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4-2006
Olfactory Bulb Neurons of Adult Zebrafish
Morphology, Distribution, Cellular Interactions
and Structural Stability following Deafferentation
Cynthia L. Fuller
Western Michigan University
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Fuller, Cynthia L., "Olfactory Bulb Neurons of Adult Zebrafish Morphology, Distribution, Cellular Interactions and Structural
Stability following Deafferentation" (2006). Dissertations. Paper 941.
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OLFACTORY BULB NEURONS OF ADULT ZEBRAFISH MORPHOLOGY,
DISTRIBUTION, CELLULAR INTERACTIONS AND STRUCTURAL
STABILITY FOLLOWING DEAFFERENTATION
by
Cynthia L. Fuller
A Dissertation
Submitted to the
Faculty of The Graduate College
in partial fulfillment of the
requirements for the
Degree of Philosophy
Department of Biological Sciences
Dr. Christine Byrd, Advisor
Western Michigan University
Kalamazoo, Michigan
April 2006
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OLFACTORY BULB NEURONS OF ADULT ZEBRAFISH: MORPHOLOGY,
DISTRIBUTION, CELLULAR INTERACTIONS AND STRUCTURAL
STABILITY FOLLOWING DEAFFERENTATION
Cynthia L. Fuller, Ph.D.
Western Michigan University, 2006
The zebrafish is becoming an increasingly popular model for studies involving
olfactory function, yet there is still much to be learned about the anatomy and
circuitry of different cell types in the olfactory bulb. This study focuses on identifying
the morphology and distribution of output neurons and intemeurons in the olfactory
bulb of adult zebrafish, Danio rerio. Furthermore, this investigation examines the
cellular interactions of the primary output neuron, the mitral cell, and addresses the
issue of neuronal plasticity by considering the structural stability of this cell type
following loss of afferent innervation.
Using retrograde tract tracing with various dextrans and live tissue culture, we
were able to label several types of output neurons in the olfactory bulb including
mitral cells, ruffed cells, and ganglion cells of the terminal nerve. Mitral cells were
the most numerous output neurons in the olfactory bulb. These cells, located
primarily in the glomerular layer and superficial internal cell layer, had variable­
shaped somata that ranged in size from 5-20pm in diameter and 22-156pm2 in surface
area. These cells typically had a single dendritic tuft, although some cells possessed
multiple primary dendrites. Even mitral cells with multiple dendrites appeared to
contact a single glomerulus, a finding that suggests olfactory coding in these teleosts
may be more similar to mammals than previously suggested. This information
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provides further background for olfactory coding and processing studies in this key
model system.
Another focus of this study was to examine the structural integrity of mitral
cells following denervation of the olfactory nerve. A comparison of dendritic
complexity at 3 to 5 months post-deafferentation showed that mitral cell morphology
was affected by loss of afferent input. This investigation demonstrated that
innervation was required in order to maintain dendritic complexity by 8 weeks after
injury, but did not appear to alter significantly major dendritic processes even 5
months following ablation. This finding suggests that mitral cells are extremely stable
structures that may be capable of reforming synaptic contacts if afferent targets are re­
established following injury.
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Copyright by
Cynthia L. Fuller
2006
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ACKNOWLEDGMENTS
I would like to thank all of my colleagues, family, and friends for their
support. W ithout them this dissertation would not have been completed. First and
foremost I would like to thank my advisor, Dr. Christine Byrd, for all of her help and
guidance over the last five years. She has been a wonderful mentor and friend. Her
leadership has taught me so much and for that I am so very grateful. I also would like
to thank my committee members Drs. John Jellies, John Spitsbergen and Peter
Hitchcock for their time and advice. Dr. Jellies, you are by far the best teacher I ’ve
ever known. I am so thankful that I ’ve had the opportunity to be one of your students.
Dr. Spitsbergen, thank you for always being there to help me find my way through
the maze of graduate school. Dr. Hitchcock, your advice has been invaluable. Thank
you for taking the time to be on this committee. To my fellow graduate students past
and present, especially Stephanie, Eric, Bobbie, Jolene, Berta and Ken (the honorary
graduate student), thank you so very much just for being there. You all have been
such supportive friends and I sincerely have enjoyed our time together. To our
futures!
Finally, I would like to thank my family. I never could have made it without
the love and support of my parents, my brother, and my husband, Jason. You’ve all
been so special to me, cheering me on even when you couldn’t understand what I was
going through. Y ou’ve helped me to keep my eyes on the road ahead with my goals
in mind. I love you all so much for that and to you I dedicate this work. Thank you!
Cynthia L. Fuller
ii
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TABLE OF CONTENTS
ACKNOWLEDGMENTS..............................................................................................
ii
LIST OF TA B LES..........................................................................................................
vi
LIST OF FIGURES...........................................................................................
vii
CHAPTER
I. INTRODUCTION..............................................................................................
1
General Introduction..................................................................................
1
The Zebrafish..............................................................................................
1
The Olfactory System ...............................................................................
3
Olfactory Bulb Structure...........................................................................
6
Olfactory Bulb Circuitry...........................................................................
7
D eafferentation..........................................................................................
9
Goals of This D issertation........................................................................
11
B. IDENTIFYING THE MORPHOLOGY AND DISTRIBUTION OF
JUXTAGLOMERULAR CELLS IN THE OLFACTORY BULB
USING A TRANSGENIC ZEBRAFISH LIN E............................................
12
Introduction.................................................................................................
12
Materials and M ethods..............................................................................
13
Results..........................................................................................................
15
Discussion...................................................................................................
16
iii
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Table of Contents—continued
CHAPTER
III. IONOTROPIC GLUTAMATE RECEPTOR SUBUNIT 4 IN THE
OLFACTORY BULB OF ADULT ZEBRAFISH: DISTRIBUTION
AND EXPRESSSION FOLLOWING LOSS OF INNERVATION
22
Introduction.................................................................................................
22
Materials and Methods..............................................................................
25
Results..........................................................................................................
30
Discussion...................................................................................................
37
IV. THE MORPHOLOGY AND DISTRIBUTION OF RUFFED CELLS
IN THE OLFACTORY BULB OF ADULT ZEBRAFISH........................
47
Introduction.................................................................................................
47
Materials and Methods..............................................................................
48
Results..........................................................................................................
51
D iscussion...................................................................................................
52
V. THE MORPHOLOGY AND DISTRIBUTION OF MITRAL CELLS
IN THE OLFACTORY BULB OF ADULT ZEBRAFISH........................
59
Introduction.................................................................................................
59
Materials and M ethods..............................................................................
61
Results..........................................................................................................
69
Discussion...................................................................................................
79
VI. THE EFFECTS OF DEAFFERENTATION ON MITRAL CELLS:
AN EXAMINATION OF MORPHOLOGY FOLLOWING LOSS OF
AFFERENT INNERVATION.........................................................................
91
Introduction.................................................................................................
91
Materials and M ethods..............................................................................
93
iv
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Table of Contents—continued
CHAPTER
Results..........................................................................................................
96
Discussion
98
.........................................................................................
VH. DISCUSSION AND FUTURE DIRECTIONS.............................................
104
Major Contributions of This W o rk ...........................................................
104
General D iscussion......................................................................................
104
Future D irections.........................................................................................
110
Final T houghts.............................................................................................
113
REFERENCES..................................................................................................................
115
APPENDICES
A.
Results from Additional Examinations of Mitral Cell and Olfactory
Sensory Neuron Interactions..............................................................................
136
B.
Results from Mitral Cell Studies in Other Teleosts.........................................
140
C.
Animal Care and Use Committee Approval Forms.........................................
144
v
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LIST OF TABLES
1.
Summary of Ruffed Cell and Mitral Cell and Mitral Cell Soma
M easurements.....................................................................................................54
2.
Number of Ruffed Cells in Reference to Labeled Output Neurons...........55
3.
Comparisons of Soma Cross-sectional Areas, Lengths, and Widths for
All Populations of Mitral C ells....................................................................... 76
vi
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LIST OF FIGURES
1. A schematic representation summarizes potential intracellular signal
transduction pathways used in olfactory processing......................................6
2. A hematoxylin and eosin-stained horizontal section of a zebrafish
olfactory bulb is shown with a list of the cell types present in the
corresponding layers of the b u lb ....................................................................... 8
3. A schematic representation of the zebrafish olfactory bulb shows the
circuitry of cellular interactions...................................................................... 10
4. A schematic diagram shows the steps taken to develop the transgenic
zebrafish as well as a diagram of the sCMV-dsRED expression cassette.. 13
5. The dsRED expression pattern was observed in several regions of the
f is h .......................................................................................................................17
6. The transgene was expressed in a subset of cells in the olfactory bulb...... 18
7. dsRED-positive cells labeled with a neuronal marker...................................19
8. Cells that were dsRED+ were not output neurons.........................................20
9. A few dsRED cells were positive for tyrosine hydroxylase........................ 21
10. Control experiments were used to confirm the specificity of the
antibody in zebrafish tissue..............................................................................32
11. Ionotropic GluR4 is expressed in the adult zebrafish olfactory b u lb ......... 33
12. Coronal sections taken at intervals through the olfactory bulb confirm
the strongest labeling is seen in the glomerular layer...................................34
13. Immunocytochemical techniques help to identify iGluR4-expressing
cell types............................................................................................................. 36
14. Quantitative analysis illustrates changes in iGluR4 immunoreactivity in
the whole olfactory bulb................................................................................... 38
vii
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List of Figures—continued
15. Quantitative analysis shows changes in iGluR4 immunoreactivity
following deafferentation in four bulb quadrants......................................... 39
16. Zebrafish ruffed cells are visualized under high magnification using
Golgi-Kopsch and retrograde tract-tracing techniques................................ 53
17. A phylogenetic tree of the teleost line exhibits the interrelationships of
the primary fish discussed in this study: the zebrafish, goldfish, catfish
and sea e e l.......................................................................................................... 56
18. Low magnification and high magnification images of hematoxylin and
eosin-stained horizontal sections of a zebrafish olfactory bulb are
show n..................................................................................................................73
19. Various methods of labeling revealed the typical structure of an adult
zebrafish mitral cell: a soma of variable-shape, a single primary
dendrite terminating in fairly discrete dendritic tufts near the cell
body, and a single axon projecting toward the medial or lateral
olfactory tra c t.....................................................................................................74
20. The morphologies of multi-dendritic mitral cells also were seen using
the retrograde tract-tracing technique............................................................. 75
21.
The distribution of MOT-projecting or LOT-projecting mitral cells was
examined in the horizontal and coronal p lan es............................................ 77
22. Mitral cells were binned according to the cross-sectional area of their
cell bodies at the largest p o in t......................................................................... 78
23.
The morphology and distribution of mitral cells was compared to other
neurons in the olfactory bulb............................................................................81
24. Mitral cells sizes overlapped somewhat with tyrosine hydroxylasepositive (TH+) cells...........................................................................................82
25. Afferent innervation directly influences the maintenance of the
olfactory bulb in adult zebrafish......................................................................94
26. A stepwise diagram shows the process of dendritic analysis used to
obtain estimates of dendritic complexity in control and deafferented
olfactory bulbs....................................................................................................96
viii
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List of Figures—continued
27.
Retrograde labeling revealed both Type I and Type II mitral cell
morphologies......................................................................................................99
28.
Mitral cell morphology examined in animals at 3 weeks, 6 weeks, and 8
weeks, and 20 weeks post-deafferentation showed that elaborate
dendritic processes still existed in the animals even 2 months after loss
of afferent innervation.....................................................................................100
29.
A comparison of dendritic complexity at 3 to 20 weeks postdeafferentation showed that mitral cell morphology was affected by
loss of afferent input........................................................................................ 101
30.
A comparison of dendritic tuft area at 3 to 20 weeks postdeafferentation showed that mitral cell tuft areas were not affected
by loss of afferent input.................................................................................. 102
31.
