012) Thermal degradation of HA could cause an

increased in HA fragments, hence, resulting in a more potent Schreckstoff Together,

these studies may also imply that HA may be a component of Schreckstoff

Many human diseases are associated with destructive actions of free radicals,

degrading essential tissue or related components Observed decrease of HA

molecular weight in the synovial fluid of patients suffering from inflammatory joint

diseases led to in vitro studies of HA fragmentation by ROS Superoxide-treated HA

solutions decreased in viscosity, but regained so after addition of ROS scavenger,

superoxide dismutase (McCord, 1974; Greenwald and Moy, 1980) A noteworthy

reminder is that in the process of lesioning fish skin for Schreckstoff production, free

radicals are also produced (Niethammer et al., 2009) These free radicals may have

fragment HA, making Schreckstoff more potent than crude skin extract Hence, this

indicates that HA itself may act as an odor and could also be present in Schreckstoff.

These compelling suggestions make HA an attractive candidate component of the

alarm substance Currently, there are no reports of HA as an odor in vertebrates

Before we seek to answer if HA is an odor in fish, we must first comprehend how do

fish detect different chemical cues How does the olfactory system work? What

sensory neurons are responsible for detecting these various cues? How do these

neural activities from the olfactory system translate to physiological and behavioral

outcomes? I would hereafter attempt to answer these questions by describing the

anatomy and functional organization of the olfactory system in details

A myriad of chemical signals pervade the aquatic environment of fishes, activating

the chemosensory systems and evoking various physiological and behavioral

responses In particular, the fish olfactory system is highly elaborated to recognize

and distinguish among the plethora of soluble chemical odors, transmitting their

information to the brain and triggering correct innate behaviors crucial to survival

Other odors besides Schreckstoff, such as steroids, prostaglandin F2α and bile acids, mediate communication within conspecifics in a group, either through social

interaction or informing them of impending danger Steriods and prostagladin F2α are hormones produced in gonads and released in urine, and were shown to trigger

species and sex specific reproductive behavior (Hara, 1994) Conversely, mixtures of

bile acids, which are bio-active steriods secreted by the liver and released in the

urine, have been implicated in migration to spawning sites in lampreys (Sorensen et al., 2005) Amino acids and nucleotides, such as L-Lysine (L-Lys) and adenosine-5’-triphosphate (ATP) respectively, indicate the presence of food and its freshness

(Lindsay, 2004) Their presence induces appetitive swimming behavior characterized

by increased number of turns and swimming speed

1.5.1 Advantages of zebrafish as a model system

Advances in understanding the function of olfactory systems have intensified over

the last decade, with zebrafish becoming an increasingly popular vertebrate model

for olfactory research Zebrafish are advanced freshwater teleosts that inhabit still or

slow waters in Southeast Asia Known for their high fecundity and rapid development,

George Streisinger and colleagues first discovered their scientific potential as an

alternative vertebrate model in 1981 (Streisinger et al., 1981) Their small brain is

also amendable to various genetic engineering techniques used in other model

systems, such as CRISPR/Cas9 system (Hwang et al., 2013; Auer et al., 2014) and

TALENS (Huang et al., 2011; Sander et al., 2011) Embryonic and early larval-stage

zebrafish are also optically transparent, permitting optical access to visualizing their

central nervous system Furthermore, recently developed whole brain and single cell

functional imaging techniques took advantage of these unique characteristics,

enabling activity from hundreds of neurons to be monitored at once for rapid and

economical in vivo neuronal network analysis, in living embryos whereby genetically

engineered, highly sensitive calcium indicators are expressed (Higashijima et al.,

2003; Ahrens et al., 2013; Akerboom et al., 2013; Panier et al., 2013; Freeman et al.,

2014; Miyasaka et al., 2014) Collectively, these advantages make zebrafish an

excellent model for a deeper understanding of the olfactory system in vertebrates

1.5.2 Anatomy of the zebrafish olfactory system

Unlike their mammalian counterparts, zebrafish possess only a single type of

olfactory organ It lies on the dorsal side of the head, anterior to the eyes, where they

are most exposed to moving water (Hansen and Zielinski, 2005) (Figure 1-2) Odor

cues present in the water enter the nasal cavities through a funnel-shaped anterior

nostril and exit through a posterior nostril The multi-lamellar, rosette-shaped

olfactory epithelium lies between these two nostrils, whereby lamellae project

outwards from the middle of the rosette, known as the midline raphe The older and

larger lamellae are arranged radially at the caudal end of the organ, with the young

lamellae being formed rostrally at both sides of the midline raphe

The olfactory sensory and non-sensory neurons are strictly segregated in the

olfactory epithelium with sensory neurons located in the medial of each lamella

(Figure 1-2) (Baier et al., 1994) Non-sensory neurons consist of supporting cells,

basal cells, globlet cells and ciliated cells, which facilitates the movement of odors

towards the center of the epithelium (Hansen and Zeiske, 1998) As of now, four

classes of OSNs have been identified: ciliated and microvillous which are two major

types of OSNs, as well as the lesser abundant crypt neurons (Hansen et al., 2005)

Not much is known about the recently identified fourth class of OSNs, the kappe

neurons, which is even rarer than crypt neurons (Ahuja et al., 2014) OSNs are

unique compared to cells in the visual or auditory system First, they are randomly

dispersed throughout the olfactory epithelium Second, they are constantly renewed

throughout adult life or following chemical lesion of the olfactory epithelium, to repair

the damage caused to OSNs by being exposed to the environment (Cancalon, 1982)

1.5.3 Types of OSNs

Their morphology, molecular markers and receptor classes distinguish the four

classes of OSNs The distinct spatial position of the soma of the different populations

of OSNs, along with their shape, can be visualized through either applications of the

lipophilic tracer carbocyanine dye (DiI) in the olfactory bulb (Baier et al., 1994) or

neuronal marker immunoreactivity (Jesuthasan and Mathuru, 2008) Ciliated OSNs

are characterized by their long dendrites with a basal cell body, extending several

long cilia into the lumen of olfactory epithelium On the contrary, microvillous neurons

are located at an intermediate position, have shorter dendrites and emanate tens of

microvilli into the lumen Crypt neurons have a large globular cell body, with a

distinctive crypt bearing cilia projecting towards the lumen (Germanà et al., 2007)