A chart showing cell types in the olfactory bulb of adult zebrafish
describes the features of these cells including their distribution
and size..............................................................................................................108
ix
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CHAPTER I
INTRODUCTION
General Introduction
Neurology has become an increasingly recognized discipline in today’s world
because of the number of degenerative disorders, ailments, and injuries that involve
loss of brain function in humans. W hether researchers examine the molecular biology
of the mammalian hippocampus, the physiology of the avian auditory system or the
anatomy of the fish olfactory bulb, these studies contribute to our general
understanding of the brain as a whole. Further, with recent advances in disease
models, the scientific community is better able to examine the effects of injury on the
anatomy and physiology of specific areas in the brain. Collectively, these studies
contribute to our ability to understand the function and, more importantly, the
recovery of the human brain following injury or disease. Such foundations continue
to lead to advances in treatments for conditions such as Parkinson’s, Alzheimer’s,
stroke, and epilepsy, to name a few.
The Zebrafish
The zebrafish, Danio rerio, is a popular model in the scientific community
and has been used in a number of disciplines including: cellular biology (Kim et al.,
1997), molecular biology (Weth et al., 1996), genetics (Ardouin et al., 2000),
histology (Byrd and Brunjes, 1995), neurobiology (Fetcho and Liu, 1998; Byrd and
Brunjes, 2001), developmental biology/embryology (Dynes and Ngai, 1998; Brown et
al., 2000), behavior (Fetcho and Liu, 1998), and physiology (Friedrich and
Korsching, 1998), to name a few. There are a number of reasons why the zebrafish is
1
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a good model system. For example, this animal is easy to maintain, fairly inexpensive
to purchase, and reproduces efficiently. Furthermore, zebrafish eggs and larvae are
transparent, which makes them strong candidates for developmental studies. There
also is substantial existing knowledge regarding this species and since some zebrafish
genes are homologous to human genes (Morizot, 1991; Ardouin et al., 2000), studies
suggest that once large numbers of genes have been mapped in zebrafish it will be
possible to determine where those genes are located in the human genome
(Postlethwait et al., 1999). As a result, the data generated from studies on this fish
could have enormous influence on disease research in humans.
The zebrafish is also an exceptional model for studies of the olfactory system
(Baier et al., 1994; Byrd, 1995; Dynes and Ngai, 1998; Whitlock and Westerfield,
1998; Friedrich and Laurent, 2001; Edwards and Michel, 2002). First, the olfactory
epithelium in this animal is much more accessible than the mammalian olfactory
epithelium, which is surrounded by a bony nasal cavity. This allows more ease with
experimental manipulations to the nose and is much less invasive than similar
procedures in mammals. As a result, fish are able to recover quickly and have high
survival rates after injury. In addition, gaining access to the olfactory bulb and brain
is less difficult in the zebrafish because the casing surrounding these structures is
much thinner and can be removed with forceps, unlike mammals and other larger
teleosts, where micro-rongeurs are often required to chip away at this housing and can
cause damage to the underlying tissue.
While numerous studies have begun to characterize the anatomy of the
olfactory system in this species (Baier and Korsching, 1994; Byrd, 1995; Edwards
and Michel, 2002), there is still little known about the structure and function of the
different cell types within the olfactory bulb. In order to gain further understanding of
2
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the way zebrafish process olfactory information, many researchers have focused on
the functional aspects of the olfactory system, such as odor representation and
stimulation (Corotto et al, 1996; Freidrich and Korsching, 1998; Friedrich and
Laurent, 2001; Edwards and Michel, 2002; Lipschitz and Michel, 2002; Michel et al,
2003). While some of these studies have touched upon characteristics of zebrafish
bulbar neurons, a complete analysis is lacking. Thus, this study will examine
olfactory bulb neurons in detail to provide further insight into information processing
and potential plasticity in this key model system.
The Olfactory System
The olfactory system is excellent for studies involving plasticity of the
nervous system. The adult olfactory bulb is among the most plastic structures in the
brain. Olfactory sensory neurons in the nose send axons to the olfactory bulb where
they synapse with bulbar neurons. There is normal turnover of these sensory neurons
in the olfactory epithelium, which results in the need for continual identification of
new targets and formation of new synapses within the bulb (for review, see Farbman,
1992). Furthermore, the olfactory bulb itself undergoes continuous neurogenesis
(Altman, 1969; Kaplan and Hinds, 1997; Burd and Sein, 1998; Peretto et al., 1999;
Byrd and Brunjes, 2001; Iwai et al., 2003, Stenman et al., 2003), which adds to the
plasticity of this sensory system. Consequently, the olfactory system is a logical
model for studies involving restructuring and recovery after injury or disease.
The olfactory system has been studied in a variety of animals including
mammals (Farbman and Margolis, 1980; Graziadei et al., 1980; Greer and Shepherd,
1982; Hinds et al., 1984; Shipley and Adamek, 1984; Menco and Farbman, 1985;
Mori et al., 1985; Meredith, 1986; Wilson and Leon, 1988; Guthrie and Leon, 1989;
3
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Baker, 1990; Finger et al., 1990; Buck and Axel, 1991; Brunjes, 1994; Puche et al.,
1996; Sullivan et al., 1996; Menco and Jackson, 1997; Mombaerts, 1999), birds
(Graziadei and Bannister, 1967; Wenzel and Sieck, 1972; Clark and Smeraski, 1990),
reptiles (Halpem and Kubie, 1980; Wang and Halpem, 1988; Lanuza and Halpem,
1998), amphibians (Graziadei and Metcalf, 1971; Getchell, 1974; 1977; Silver et al.,
1988; Byrd and Burd, 1991; Burd, 1992; Byrd and Burd, 1993; Firestein et al., 1993;
Hansen et al., 1998; Higgs and Burd, 1999), fishes (Byrd and Caprio, 1982; Kosaka
and Hama, 1982; Satou, 1990; Hansen and Zeiske, 1993; Ngai et al., 1993; Restrepo
et al., 1993; Baier et al., 1994; Byrd and Brunjes, 1995; Kang and Caprio, 1995;
Friedrich and Korsching, 1997; Byrd and Brunjes, 2001; Sorensen et al., 1998;
Whitlock and Westerfield, 1998; Byrd, 2000; Edwards and Michel, 2002) and a
number of invertebrates (Kaissling, 1986; McClintock and Ache, 1989; Tolbert and
Oland, 1989; Vogt et al., 1989; Michel et al., 1991; Emery, 1992; Bargmann et al.,
1993; Chase and Tolloczko, 1993; Krull et al., 1994; Carlson, 1996; Kaissling, 1996;
Christensen and Hildebrand, 1997; Oland et al., 1998).
In vertebrates, this sensory system is generally comprised of a peripheral,
receptive olfactory epithelium, the olfactory bulb, the olfactory tracts, and the
olfactory cortical areas that process information. In most terrestrial vertebrates, the
system also includes a vomeronasal organ and an accessory olfactory bulb (Farbman,
1992).
This organ, which is noted for its connections to pheromonal-processing,
apparently is absent in fish, birds, and higher primates (Farbman, 1992). The
olfactory epithelium is comprised of a mucous membrane that is housed in the nasal
cavity and comes into contact with the external environment, as well as the lamina
propria, which is an underlying layer made up of connective tissue, blood vessels, and
glands (Farbman, 2000).
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The major cell type in the olfactory epithelium is the sensory neuron, which
comprises about 70-80% of the total cells (Farbman, 2000). These sensory neurons
extend their dendrites to the surface of the nasal epithelium where they receive
information about odors from the outside environment via olfactory receptors. Thus,
they also are termed olfactory receptor neurons. It should be noted that primary
sensory neurons vary in dendritic morphology. In most fish, for example, there are at
least two morphologies of sensory neurons including ciliated and microvillar
(Laberge and Hara, 2001). It has been implied that microvillar neurons respond to
pheromones while ciliated neurons detect amino acids (Zielinski and Hara, 1988,
Zippel et al., 1997; Hansen et al., 2003).
It also has been suggested that the four different classes of odorants detected
by fish, including amino acids, gonadal steroids, bile salts, and prostaglandins (Hara,
1994), are detected by different transduction mechanisms (Laberge and Hara, 2001).
Whereas the current consensus is that most receptors transmit odorant-binding
information via a second messenger pathway (for review, see figure 1), it also is
known that different types of sensory neurons can express different families of
receptor molecules and G-proteins (Jones and Reed, 1989; Buck and Axel, 1991;
Shinohara et al., 1992; Jia and Halpem, 1996; for review, see Mori et al., 2000;
Hansen et al., 2003). Thus, there also is agreement upon the variation that exists
between these signal transduction cascades and the mechanisms involved in odorant
perception.
5
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t
t
ODORANT - ^ 4
N*',
&
ODORANT BINDING PROTEIN
Figure 1. A schematic representation summarizes potential intracellular signal transduction pathways
used in olfactory processing. There are several suggested pathways for odorant perception, but the
most commonly agreed upon pathway includes a receptor protein (seen here docking with an odorant
attached to an odorant binding protein), a GTP-binding protein (Gl, G0if for example), an adenylyl
cyclase (AC) and a gated cation channel that is activated by adenosine 3’,5’-cyclic monophosphate
(cAMP; Ache and Restrepo, 2000). The other suggested pathway includes a different receptor, a
different GTP-binding protein, a phospholipase C (PLC) and a gated cation channel activated by
inositol 1,4,5-triphosphate (IP3; Ache and Restrepo, 2000). These initial pathways also can trigger
secondary pathways such as calcium-activated chloride channels or calcium-activated potassium
channels that carry some of the transduction current (Ache and Restrepo, 2000).
Olfactory Bulb Structure
Another form of variation that exists in the olfactory system is exhibited in the
differences between olfactory bulb structures in different animals. One significant
distinction between the olfactory systems of lower vertebrates and mammals is in the
layering of the olfactory bulb. Mammals have a laminar olfactory bulb with wellcharacterized cell types in each discrete layer (Shepherd, 1972). The organization of
the teleost olfactory bulb, in contrast, is much less obvious (Figure 2). It does,
however, contain several diffuse layers: the olfactory nerve layer (ONL), the
glomerular layer (GL), the mitral cell layer (MCL), and the granule cell layer (GCL;
Oka et al., 1982; Satou, 1990; Byrd and Brunjes, 1995). Because the MCL and GCL
6
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overlap substantially, it is hard to distinguish them. As a result, they are collectively
termed the internal cell layer (ICL; Byrd, 2000). Furthermore, unlike mammals fish
have both a lateral and medial olfactory tract where axons from mitral cells bundle
and project to the brain.
It has been suggested that the medial tract processes
pheromonal information and mediates reproductive behavior while the lateral tract
conveys information about food (Satou et al., 1983; Stacey and Kyle, 1983; Demski
and Dulka, 1984; Satou et al., 1984; Kyle et al., 1987; Sorensen et al., 1991;
Hamdani, Alexander and Doving, 2001; Hamdani, Kasumyan and Doving, 2001;
Weltzien et al., 2003). As a result, differences between the output neurons projecting
to the medial or the lateral olfactory tract could provide important information about
olfactory coding in the teleost olfactory bulb.
Olfactory Bulb Circuitry
The olfactory receptor neurons project their axons to glomeruli in the
olfactory bulb. Glomeruli are spherical regions of neuropil containing numerous
neuronal processes and synapses. In glomeruli the axons of these primary olfactory
receptor neurons synapse with the dendrites of secondary neurons, such as
juxtaglomerular cells or mitral cells (Figure 3). The juxtaglomerular cells are local
intemeurons, located in the glomerular layer of the olfactory bulb. Mitral cells are
projection neurons that carry information out of the olfactory bulb to the main
olfactory cortical areas, including the anterior olfactory nucleus, the olfactory
tubercle, the posterior pyriform cortex, the amygdala, the entorhinal cortex, as well as
parts of the hippocampus and hypothalamus.
7
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Cell Type
Location in
Olfactory Bulb
Mitral Cells
GL/ICL
Juxtaglomerular
Cells
GL/1CL
Granule Cells
ICL
Short Axon
Cells
Unknown in zebrafish
RufTed Cells
GL/ICL
Glial Cells
ONL/GL/ICL
Terminal Nerve
Cells
ONL/GL
Figure 2. A hematoxylin and eosin-stained horizontal section o f a zebrafish olfactory bulb is shown
with a list o f the cell types present in the corresponding layers of the bulb. The zebrafish olfactory bulb
is diffusely organized and can be divided into three principal laminae: the olfactory nerve layer (ONL),
the glomerular layer (GL), and the internal cell layer (ICL). Axons o f output neurons exit the bulb via
two distinct paths, the medial (MOT) and lateral (LOT) olfactory tracts. Scale bar = 50pm.
Mitral cells can contact a group of cells known as the granule cells, which are
intemeurons located in the deeper layers of the bulb (Figure 3). Also, mitral cells can
contact another type of output neuron, the ruffed cell, through dendro-dendritic
synapses (Figure 3). Juxtaglomerular cells, granule cells, and ruffed cells modify the
output of the mitral cells. So mitral cells represent a functionally important class of
neurons that integrate chemical signals. Consequently, this study will attempt to
further examine mitral cells in the teleost system.
8
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Deafferentation
A portion of this project focuses on examining alterations in the morphology
and plasticity of neurons following loss o f afferent input. Several studies have shown
that changes in afferent input from the olfactory organ can result in drastic changes to
the olfactory bulb. For example, ablation of the nasal epithelium in zebrafish (Byrd,
2000) and unilateral naris closure in rat pups (Brunjes, 1985) result in reduction of
bulb size and loss of volume. Additional studies suggest that unilateral olfactory
deprivation results in modification of olfactory bulb anatomy, chemistry, and function
in vertebrates (Wilson and Wood, 1992; Brunjes, 1994; Fuller et al., 2005). These
findings support the fact that reorganization of the olfactory bulb occurs following
activity blockade or removal of the olfactory organ.
Many investigators have used deafferentation to examine injury. Researchers
have used chemical deafferentation of the nasal epithelium as a means o f testing
permanent and temporary injury as well as recovery in mammals (Nadi et al, 1981).
Other methods by which loss of afferent input has been analyzed include removal of
olfactory epithelium precursors via surgery and transaction of the nerve (Matthew and
Powell, 1962; Sanes et al, 1977; Graziadei, 1980; Stout and Graziadei, 1980; Meisami
and Hamedi, 1986; Oland and Tolbert, 1987; Byrd and Burd, 1993). In all of the
above studies, loss of afferent input has caused drastic changes in the development,
the anatomy, or the function of the bulb or antennal lobe.
9
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#
s«M
M
C
«R
#
s Ruffed Cell
O = Granule Cell
0
s Juxtogfonwulir
CM
• / • s Ott*clcxy Stntory
Nm v o ir
ICL
LOT
WOT
Figure 3. A schematic representation o f the zebrafish olfactory bulb shows the circuitry o f cellular
interactions. The olfactory sensory neurons, or primary sensory neurons, are located in the olfactory
epithelium and project their axons into the olfactory bulb. Here, they make synaptic contacts with the
primary output neurons o f the bulb, the mitral cells, and the intemeurons, juxtaglomerular cells. Other
cell types including the mffed cell and the granule cell can contact mitral cells and modify their output
to higher brain centers.
Deafferentation has been used as source for examining injury in other systems
as well. For example, it has been noted that lesions to photoreceptor axons have
caused changes in the formation of the optic ganglion in invertebrates (Power, 1943;
Meyerowitz and Kankel, 1978). In the vertebrate visual system, reduction of afferent
input resulted in an alteration of molecular activity (Wong-Riley, 1979; LeVay et al,
1980; Zhang et al, 1995; Pires et al, 1998; Yan and Ribak, 1998). Additionally,
investigators have evaluated the role of afferent input and injury in the auditory
system (Levi-Montalcini, 1949; Parks, 1979; Garden et al, 1995; Zheng et al, 1998;
Edmonds et al, 1999; Russell and Moore, 1999) and motor-cortical region o f birds
(Johnson et al., 1997). Thus, results from an olfactory deafferentation study may be
10
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applicable to other sensory systems in other models.
Goals of This Dissertation
The following dissertation examines the olfactory system in the adult
zebrafish, Danio rerio. The first goal of this study is to characterize the morphology
and distribution of both output neurons and intemeurons in the olfactory bulb of
untreated animals, employing various methods of visualization. In addition, cellular
interactions between the primary and secondary neurons of the olfactory bulb are
addressed. Finally, the plasticity of this central nervous system structure is examined
by analyzing changes in protein immunoreactivity and cellular morphology following
peripheral injury and subsequent loss of afferent innervation.
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CHAPTER II
IDENTIFYING THE MORPHOLOGY AND DISTRIBUTION OF
JUXTAGLOMERULAR CELLS IN THE OLFACTORY BULB
USING A TRANSGENIC ZEBRAFISH LINE
(Collaboration with Drs. Dan Goldman and Steven Suhr, University of Michigan)
Introduction
The cytomegalovirus (CMY) gene promoters are among the most potent
mammalian enhancer elements known and often are used as promoters in the
development of transgenic animals because of their ubiquitous expression. In this
study, a simian CMV promoter was used to drive the expression of a fluorescent
reporter protein, dsRED. The construct, which was injected into zebrafish eggs, was
comprised of the CMV promoter, a dsRED protein-encoding region, and an SV40
polyA signal that was added to prevent rapid degradation of the transcript (Figure 4).
These injections yielded a transgenic population that showed dsRED expression in a
highly specific and consistent pattern. The expression was limited primarily to
portions of the nervous system including cells in the retina, cerebellum, spinal cord
and olfactory bulb. The focus of this study was to establish the identity of the
olfactory bulb cells expressing the transgene and to use this information as a basis for
determining the morphology and distribution of specific cell types in the olfactory
bulb of adult zebrafish.
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A
1-
3.
?.
4.
5.
Figure 4. A schematic diagram shows the steps taken to develop the transgenic zebrafish as well as a
diagram o f the sCMV-dsRED expression cassette. A) 1. The dsRED expression plasmid was only one
o f several constructs injected into the zebrafish eggs. 2. The mix o f plasmids was introduced into fish
eggs at the single-cell stage by microinjection. 3. Fish that had the potential for germ cell transmission
were identified by the red fluorescence in FO animals. 4. Positive FOs were mated and positive FI
progeny also were identified by fluorescence. 5. Positive FIs were screened for the presence of the co­
injected plasmid by PCR analysis o f tail DNA. These animals were out-crossed to wild-type fish to
perpetuate the lines. B) The expression vector was comprised o f a 990bp sCMV promoter, a dsRED
protein-encoding region, and an SV40 polyA signal.
Materials and Methods
Transgenic Fish
Simian CMV-dsRED transgenic lines were created by the injection of plasmid
constructs into fertilized fish eggs by standard methods (Figure 1). Positive FO fish
were identified by the presence of dsRED expression at 10 days post-fertilization.
These animals were mated to produce an FI population, which also was screened at
10 days post-fertilization for dsRED expression. Positive FIs were out-crossed to
wild-type fish to perpetuate the transgenic line.
Immunocytochemistry
Adult zebrafish were
transcardially
with
over-anesthetized with tricaine
phosphate-buffered
saline
(PBS)
and perfused
followed
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by
4%
paraformaldehyde in PBS. The fish were then incubated overnight in the fixative
solution. Following dissection, brains were embedded in gelatin, sectioned at 1020pm on a cryostat, and mounted on positively charged slides. Olfactory bulb
sections were blocked with 3% normal goat serum and 0.4% triton X-100 in PBS and
treated with primary antibody (anti-tyrosine hydroxylase, ImmunoStar, 1:1000 in
blocker, or anti-HuC/HuD, Molecular Probes, 1:100 in PBS with 1% BSA) for 24
hours. After rinsing in PBS, slides were incubated in biotinylated secondary antibody
(goat anti-mouse IgM, Jackson ImmunoResearch, 1:100 in blocking solution) for 1
hour, and antibodies were visualized using fluorescent avidins (Molecular Probes,
1:200 in PBS). Other tissue sections were either treated with DAPI, a DNA-labeling
fluorescent dye that serves as a general nuclear marker, or the red/green cone
photoreceptor antibody, FRET 43 (Zpr-1, University of Oregon monoclonal bank,
1:200 in PBS). Neuromast staining along the lateral line was completed by immersing
whole fish in 5mM 4-(4-diethylaminostyryl)-jV-methylpyridinium iodide (4-di-2-Asp,
Sigma) for 5 minutes in tank water. Fish were then anesthetized and imaged with a
Leica FLUO microscope.
Olfactory Tract Tracing
For olfactory tract-tracing, fish were anesthetized with 0.03% MS222 (3amino benzoic acid ethyl ester, Sigma) and perfused transcardially with phosphate
buffered saline (PBS). The brains were dissected, and the fluorescent dextran
microemerald (3000 MW, diluted 5mg/ml in PBS, Molecular Probes) was injected
into the medial or lateral olfactory tracts or into both tracts of each olfactory bulb
using a PV 800 Pneumatic Picopump (World Precision Instruments). The brains were
then transferred to a sterilized filter in a six-well culture dish (Costar) and incubated
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at 28.5°C and 1.5% CO2 in artificial fish cerebrospinal fluid (lOOmM NaCl, 2.46mM
KC1, ImM MgCl2-6H20 , 0.44mM NaH2P 0 4H20 ,
1.13mM CaCl2H20 , 5mM
NaHCOs) for approximately 4 hours (Tomizawa et al., 2001). Following fixation in
4% paraformaldehyde for 24 hours, horizontal cryostat sections at 20-50pm were
obtained and mounted on positively charged slides. The tissue was coverslipped and
viewed on a Nikon E600 Eclipse microscope or a Zeiss LSM 510 confocal
microscope.
Results
General dsRED Expression
Expression of the dsRED transgene was seen in a number of zebrafish
structures including the olfactory bulb, the retina, the spinal cord, the cerebellum, the
adrenal gland, and nerve fibers that innervate the lateral line (Figure 5). Expression
patterns were first visible in the olfactory bulb at 5 days post-fertilization (dpf),
making this the first structure in which cells expressed the transgene. By lOdpf, cells
in the eye and spinal cord were apparent and at 15-30dpf, fibers of the lateral line, the
adrenal gland and Purkinje cells in the cerebellum were noticed (Figure 5).
Expression in the Olfactory Bulb
Initial screenings of transgene expression in the olfactory bulb showed
dsRED-positive cells in the superficial internal cell layer (ICL) and in the glomerular
layer (GL; Figure 6A). The labeled cell bodies had teardrop-shaped somata that
ranged in size from 5- 10pm in diameter (Figure 6B). The processes were visible
protruding from the cell bodies and terminated as a tuft in the glomerular layer
(Figure 6C-D).
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In order to determine the cell type that was being labeled, it was first
necessary to establish whether the cells were a neuronal population, a non-neuronal
population, or both. Based on labeling experiments with the neuronal marker
HuC/HuD, the cells expressing the transgene were neurons (Figure 7). However,
retrograde tract tracing experiments with a fluorescent dextran confirmed that the
cells were not output neurons (Figure 8). Another neuronal cell type found in the GL
and the superficial ICL is a group of intemeurons known as juxtaglomerular cells.
These cells are commonly labeled with an antibody against tyrosine hydroxylase,
which is a potential indicator of dopaminergic neurons. Labeling with anti-tyrosine
hydroxylase yielded co-localization of the markers in some, but not all cell bodies
(Figure 9).
Discussion
The creation of this transgenic line is interesting in that unlike many other
CMV promoters that exhibit universal expression, the expression o f this promoter is
confined almost exclusively to the nervous system. Further, this particular transgenic
line is beneficial in that the trangene is expressed early in development, which allows
tracking of specific cell populations from very early stages through adulthood. The
focus of this study was to establish the identity of the cells expressing dsRED in the
olfactory bulb of adult zebrafish. Based on HuC/HuD labeling, the cells are neurons.
While these cells are found in the same location as mitral cells, they are not output
neurons since retrograde labeling of the olfactory tracts with a fluorescent dextran
labels a different subset of bulbar neurons. Although, it also is possible that not all of
the axons were labeled when the tract tracing experiments were being completed.
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Figure 5. The dsRED expression pattern was observed in several regions o f the fish. A) A fluorescence
image o f a zebrafish whole mount showed high levels o f expression in the olfactory bulb. B) UV/blue
subtype cones were dsRED positive. The FRET43 antibody (Zpr-1, University o f Oregon), which is
specific for the red/green cone subtype, was viewed with FITC (green). C) A low magnification view
o f the zebrafish flank showed positive cells in the spinal cord (arrows). D) Purkinje cells in the
cerebellum also were labeled by the transgene. E) The adrenal gland located just rostral to the dorsal
fin showed distinct expression. The fluorescent blue marker, DAPI, showed staining o f nuclei. F)
Nerve fibers innervating the lateral line were dsRED positive. Green channel fluorescence represented
4-di-2-Asp staining of the lateral line neuromasts, and yellow was indicative of colocalization (arrow).
Additionally, it is possible that the dextran was not allowed ample time to
travel and did not label all of the cells bodies. On the other hand, previous work in
our laboratory has shown that cells in the most rostral regions of the olfactory bulb
can be labeled in the tracing time used for this study (data not shown).
Another population of cells distributed in the same vicinity as mitral cells is
the juxtaglomerular cells. These cells, which can range from 5-10pm in diameter, are
local intemeurons that modify the output of mitral cells. Since these intemeurons
interact closely with mitral cell dendrites, they also are distributed throughout the
superficial internal cell layer and the glomerular layer.
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A
B
ONL
GL
ICL
f---------»
50 pm
1 0 |im
D
C
Figure 6. The transgene was expressed in a subset o f cells in the olfactory bulb. A) In the olfactory
bulb, dsRED-positive cells were observed in the superficial ICL and the GL but not the ONL. B) A
high magnification image o f dsRED-expressing cell bodies showed that the cells were approximately
5-1 Opm in diameter. C and D) Cellular processes also showed the presence o f the reporter protein.
Based on immtmocytochemistry experiments with the antibody against
tyrosine hydroxylase (TH), it is apparent that many of the dsRED positive cells also
are TH positive. Because TH is used as a marker for intemeurons in this species
(Edwards and Michel, 2002), we conclude that the majority of the dsRED expressing
cells are intemeurons.
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Figure 7. dsRED-positive cells labeled with a neuronal marker. A) Anti-HuC/HuD labeling o f the
zebrafish olfactory bulb showed that neurons were distributed throughout the ICL and GL. B and C)
The somas o f cells expressing dsRED were positive for anti-HuC/HuD.
Based on the distribution of the cells in the glomerular layer, we conclude that
these cells are juxtaglomerular intemeurons. Some, but not all, dsRED cells were TH
positive. It is possible that some of the dsRED positive cells that are TH negative are
a different subpopulation of juxtaglomerular cells that label with a protein other than
TH. In some mammalian studies, periglomerular cells (the presumptive equivalent of
juxtaglomerular cells in fish) label with calcium binding proteins such as calbendin or
calretnin (Crespo et al., 1997). Thus, the dsRED cells that did not label with TH still
may be juxtaglomerular cells. Further studies are necessary to establish this
possibility.
This study is important in that it begins to reveal the morphology and
distribution of juxtaglomerular cells in the adult zebrafish olfactory bulb. This cell
type is often considered equivalent to the granule cell intemeurons o f the internal cell
layer, but its morphology has never been established independently in this animal. In
addition, this transgenic line may provide an important clue as to the development of
juxtaglomerular cells in zebrafish from very early stages following fertilization
through adulthood.
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D
F
10 |im
Figure 8. Cells that were dsRED+ were not output neurons. Cells showing dsRED expression (A) were
seen in the GL and ICL of the olfactory bulb as were output neurons labeled with microemerald
applied to the olfactory tracts (B). C) Overlay o f the two channels, however, showed that dsRED cells
were not output neurons. D) A higher magnification image of dsRED and microemerald again
suggests that the dsRED cells are not mitral cells since they do not extend axons to the medial or
lateral olfactory tracts although both mitral cells (E) and dsRED cells (F) exhibited a similar size and
morphology.
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B
X
1 0 (Jm
Figure 9. A few dsRED cells were positive for tyrosine hydroxylase A) Typical tyrosine hyrdoxylase
antibody labeling, shown here with diaminobenzidine, identified numerous cells and processes
scattered throughout the bulb. B and C) Some dsRED-positive cells showed tyrosine hydroxylase
immunoreactivity (arrows), but others did not label with the antibody. D) Processes of dsRED-positive
cells and TH-positive cells were tightly interspersed throughout the glomerular layer.
This study was supported by NIH-NIDCD grant DC04262 (CAB) and the Hereditary
Disease Foundation: Cure Huntington’s Disease Initiative (STS).
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CHAPTER IE
IONOTROPIC GLUTAMATE RECEPTOR SUBUNIT 4 IN THE OLFACTORY
BULB OF ADULT ZEBRAFISH: DISTRIBUTION AND EXPRESSION
FOLLOWING LOSS OF INNERVATION
Introduction
The adult olfactory bulb is an extremely plastic structure. There is normal
replacement of the olfactory sensory neurons in the olfactory epithelium (for review,
see Farbman, 1992) as well as continuous neurogenesis in the olfactory bulb (Altman,
1969; Kaplan and Hinds, 1977; Burd and Sein, 1998; Peretto et al, 1999; Byrd and
Brunjes, 2001; Iwai et al, 2003, Stenman et al, 2003). Due to this constant turnover,
there is a need for continual identification of new targets and formation of new
synapses within the bulb (for review, see Farbman, 1992). In addition to the natural
plasticity of this region in adult animals, deafferentation studies show the ability of
the adult olfactory bulb to respond to changes in afferent input. For example, ablation
of the olfactory organ in fish (Byrd, 2000; Poling and Brunjes, 2000), removal of the
nasal
epithelium
in
rabbits
(Matthews
and
Powell,
1962)
and
chemical
deafferentation in mice (Margolis et al., 1974; Harding et al., 1978) result in
reduction of bulb size. Other studies have shown that olfactory deprivation results in
changes in mRNA and protein expression (Ehrlich et al., 1990; Stone et al., 1991;
Ferraris et al., 1997; Casabona et al., 1998; Oberto et al., 2001) as well as enzymatic
activity (Nadi et al., 1981; Baker et al., 1984). Furthermore, numerous studies suggest
that loss of afferent input results in decreased cell number (Henegar and Maruniak,
1991; Meisami and Safari, 1991; Byrd, 2000) and changes in cellular morphology
(Pinching and Powell, 1971) in the deprived bulb of adult animals.
Many studies have shown that glutamatergic systems influence synaptic
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plasticity (Bliss and Collingridge, 1993; Gean et al., 1993; Bear, 1996; Carroll et al.,
1999). Glutamate is the primary excitatory neurotransmitter in the vertebrate central
nervous system (for review, see Johnson, 1972) and is the chief excitatory
neurotransmitter in the vertebrate olfactory system as well. Glutamate is used by
olfactory sensory neurons to communicate with the primary output neurons of the
olfactory bulb, the mitral cells, and the intemeurons at the first level of lateral
interaction, the periglomerular cells (Sassoe-Pognetto et al., 1993; Berkowicz et al.,
1994; Ennis et al., 1996). Mitral cells also use glutamate to communicate with granule
cells, the intemeurons involved in the second level of lateral inhibition in the
olfactory bulb (Jacobson et al., 1986; Trombley and Westbrook, 1990). Glutamate
can activate both metabotropic (mGluR 1-8) and ionotropic (iGluR 1-7) receptors on
the postsynaptic cell. Metabotropic receptors are composed of one polypeptide linked
to a G-protein second messenger. The ionotropic receptors are ligand-gated ion
channels and mediate fast transmission (Berkowicz et al., 1994) they respond to the
agonists,
N-methyl-D-aspartic
acid
(NMDA),
a-amino-3-hydroxyl-5-
methylisoxazole-4-proprionic acid (AMPA), or kainic acid (KA). It has been shown
that iGluRs have a tetrameric structure (Rosenmund et al., 1998; Chen et al., 1999)
formed by a dimerization of dimers (Armstrong and Gouaux, 2000; Ayalon and
Stem-Bach, 2001).
These receptors can be formed by a combination of iGluR 1-
iGluR4 subunits (for review, see Mansour et al., 2001; Esteban, 2003).
Previous studies in mammals show that iGluRs, specifically AMPA receptors,
are expressed in a number of neurons in the olfactory bulb (Petralia and Wenthold,
1992; Molnar et al., 1993; Sato et al., 1993; Montague and Greer, 1999). Montague
and Greer (1999) propose that the response of postsynaptic cells to glutamate will
vary depending upon the differential distribution of these subunits in the synapse. In
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fish, pharmacological examinations also suggest the presence of AMPA/KA receptors
in the olfactory bulb (Edwards and Michel, 2002). These analyses reveal that AMP A
and KA receptors, activated by kainate, stimulate mitral cells, granule cells, and
juxtaglomerular/TH-positive cells (Edwards and Michel, 2002). Periglomerular cells
have not been defined in teleosts, but we refer to their presumptive equivalents in
zebrafish as juxtaglomerular cells based on their distribution and function. While
these findings are significant in that they help to define the role of glutamatergic
neurotransmission in the fish olfactory bulb, the AMPA and KA receptors are
grouped into a single category. As a result, it is unclear which specific receptor
subunits are expressed by individual cell types. In general, AMPA receptors have
been widely studied in the brain and spinal cord, but investigations into their precise
distribution in the olfactory bulb has been limited.
The ionotropic AMPA receptor, iGluR4, is of particular interest because it is
expressed in high levels where cells display fast kinetics and rapid desensitization
(Moyner et al., 1991; Raman et al., 1994; Rubio and Wenthold, 1997; Silver et al.,
1992). Localization of this subunit has been examined specifically in the central
cervical nucleus of rat (Ragnarson, 2003), the outer plexiform layer of goldfish
(Schultz, 1997), retinal cells of chick embryos (Cristovao and Carvahlo, 2003), and
the human cerebral cortex (Ong and Garey, 1996). It is expressed in auditory cells
that need to communicate signals for pinpointing sound (Raman et al., 1994) and is
preferentially expressed in motor neurons of the spinal cord (Tomiyama et al., 1996)
where prompt neurotransmission has been documented (Smith et al., 1991). In turn, it
may be important for specific neurons in the olfactory bulb to express this receptor
subunit, where rapid response may prove significant in detection of food, predators,
or mates.
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The present study examines the distribution of the AMPA-type receptor
subunit, iGluR4, in the olfactory bulb of adult zebrafish, Danio rerio. It establishes
that olfactory
sensory input plays
a role in the maintenance of iGluR4
immunoreactivity in this plastic system. Our results demonstrate a specific laminar
and cellular distribution of iGluR4 and distinct changes in the localization or
expression of this protein following loss of afferent input.
Materials and Methods
Western Blot Analysis
In order to confirm that the ionotropic GluR4 antibody made against rat
protein was not crossreactive with multiple proteins in the zebrafish olfactory bulb,
Western blot analysis was performed. Adult zebrafish brains (n=10) were dissected
out of anesthetized, live animals, frozen on dry ice, and macerated in lysis buffer
(Tris-HCl
20mM,
Sucrose
10%,
EDTA
ImM,
SDS
2%,
phenylmethanesulfonylfluoride ImM and Protease Inhibitor Cocktail 1:100, all from
Sigma). A rat brain (Sprague Dawley, 4 weeks of age) also was examined, using the
above protocol, in order to compare protein size in both animals. The rat was
anesthetized by carbon dioxide asphyxiation followed by thoracotomy. It was being
used for another study and the brain was a generous gift of Dr. John Spitsbergen. The
samples were loaded into a 10% Tris-Glycine gel (Invitrogen) and the iGluR4
blocking peptide (1:1000 in lysis buffer, Chemicon) also was loaded in order to
confirm binding specificity of the peptide to the antibody (data not shown). The gel
was run at 200Vfor 1 hour. Proteins were then transferred to an Immobilon-P PVDF
transfer membrane (Millipore) for 2 hours and blocked with blocking agent (5% in
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distilled water, ECL Western Blot Analysis System, Amersham Biosciences). The
membrane was incubated in primary antibody (anti-ionotropic GluR4, Chemicon,
1:1000 in blocking agent) for 12 hours at 4°C and treated with secondary antibody
(goat anti-rabbit IgG, Amersham Biosciences, 1:2500 in blocking agent) for 1 hour.
The membrane was then treated with a peracid and luminol mixture (1:1, ECL
Western Blot Analysis System, Amersham Biosceinces), exposed to photographic
film (OMAT, Kodak) for 3 minutes and developed.
Tissue Processing
Adult male and female zebrafish, at least 2.5cm in length, were obtained
commercially from local suppliers. They were maintained in 28.5°C aerated,
conditioned fish water and fed freshwater flake food (Wardley Corporation) twice
daily. All animal care protocols and experimental procedures were approved by the
Institutional Animal Care and Use Committee.
Fish were anesthetized with 0.03% MS222 (3-amino benzoic acid ethyl ester,
Sigma) and perfused transcardially with phosphate buffered saline (PBS) followed by
4% paraformaldehyde. The animals were placed in 4% paraformaldehyde overnight
for further fixation of the tissue. The brains were dissected, washed in PBS,
dehydrated, and embedded in paraffin blocks. Ten-micron horizontal or coronal
sections of the olfactory bulbs were obtained and mounted on positively charged
slides.
Immunocytochemistry
Sections were rehydrated, treated with 3% hydrogen peroxide to remove
endogenous peroxidases, and blocked with 3% normal goat semm and 0.4% Triton
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X-100 in PBS. They were treated with a polyclonal antibody (anti-ionotropic GluR4,
1:100 in blocking solution, Chemicon) for 24-48 hours at 4°C, rinsed, and incubated
in biotinylated secondary antibody (goat anti-rabbit IgG, 1:100 in blocking solution,
Jackson ImmunoResearch). They were then treated with avidin-biotin complex
(Vectastain Elite ABC Kit, Vector) and visualized using diaminobenzadine (DAB
substrate kit, Vector). Intensity profiles were examined using the Metamorph
program. For double-label fluorescence techniques, slides were treated sequentially
with primary antibody (polyclonal anti-ionotropic GluR4, 1:100 in blocking solution,
Chemicon or monoclonal anti-tyrosine hydroxylase, 1:1000 in blocking solution,
ImmunoStar) for 24 hours at 4°C, rinsed in PBS, and incubated in a fluorescently
labeled secondary antibody (goat anti-rabbit Alexafluor 594, 1:100 in PBS, Molecular
Probes or goat anti-mouse Alexafluor 488, 1:100 in PBS, Molecular Probes) for 1
hour. Following staining, the slides were coverslipped with glycerol and PBS (50:50)
and viewed on a Zeiss LSM 510 confocal microscope using a multitrack overlay with
narrow band filters. A total of five fish were examined in double label experiments.
Controls
Negative controls, in which the primary antibody was omitted in order to
account for the possibility of non-specific staining, yielded no staining in both
Western blot analysis and immunocytochemistry. Positive controls were completed
using anti-keyhole limpet hemocyanin (KLH, 1:100 in blocking solution, Sigma) to
confirm the immunocytochemical staining method. In addition, antibody specificity
controls were done by pre-absorbing the diluted antibody with the iGluR4 blocking
peptide (1:100, Chemicon). This control showed no staining in both Western blot
analysis and immunocytochemistry. In order to confirm that the iGluR4 antibody was
27
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not binding to the tyrosine hydroxylase protein, an additional western blot analysis
was performed as described above; anti-tyrosine hydroxylase (TH, Immunostar,
1:2000 in blocking agent) was used as the primary antibody and goat anti-mouse
(Amersham Biosciences, 1:2500 in blocking agent) served as the secondary antibody
(data not shown). Also, to verify the results of the tissue blocking experiment, an
additional control was completed whereby anti-KLH was pre-absorbed with the
iGluR4 blocking peptide (1:100). This experiment resulted in the standard KLH
staining pattern on tissue sections suggesting that the blocking peptide did not prevent
labeling in a non-specific manner.
Deafferentation
To
examine the effects of afferent removal on glutamate receptor
immunoreactivity in the bulb, peripheral deafferentation was performed as described
previously (Byrd, 2000). The fish were anesthetized using 0.03% MS222 until they
no longer responded to a tail pinch, and the right olfactory organ was extirpated using
a small-vessel cautery iron. The left olfactory organ remained intact for use as an
internal control for comparison. Sham-operated control animals (n=4) received a
wound to the tissue between the olfactory organs that was similar in size to that of the
deafferented fish in order to control for non-specific wound responses. Fish were
returned to
aquarium water containing the antibiotic Kanacyn
(O.Olmg/ml,
Aquatronics) and were allowed to survive for 24 hours (n=7), 48 hours (n=5), 1 week
(n=5), or 3 weeks (n=5). Control fish (n=5) were maintained in a separate aquarium
and also were treated with Kanacyn (O.Olmg/ml, Aquatronics). After the appropriate
survival period, the deafferented, sham-operated, and control fish were processed as
described above.
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Quantitative Analysis
In order to quantify ionotropic GluR4-immunoreactivity, digital images were
obtained using a Nikon E600 microscope and Spot Advance software. The color
images were converted to grayscale images, and the average gray values of 3
midbulbar sections of left and right olfactory bulbs were determined using the ImageJ
program provided by NIH. For each animal, the largest sections from right and left
bulbs were chosen independently and analyzed in five measures: whole bulb, rostral
medial quadrant, caudal medial quadrant, rostral lateral quadrant, and caudal lateral
quadrant. In addition, measurements were recorded from a section 30pm to the right
and 30pm to the left of the largest section in order to accurately reflect staining in
different planes of the olfactory bulb.
To account for minor variations in tissue processing and background staining,
gray values for each section were converted to optical density (OD) values by the
formula: OD= log (intensity of background/intensity of area of interest). The
background intensity was established by measuring the average gray level o f a non­
staining telencephalic region similar in size to the area of interest. The optical
densities for the three midbulbar sections were then used to calculate an average OD
value for the right and left bulb of each fish. Percentage differences between the right
and left bulbs were calculated using the following formula: Average OD Right BulbAverage OD Left Bulb/Average OD Right Bulb x 100%. The percentages for all
animals in a given time point were averaged. Paired Two-Sample T-tests were used to
examine differences between deafferented and internal control bulbs at each time
point. Also, a one-way ANOVA and Fisher’s test for multiple comparisons were used
to determine if there was a significant difference in iGluR4-immunorectivity in
control and deafferented tissue across and between all time points. P-values less than
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0.05 were considered significant.
To determine the size of immunoreactive cell bodies in the olfactory bulb,
high magnification digital images were obtained from the olfactory bulbs of adult
animals, and the diameters of somata were measured using the Spot Advance
Software line measurement tool.
Results
Antibody Specificity and Controls
The specificity of the antibody to the iGluR4 protein in zebrafish and rat brain
was analyzed using Western immunoblots (Figure 10A). In zebrafish and rat brain
homogenates, the antibody to iGluR4 showed an immunoreactive band with a
presumptive molecular mass just above lOOkDa. In addition, a labeled band
frequently appeared around 50kDa in both the rat and the zebrafish homogenates. On
tissue sections, a control in which the primary antibody was omitted (10B) resulted in
no staining and, when the iGluR4 antibody (10C) was pre-absorbed with the iGluR4
peptide (10D), the labeling was blocked. In order to confirm that blocking was
specific, a positive control was run whereby anti-keyhole limpet hemocyanin (KLH)
was incubated with the iGluR4 peptide. In this case, the KLH antibody showed the
same pattern of immunoreactivity on tissue sections as the control KLH (compare
Figure 10E and 10F). This confirms that the iGluR4 peptide is specific to the iGluR4
antibody and does not cause non-specific blocking. Based on the results of the peptide
blocking experiments, consistency of band migration between species, and other
investigations that report similar results at both 50kDa and lOOkDa (Wenthold et al.,
1992), we conclude that the antibody is specific to iGluR4.
30
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Laminar Distribution of iGluR4 in Zebrafish Olfactory Bulbs
Distribution of the AMPA receptor subunit iGluR4 in the adult zebrafish
olfactory bulb was examined using immunocytochemistry. Ionotropic GluR4
immunoreactivity displayed a laminar pattern in the olfactory bulb. Antibodies to this
receptor subunit showed the greatest amount of labeling in the glomerular layer (GL,
Figure 11 A, B), with dendrites of several cells showing prominent immunoreactivity
(Figure 11B, C). There was lesser staining in the internal cell layer (ICL), and
virtually no staining in the olfactory nerve layer (ONL, Figure 11 A, B). A 10pm
section of the bulb was analyzed using Metamorph Intensity Profile (Figure 11D, E).
This program utilizes optical density to establish intensity distributions where the
maximum height of the profile is correlated with the lightest area of the tissue, or in
this case, reduced immunoreactivity. The highest level of labeling was found in the
GL and the lowest level in the ONL (Figure 11D, E). Ionotropic GluR4
immunoreactivity also was examined in coronal sections in order to examine further
the laminar distribution seen in the horizontal plane. These analyses showed a similar
pattern of iGluR4 immunoreactivity with the most abundant staining seen in the GL
(Figure 12A,B) and lesser staining observed in the ICL (Figure 12C).
Cellular Distribution of iGluR4 in the Olfactory Bulb
With regard to cellular distribution, numerous somata were labeled throughout
the glomerular region (Figure 11A-C, 12B-C, arrows). Cell bodies ranged in size
from 4pm to 8pm in diameter, and their processes were visible in the glomerular
layer (Figure 1 IB, C).
31
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B
Rat
ZF
120 kDa
100 kDa
No Primary
0NL
$
-f.Nf
'a.-
.’
‘1
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Figure 10. Control experiments were used to confirm the specificity o f the antibody to zebrafish tissue.
(A): Western blot analysis o f zebrafish and rat brain tissue, using an antibody to ionotropic glutamate
receptor subunit 4, iGluR4, shows an immunoreactive band at 108kDa in both species. (B): A control
in which the primary antibody was excluded yields no staining when treated with goat anti-rabbit
secondary antibody and diaminobenzadine. (C): Ionotropic GluR4 (iGluR4) labels somata (arrows) and
shows high immunoreactivity in the glomerular layer (GL) o f the adult zebrafish olfactory bulb. There
is no label in the olfactory nerve layer (ONL). (D): Immunoreactivity is blocked completely when the
iGluR4 antibody is pre-absorbed with the iGluR4 peptide (iGluR4 pep). (E): An antibody against
keyhole limpet hemocyanin (KLH) is used as a positive control in the adult zebrafish olfactory bulb
due to its distinct and consistent labeling of the olfactory nerve layer in fish (arrow, for example). (F):
When the KLH antibody is incubated with the iGluR4 peptide at the same concentration used in the
anti-iGluR4 blocking experiment, normal anti-KLH staining is observed. Scale bar = 80pm (B-F).
32
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Figure 11. Ionotropic GluR4 is expressed in the adult zebrafish olfactory bulb. (A): A low
magnification view shows the heaviest immunoreactivity in the glomerular layer (GL), lighter
expression in the internal cell layer (ICL), and very little to no immunoreactivity in the telencephalon
(TEL) and olfactory nerve layer (ONL). Even at this magnification, cell bodies are discemable (arrow).
(B): At higher magnification, laminar distribution is seen more clearly when staining time is reduced.
Numerous cell bodies (arrow) and abundant processes (arrowhead) are labeled in the GL. Some
immunopositive cells and processes also are found in deeper regions o f the bulb. (C): The labeled
soma o f a single cell, found in the GL, exhibits iGluR4 immunoreactivity in a primary dendrite and a
presumptive axon, as well. (D): This Metamorph Intensity Profile, which corresponds directly to the
bulb segment in E, reveals that the greatest iGluR4 immunoreactivity is in the GL and the least iGluR4
immunoreactivity is in the ONL. The lowest points on the profile are representative of the darkest
areas o f the tissue or areas o f highest optical density and greatest immunoreactivity. The highest points
on the profile indicate regions that are much lighter and have low optical density and low
immunoreactivity. (E): A strip o f the adult zebrafish olfactory bulb corresponding to the profile data in
D is shown. Scale bar = 100pm (A), or 40pm (B), or 25pm (C) or 15pm (E).
33
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Figure 12. Coronal sections taken at intervals through the olfactory bulb confirm the strongest labeling
is seen in the glomerular layer. (A): The GL in the most rostral portion of the bulb shows the heaviest
staining. The ONL around the perimeter of the bulb shows no label. (B): The boundary between the
GL and the ICL exhibits a number of cell bodies (arrows) (C): The difference in the laminar staining
pattern becomes more obvious in more caudal regions, with the ICL showing less immunoreactivity
than the superficial GL. Again, cells can be seen easily in the glomerular region (arrow). d=dorsal,
m=medial, v=ventral, 1= lateral. Scale bar = 100pm (A-C).
Olfactory bulbs were examined using double-label fluorescence techniques with the
iGluR4 antibody as well as an antibody to tyrosine hydroxylase (TH). TH, the ratelimiting enzyme in catecholamine synthesis, is expressed in a subset of neurons found
in the glomerular layer of the adult zebrafish olfactory bulb (Byrd and Brunjes, 1995;
Edwards and Michel, 2002).
TH-positive cells and iGluR4-positive cells,
individually, gave similar immunoreactive profiles (compare Figures 13A, B). Also,
double-label techniques showed colocalization between TH and iGluR4-expressing
cells (Figure 13C-E). However, some iGluR4-immunoreactive cells were THnegative (Figure 13E, F, arrow) and some TH-immunoreactive cells were iGluR4negative (Figure 13E, arrowheads). These cells had the same size and morphology as
the TH-positive/iGluR4-positive cells and also were distributed in the glomerular
region of the bulb. Due to the similarity in the expression patterns of antibodies to the
two proteins, Western blot analysis of zebrafish brain was completed using antibodies
to iGluR4 and TH. The two proteins each displayed bands of different molecular
weight suggesting that they were not crossreactive with one another in the
34
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fluorescence stain (data not shown).
Effects Of Deafferentation On iglur4 Immunoreactivity
Ionotropic GluR4 immunoreactivity in the adult zebrafish olfactory bulb was
examined following removal of afferent input. The whole bulb, excluding the
olfactory nerve layer, was analyzed by averaging the OD values of three right and left
midbulbar sections in at least 3 fish at a given timepoint. In unoperated, control
animals, there was no significant difference between the labeling of iGluR4 in the
right and left olfactory bulbs (P=0.719, Figure 14A). In sham-operated animals,
where a small wound was made to the tissue between the right and left nares, iGluR4staining intensity remained unchanged between the right and left bulb (P=0.821,
Figure 14B). At 24 hours post-deafferentation, however, there was a significant
. difference in iGluR4 immunoreactivity between the right, unoperated bulb and the
left, treated bulb (P=0.018, Figure 14C). At this timepoint, the iGluR4 label was
noticeably diminished in the deafferented bulb. This decrease appeared to occur
primarily in the processes (compare Figure 14G,H), as iGluR4-positive cell bodies
were still apparent; however, overall the number of immunoreactive cells appeared
less than in control animals. By 48 hours after surgery, the right bulb still showed a
trend of reduced immunoreactivity (Figure 14D), but statistically there was no
difference between the treated and untreated bulbs (P=0.054). At 1 and 3 weeks postdeafferentation, iGluR4 staining had returned to normal levels (P=0.343, Figure 14E,
F). A one-way ANOVA showed a significant difference in immunostaining between
the right and left olfactory bulbs across all time points (P=0.048), and Fisher’s test for
multiple comparisons confirmed that the only significant difference was between the
24-hour time point and all others.
35
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Figure 13. Immunocytochemical techniques help to identify iGluR4-expressing cell types. (A): Cell
bodies and processes labeled with iGluR4 are found predominantly in the GL and the superficial ICL,
a similar distribution pattern to tyrosine hydroxylase (TH)-immunoreactivity (B). Ionotropic GluR4
(C) and TH (D), observed with immunofluorescence, demonstrate the same labeling patterns seen with
diaminobenzadine. (E): Double labeling with iGluR4 (red) and TH (green) shows almost complete
overlap o f the two antibodies. Notice that some iGluR4-positive cells (arrow) are not TH-positive and
some TH-positive cells (arrowheads) are not iGluR4-positive. (F): A high magnification image
confirms that not all iGluR4 cells are TH-positive (arrow). Scale bar =50pm (A, B) or 40pm (C-E) or
10pm (F).
To
analyze further the
effects
of sensory input loss
on iGluR4
immunoreactivity, the distribution of the AMPA receptor subunit was examined in
four quadrants of the olfactory bulb in the horizontal plane: rostral medial, rostral
36
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lateral, caudal medial, caudal lateral. These quadrants were chosen based on
glomerular distribution and provided a delineation between the medial and lateral
portions of the bulb as well as the rostral and caudal portions of the bulb. Again, no
difference was seen between the right and left bulbs o f control or sham animals in any
quadrant (Figure 15A-D). At 24 hours post-surgery, both the rostral medial and
rostral lateral quadrants showed a decreased labeling of iGluR4 (Figure 15A, B). Both
caudal quadrants did not show this loss of immunoreactivity at the 24-hour timepoint
(Figure 15C, D). Forty-eight hours, 1 week, and 3 weeks after loss of sensory input,
iGluR4 staining had returned to initial levels in all quadrants (Figure 15A-D).
Fisher’s test for multiple comparisons confirmed that the only significant difference
in the rostral areas of the bulb was between the 24-hour time point and all others.
Discussion
These results show the distribution of the AMPA-type receptor subunit
iGluR4 in the normal and deafferented adult zebrafish olfactory bulb. The specificity
of the antibody and the validity of the staining method were confirmed using several
controls including Western blots and pre-absorption with the iGluR4 peptide in tissue
sections. Western blots of rat and zebrafish brain homogenates show bands with
presumptive molecular weights of 108kDa and 51kDa. Ionotropic GluR4 has an
apparent molecular mass of 108kDa in rat (Wenthold et al., 1992) and goldfish
(Schultz et al., 1997). The two bands in our immunoblots migrate equally in both rat
and zebrafish, and other researchers have found a 51kDa band in addition to the
108kDa band (Wenthold et al., 1992). In our experiments, omission of the primary
antibody and pre-absorption of the iGluR4 antibody with the iGluR4 peptide yield no
staining on Western blots.
37
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100
80
60
4020
-
0 ■
-
20-
-40
-60
-
-
80-
100
-
Control
Sham
24H
48H
1WK
3WK
Figure 14. Quantitative analysis illustrates changes in iGluR4 immunoreactivity in the whole olfactory
bulb. The graph shows the mean percent difference (± SEM) in optical density values between the left,
control bulb and the right, treated bulb. Control (n=5), sham-operated (n=4), and deafferented 24 hour
(n=7), 48 hour (n=5), 1 week (n=5), and 3 week (n=5) olfactory bulbs were analyzed. There is a
significant difference between the two bulbs at 24 hours post-deafferentation (*P<0.05).
Photomicrographs below each bar show iGluR4 immunoreactivity in the right bulbs of control and
deafferented animals. Control (A) and sham-operated (B) animals show high levels o f iGluR4 label in
the olfactory bulb. Twenty-four hours after deafferentation (C) the extent o f labeling is noticeably
diminished, particularly in the medial bulb regions. At 48 hours (D), lweek (E), and 3 weeks (F) postdeafferentation, iGluR4 immunoreactivity in the olfactory bulb has returned to normal. (G): In a
control animal, the glomerular layer shows heavy labeling in the cell bodies and processes. (H):
Twenty-four hours after deafferentation immunoreactivity in the cell bodies is still apparent, but
labeling in the processes has significantly decreased. Scale bar = 100pm (A-F) or 15 pm (G, H).
38
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R ostral M edial
A
R ostral L ateral
B
iso
150
100
100
so
50
*
0 _j_
]
0
flip
-50
-50
-100
-100
-150
-150
SH
24H
48H
1WK 3WK
C
D
C a u d a l M edial
24H
48H
1WK 3WK
C au d al L ateral
150i
150
100
100
50
50
o
0
-50
-50
-100
-100
-150
SH
-150
SH
24H
48H
1WK 3WK
C
SH
24H
48H
1WK
3WK
Figure 15. Quantitative analysis shows changes in iGluR4 inununoreactivity following deafferentation
in four bulb quadrants. There is a significant difference between the control bulbs and the treated bulbs
at 24 hours in the rostral medial quadrant (A) and rostral lateral quadrant (B), but not in the caudal
medial quadrant (C) or the caudal lateral quadrant (D). The number o f animals analyzed is the same as
in Figure 5. *P<0.05.
Finally, a GenBank BLAST (Pubmed) query of the protein sequence to which
the antibody was made does not generate matches for any other proteins (data not
shown). Together, these results suggest that the iGluR4 antibody is specific to iGluR4
in zebrafish and that the smaller band is likely due to proteolytic breakdown of the
39
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antigen that can occur with rat tissue as well.
Ionotropic GluR4-immunoreactivity in tissue sections is highest in the rostral
portion of the bulb in the GL, with lesser labeling in the ICL and no labeling in the
ONL. Further, iGluR4-positive cells co-localize with TH-positive cells. These data
suggest that the expression of iGluR4 occurs primarily in the TH-positive
juxtaglomerular (JG) cells of the olfactory bulb and in many processes in the
glomerular region. However, some iGluR4-immimoreactive cells located in the
glomerular layer were TH-negative. Edwards and Michel (2002) describe two
populations of cells in this region that are smaller than mitral cells: one is TH-positive
and the other is TH-negative. Both cell types contain low glutamate levels and high
GABA levels and are different from the granule cells of the ICL. It is possible that the
iGluR4-positive cells that did not show TH-immunoreactivity are equivalent to the
TH-negative JG cells observed by Edwards and Michel. The TH-positive cells are
likely to be the equivalent of periglomerular cells in other animals. However, since
periglomerular cells have not been defined specifically in teleosts, we identify them
as JG cells and otherwise define them based on their TH expression pattern. The
distribution of this AMPA subunit is consistent with the idea that iGluR4 is
preferentially expressed in areas of fast kinetics and rapid desensitization.
Juxtaglomerular/periglomerular cells are inhibitory intemeurons that receive input
from the olfactory nerve and apical dendrites of projection neurons and modify the
output of mitral cells through release of inhibitory neurotransmitter (for review, see
Farbman, 1992). Rapid activation of intemeurons is likely involved in proper
localization and identification of odorants as it would prevent the brain lfom
receiving equal input from a large number of mitral cells corresponding to a greater
surface area on the olfactory epithelium.
40
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In mammals, iGluR4 is expressed preferentially in the olfactory bulb layers.
Previous studies have described its presence in the neuropil of the external plexiform
layer (Petralia and Wenthold, 1992; Montague and Greer, 1999) and the glomerular
layer (Petralia and Wenthold, 1992; Martin et al., 1993, Montague and Greer, 1999),
a distribution similar to that of teleosts. This distribution has further been examined
on the ultrastructural level and is defined as secondary dendrites of mitral/tufted cells
in the external plexiform layer and periglomerular and mitral/tufted cell processes in
the glomerular layer (Montague and Greer, 1999).
Due to the diffuse laminar
structure of the zebrafish olfactory bulb, distinguishing between mitral cell and JG
cell processes is difficult. As a result, the heavy glomerular distribution of the iGluR4
protein seen in this animal could consist of processes from JG cells, mitral cells, and
astrocytes. The mammalian olfactory bulb appears to show little iGluR4
immunoreactivity in the mitral cell layer and the granule cell layer (Petralia and
Wenthold, 1992; Montague and Greer, 1999), which are equivalent to the zebrafish
ICL. Unlike in teleosts, however, iGluR4 in mammals also is expressed in the ONL
(Petralia and Wenthold, 1992; Montague and Greer, 1999).
With regard to cellular localization, iGluR4 expression in mammals has been
shown in mitral cells (Montague and Greer, 1999), granule cells (Petralia and
Wenthold, 1992), astrocytes (Martin et al., 1993; Montague and Greer, 1999), and in
periglomerular cells (Petralia and Wenthold, 1992; Montague and Greer, 1999).
Martin and colleagues (1993) suggest that minor differences in results could be due to
variations in synthetic peptide lengths, immunocytochemical procedures, or data
analysis and interpretation. In general, the distribution of the iGluR4 protein in
zebrafish is similar to that of mammals.
It is also possible that the minor differences in iGluR4 expression between
41
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mammals and teleosts are due to the differences in olfactory circuitry between the
two vertebrate groups. For example, several studies have shown that teleosts exhibit
distinct differences from mammals with regard to mitral cells (Oka, 1983; Dryer and
Graziadei, 1994). In teleosts such as goldfish (Kosaka and Hama, 1982) and tench
(Alonso et al., 1988) mitral cells have far-reaching dendritic projections that
reportedly contact multiple glomeruli (Nieuwenhuys, 1967; Dryer and Graziadei,
1994). In mammals, however, each mitral cell sends a single primary dendrite to a
solitary glomerulus (for review, see Dryer and Graziadei, 1994) and the axons of
olfactory receptor neurons that possess the same type of odorant receptor project to
the same glomerulus (for review, see McClintock, 2000). This suggests that odors
may be processed differently between the two groups. On the other hand, studies
examining the role of glutamatergic neurotransmission in the adult zebrafish olfactory
bulb suggest that the synaptology of the fish olfactory bulb is inherently similar to
that of mammals (Edwards and Michel, 2002). In fact, preliminary results on the
morphology of mitral cells in zebrafish suggest that these cells may be more similar
to mammalian mitral cells than previously believed, with most mitral cells, including
those with multiple primary dendrites, innervating a single glomerulus (Fuller and
Byrd, 2004). While olfactory processing is not yet understood fully, it is possible that
differences in olfactory bulb circuitry in fish and mammals call for minor variations
in the distribution of the iGluR4 subunit.
In this study, we show that iGluR4 immunoreactivity can be altered by
afferent input. Controls were used to confirm that changes in apparent protein levels
were due to loss of afferent input rather than a general immune response to the
wound. There was no significant difference in antibody labeling between control
animals that received no wound and sham-operated animals that received a wound
42
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between the right and left nares. However, there was a significant difference in
iGluR4-immunoreactivity of the deafferented and control bulbs at the 24-hour time
point. At this stage, antibody labeling in the deafferented bulb was significantly less
than that of the untreated bulb. This suggests reduced protein levels resulting lfom
changes in synthesis and/or degradation. Furthermore, quadrant analyses showed that
the rostral portion o f the bulb showed a significant reduction of labeling with
deafferentation while the caudal region did not. This is likely due to the distribution
of the glomeruli, which are located primarily in the rostral portion of the bulb. Since
the glomerular layer shows the heaviest iGluR4 immunoreactivity in control animals,
loss of staining in the neuronal processes of deafferented animals would be most
prevalent where glomeruli are denser. By 48 hours, iGluR4 staining had returned to
normal and was maintained through 3 weeks post-surgery.
Previous studies in the adult zebrafish olfactory bulb showed an increased
level of apoptosis at 24 hours post-deafferentation (Vankirk and Byrd, 2003). This
could account for the decrease in iGluR4-immunoreactivity seen at the 24-hour time
point, except that staining intensity returns to normal by 48 hours. If apoptosis were
causing the decrease in iGluR4 labeling, it is unlikely that the levels of
immunoreactivity would return to normal at 48 hours. It is equally likely that the JG
cells do not die off in large quantities at this stage, rather that their expression of
iGluR4 is down-regulated as an initial response to the loss of afferent input. Several
studies have shown decreased protein expression in response to sensory input loss
(Baker et al., 1983, Baker et al., 1993). This is a likely possibility considering a
previous deafferentation study in zebrafish that shows 24 hours post-surgery
apoptotic cells are found primarily in the internal cell layer, and only a small
percentage of these cells are neurons (Vankirk and Byrd, 2003). Therefore, the
43
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majority of the neuronal cells undergoing apoptosis at this time point are
intemeurons, based on their location and size. However, because the majority of these
intemeurons are found in the deep internal cell layer it is most likely that they are
granule cells, not juxtaglomerular cells. In another zebrafish study, significant loss of
TH-expressing cells in the olfactory bulb is seen at lweek post-deafferentation (Byrd,
2000). This study also suggests that the TH-positive cells are not degenerating, rather
they reduce expression of the TH protein. In mammals, when olfactory sensory input
is reintroduced following chemical lesion of the olfactory epithelium, TH expression
that had been reduced with loss of afferent input returns to normal levels (Baker et al.,
1983). These studies suggest that reduced iGluR4 immunoreactivity at 24 hours postdeafferentation could be due to a change in the pattern of protein expression
following loss of afferent input rather than a loss of JG cells expressing the protein.
However, it also is possible that there is degradation of the iGluR4 protein present in
the tissue rather than decreased synthesis of the peptide.
In this study, TH and iGluR4 immunoreactivity co-localize to the same cells.
Previous work in the adult zebrafish showed that there was a reduction in the number
of TH-positive cells at 1 week and 3 weeks post-deafferentation (Byrd, 2000). Our
current work suggests that changes in iGluR4 expression follow a different time
course: iGluR4 is reduced at 24 hours and has returned to normal levels by 1 and 3
weeks post-surgery. Once the axons o f the olfactory nerve are severed there are at
least two possible scenarios to describe the resulting cellular response. First, severed
axons release glutamate into the olfactory bulb causing internalization of the iGluR4
receptor subunit. Previous studies showed a higher concentration of extracellular
glutamate following brain injury (Katayama et al., 1990, Nilsson et al., 1990),
suggesting an increase in the release o f this neurotransmitter after trauma. It has
44
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already been established that iGluRs undergo endocytosis as a part of normal synaptic
trafficking (Carroll et al., 1999) and other recent studies have shown that iGluRs can
be internalized rapidly following activation by increased levels o f glutamate and
AMPA (Lissin et al., 1999). This redistribution is reversible following removal of the
agonist, which could explain why the receptors are once again expressed when the
excess glutamate clears. This could result in the return of iGluR4 immunoreactivity
seen at 48 hours post-surgery. Several studies have shown an up-regulation of protein
receptors in response to deafferentation (Gomez-Pinilla et al., 1989; Casabona et al.,
1998).
A second possibility is that loss of afferent input results in an immediate loss
of activation, which causes down-regulation of receptors in the juxtaglomerular cells.
Olfactory denervation results in the down-regulation of specific proteins (Sashihara et
al., 1996) and enzymes (Ehrlich et al., 1990). This correlates to the immediate
reduction in immunoreactivity seen at 24 hours post-deafferentation. Once a steady
state situation is attained, even though it does not include odorant-activated
responses, the glutamate receptors are produced again. This is supported by
investigations showing returned expression of mRNAs and proteins in the olfactory
bulb following loss and subsequent return of afferent input (Oberto et al., 2001).
While a similar initial response can be expected for an enzyme such as TH that aides
in dopamine production for juxtaglomerular intemeurons, the return to normal levels
would not be expected. This is due to the continued loss of sensory input and in turn,
continued reduction in the expression of the enzyme. Since the mitral cells are not
being activated, the JG neurons do not need to make a neurotransmitter that helps to
modify mitral cell output. As a result, the reduction in TH immunoreactivity and the
return of iGluR4 immunoreactivity seen at 1 and 3 weeks post-deafferentation is
45
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expected.
At this time it is unknown whether iGluR4 immunoreactivity is reduced as a
result of glutamate excitotoxicity or immediate loss of activation, but distinguishing
between the potential mechanisms that result in this change is the focus of future
studies. Our goal is to gain a better understanding of the cellular interactions involved
in afferent target regulation in this key model system.
This information was presented in a 2005 publication entitled, “Changes in glutamate
receptor subunit 4 expression in the deafferented olfactory bulb of zebrafish” in Brain
Research.
46
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CHAPTER IV
THE MORPHOLOGY AND DISTRIBUTION OF RUFFED CELLS IN THE
OLFACTORY BULB OF ADULT ZEBRAFISH
Introduction
The ruffed cell is an output neuron that has been identified in the olfactory
bulb of select fish, but not in other animal groups. It was first described in goldfish
(Kosaka and Hama, 1979a; 1979b; Kosaka, 1980) and later was recognized in at least
ten other teleosts (Kosaka and Hama, 1980; Alonso et al., 1987; Arevalo et al., 1991;
Matz, 1995). The ruffed cell is defined as a neuron whose axon has a distinct
unmyelinated portion at the initial segment with several membranous protrusions
arising lfom this region (Kosaka and Hama, 1979a; 1979b; Kosaka, 1980; Kosaka
and Hama, 1980). The remainder of the axon is myelinated (Kosaka and Hama,
1979a; 1979b; 1980). The cell bodies are ovoid or spherical in shape and range in size
from 9-30|im in diameter (Kosaka and Hama 1979b; Kosaka, 1980; Kosaka and
Hama, 1980; Alonso et al., 1987; Matz, 1995). These cells are located between the
olfactory nerve layer and the anterior olfactory nucleus as defined by Kosaka and
Hama (1979b; 1980) or throughout the mitral cell and glomerular layers as defined by
Alonso and colleagues (1987). The fish olfactory bulb is generally divided into three
or four concentric layers. The superficial region of the bulb is always termed the
olfactory nerve layer, and it contains the olfactory axons. The innermost layer is
termed either the anterior olfactory nucleus (which is poorly defined in fish, see Matz,
1995) or the internal cell or granule cell layer, which is composed, in part, of
numerous intemeurons. The region between these is considered either a single layer,
the glomerular layer including the mitral cells, or is subdivided into two general
regions, the glomerular and external plexiform layers or the glomerular and mitral
47
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cell layers. Thus, the ruffed cells described in other teleost studies are located in
regions equivalent to what we describe as the glomerular layer and internal cell layer
in zebrafish.
Kosaka and Hama (1980) suggest that the ruffed cell is generally present in all
teleost olfactory bulbs; however, until now ruffed cells have not been identified in the
olfactory bulb of zebrafish. Previous work has sought to describe the adult zebrafish
olfactory bulb (Byrd and Bmnjes, 1995; Friedrich and Laurent, 2001; Edwards and
Michel, 2002), but none of these studies identified the presence of ruffed cells. In
addition, pharmacological studies dealing with neurotransmission in the zebrafish
olfactory bulb have questioned the presence of ruffed cells as an additional output
neuron (Edwards and Michel, 2002). The goal of the present study was to establish if
ruffed cells were present in the adult zebrafish olfactory bulb. The identification of
this neuron in zebrafish has implications for olfactory coding studies in this model
system.
Materials and Methods
Animals
Adult male and female zebrafish, at least 2.5cm in length, were obtained from
local distributors. They were maintained in aquaria containing 28.5°C aerated,
conditioned fish water and fed freshwater flake food (Wardley Corporation) twice
daily. All animal care protocols and experimental procedures were approved by the
Institutional Animal Care and Use Committee.
Olfactory Tract-Tracing
For olfactory tract-tracing, fish were anesthetized with 0.03% MS222 (348
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amino benzoic acid ethyl ester, Sigma) and perfused transcardially with phosphate
buffered saline (PBS). The brains were dissected, and biotinylated dextran amine
(BDA, 3000 MW, 10% in PBS, Molecular Probes) was injected into the medial or
lateral olfactory tracts or into both tracts of each olfactory bulb using a PV 800
Pneumatic Picopump (World Precision Instruments). The brains were then transferred
to a sterilized filter in a six-well culture dish (Costar) and incubated at 28.5°C and
1.5% CO2 in artificial fish cerebrospinal fluid (lOOmM NaCl, 2.46mM KC1, ImM
MgCl2-6H20 , 0.44mM NaH2P 0 4H20 , 1.13mM CaCl2H20 , 5mM NaHC03) for
approximately 4 hours (Tomizawa et al., 2001). Following fixation in 4%
paraformaldehyde for 24 hours, horizontal or coronal cryostat sections at 20-50pm
were obtained and mounted on positively charged slides. The tissue was treated with
a fluorescent avidin (Alexafluor 488 avidin, 1:200 in PBS, Molecular Probes) for 1
hour at room temperature, rinsed, coverslipped, and viewed on a Zeiss LSM 510
confocal microscope.
Immunocytochemistry
For immunocytochemistry, BDA-stained sections were blocked with 3%
normal goat serum and 0.4% Triton X-100 in PBS. They were treated with a
monoclonal antibody (anti-HuC/HuD, 1:100 in blocking solution, Molecular Probes)
for 24 hours at 4°C. HuC/HuD is an RNA binding protein that is often used as a
general neuronal marker. The sections were then rinsed in PBS and incubated in a
fluorescently labeled secondary antibody (Alexafluor 594 goat anti-mouse IgG, 1:100
in PBS, Molecular Probes) for 1 hour. Following staining, the slides were
coverslipped with glycerol and PBS (50:50) and viewed on a Zeiss LSM 510 confocal
microscope.
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Golgi-Kopsch Technique
The Golgi-Kopsch procedure was adapted from Riley (1979) and optimized
for zebrafish olfactory tissue.
transcardially with PBS
Adult zebrafish were anesthetized and perfused
followed by
fixative
(1% paraformaldehyde,
1%
glutaraldehyde in 0.1M PBS). The brain was removed and stored in fixative at 4°C for
48 hours. The tissue was rinsed and stored in fresh dichromate solution (1.19M
potassium dichromate, 0.44M sucrose, 37% formaldehyde, all from Fisher Scientific)
at room temperature, protected from light, for 72 hours. The tissue was then rinsed
multiple times in silver nitrate solution (0.04M, Fisher Scientific) and stored in fresh
silver nitrate solution at room temperature, protected from light, for 12 to 32 hours.
Following incubation in silver nitrate solution, the tissue was dehydrated in ethanols
followed by a 1:1 solution of ether and ethanol and stored in celloidin (6g parlodion,
Mallinckrodt; 50% Ethanol, Fisher Scientific; 45% Ethyl Ether, Fisher Scientific) at
room temperature, protected from light, overnight. The brains were mounted in
celloidin, which was solidified in a chloroform chamber and stored in butanol
overnight. Sections were obtained on a sliding microtome at 100pm, dehydrated and
mounted on slides using DPX mounting medium (Aldrich Chemical Company). The
medium was allowed to dry for at least four days before the slides were analyzed
using brightfield microscopy and SPOT advanced software.
Quantitative Analysis
In order to determine the size of labeled cell bodies in the olfactory bulb, high
magnification digital images were obtained from the BDA-labeled or Golgiimpregnated olfactory bulbs of adult animals, and the height and width of somata
were measured using the Spot Advance Software line measurement tool.
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Measurements were made from 33 ruffed cells and 105 mitral cells from 8 animals.
Averages and standard errors of the mean were reported. To determine the percentage
of ruffed cells in the total output neuron population, cell counts were obtained from
the right and left olfactory bulbs of 8 fish. For each fish, the total number of labeled
output neurons for the right and left bulb was averaged, and the total number of ruffed
cells for the right and left bulbs was averaged. To obtain the percentage of ruffed cells
in a single olfactory bulb for a single fish, the average number of ruffed cells was
then divided by the average number of labeled output neurons.
Results
Ruffed cells in the adult zebrafish olfactory bulb were identified using
retrograde tract-tracing with a biotinylated dextran, and their presence was confirmed
with Golgi impregnation analysis. Their cell bodies were generally spherical or ovoid
in shape. They were neurons based on HuC/HuD co-labeling (data not shown). The
unique feature of this cell type was a significant protrusion of the membrane at the
initial segment of the axon (Figure 16A, B, arrow). Mitral cells did not exhibit these
elaborate axonal ruffs (Fig 16D). The ruffed cell protrusions usually occurred within
4pm of the cell body and varied in size from 10-30pm along the length of the axon.
The ruff typically had a spherical or cylindrical shape whose diameter ranged from
10-25 pm around the shaft of the axon. The axons of the ruffed cells, approximately
lpm in diameter, projected to either the medial or the lateral olfactory tracts. The
ruffed cells were located deep in the glomerular layer or superficial internal cell layer,
and the cell bodies appeared to be evenly distributed throughout these regions in the
entire olfactory bulb (Figure 16C). The ruffed cell dendrites projected superficially to
the glomerular layer, and the tufts were generally in close proximity to the soma.
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The soma height and width of ruffed cells were compared to the soma height
and width of the major output neurons, mitral cells, in the same animals (see Table 1).
The ruffed cell bodies ranged in size from 7-12pm in width and 8-15pm in height and
had a mean height of 11.2pm ± 0.6pm and a mean width of 9.9pm ± 0.3pm. The
mitral cells, on the other hand, had a mean height of 9.0pm ± 0.4pm and a mean
width of 8.3pm ± 0.2pm (Table 1). These data suggest that, on average, zebrafish
ruffed cells are larger than zebrafish mitral cells. Ruffed cells, however, appeared to
be far less frequent in number than mitral cells. Analysis of 8 fish revealed that ruffed
cells make up well less than 5% of output neurons (Table 2).
Discussion
We have established the presence of ruffed cells in the adult zebrafish olfactory bulb
and have described their morphology and distribution in this model system. Because
the fish olfactory bulb is not organized in discrete layers like the mammalian
olfactory bulb, identification of cell types based on distribution alone is difficult. As a
result, morphological, physiological and/or pharmacological analyses of individual
cell types are required to make an accurate identification. Our work supports previous
suggestions that ruffed cells are present in all teleost olfactory bulbs (Alonso et al.,
1987; Kosaka and Hama, 1980). Due to differences in the data reported in previous
studies, we were unable to compare all aspects of ruffed cell morphology between
zebrafish and other cyprinids.
Based on available comparisons, we find that the ruffed cells of zebrafish are
more similar to the ruffed cells of goldfish and catfish rather than sea eel.
52
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Figure 16. Zebrafish ruffed cells are visualized under high magnification using Gogli-Kopsch (A) and
retrograde tract-tracing (B) techniques. These cells have variable-shaped somata (*) and exhibit a ruff
(arrow) along the length o f the axon (arrowhead). C: Lower magnification view shows that ruffed cells
(arrows) are intermixed with mitral cells in the glomerular layer (GL) but are not prominent in the
internal cell layer (ICL). D: The mitral cell axon (arrowhead) does not exhibit substantial membranous
extensions like those of the ruffed cell. Scale bar = 10pm (A, B, D) and 25pm (C).
For example, like goldfish and catfish the ruff of the zebrafish occurs fairly
close to the cell body, unlike the sea eel whose axonal protrusions are exhibited at a
much further distance from the soma, generally 30-70pm (Kosaka and Hama, 1979b;
1980). These variations can be expected because the goldfish and the catfish are more
closely related to the zebrafish than the sea eel (Figure 17). The goldfish and the
zebrafish are close allies and even share a common lineage down to the same order
and family, while the catfish is not as closely related but does share the superorder,
Ostariophysi, and the subdivision, Euteleostei (Wulliman, 1998).
The ancestors of sea eel, on the other hand, diverged from the teleost line
before the Euteleosts. This fish is of a different subdivision, Elopocephala, and a
different superorder, Elopomorpha (Wulliman, 1998).
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Table 1. Summary of Ruffed Cell and Mitral Cell Soma Measurements
Ruffed Cells
Animal
Mitral Cells
Height (pm) Width (pm)
Height (pm)
N
Width (pm)
N
15
7.5±0.4
7.5±0.3
20
11.3±0.7
3
10.6±0.5
8.9±0.4
15
12.3±0.8
9.6±1.0
4
8.9±0.5
8.1±0.5
12
ZF4
9.3±0.9
8.7T0.3
3
8.5±0.3
7.7±0.3
15
ZF5
11.5±0.5
10±2.0
2
8.0±0.6
8.7±0.7
6
ZF6
9.0
9.0
1
9.0±0.4
8.7±0.7
6
ZF7
10.3±0.9
10. 0± 1.0
3
10.1±0.4
8.7±0.3
20
ZF8
14.0±1.0
10.5±1.5
2
9.7±0.5
8.4±0.4
11
Average
11.2±0.6
9.9±0.3
33
9.0±0.4
8.3±0.2
105
ZF1
11.4±0.4
9.8±0.3
ZF2
12.3±0.3
ZF3
The variations seen in the ruffed cell morphology between the sea eel and the other
fish is likely due to the evolutionary divergence between the species. There are
similarities between the goldfish, catfish and sea eel with regard to the initial part of
the ruff, which forms a spherical field about 20-50pm in diameter (Kosaka and Hama,
1979a; 1979b; Kosaka, 1980; Alonso et al., 1987). This initial segment also is seen in
the zebrafish, but its diameter ranges from 10-25 pm. Thus, the ruffed cell appears to
be unique to fish, but morphologies may vary between types of fish.
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Table 2. Number o f Ruffed Cells in Reference to Labeled Output Neurons
Animal
Ruffed Cells
Percentage of Output Neurons
that are Ruffed Cells
ZF1
24/597
4.0%
ZF2
3/338
0.9%
ZF3
4/234
1.7%
ZF4
3/362
0.8%
ZF5
3/231
1.3%
ZF6
1/270
0.4%
ZF7
3/402
0.7%
ZF8
2/185
1.1%
We found that ruffed cells are much less common than mitral cells, but it is possible
that ruffed cells make up a larger proportion of total output neurons in the zebrafish
olfactory bulb than reported here. We applied rigorous standards when identifying the
ruffed cells in the zebrafish olfactory bulb. Because the membranous protrusions of a
ruff are similar in morphology to some orientations of a dendritic tuft, we included a
cell as ruffed only if we could see the axon projecting from the cell body as well as
the projection of the axon through the “ruffed” zone. The low number of ruffed cells
identified in this study also could be a result of several factors including incubation
time of the tracer substance or variability in injection location. However, a large
number of output neurons and their dendrites filled with the tracer, so it is unlikely
that further incubation would have allowed for a much greater number o f ruffed cells
to be filled.
55
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O steichthyes
(Class)
A ct in op terygii
(Subclass)
Neop terygii
(In fra class)
A nguilia fo rm es
(Ord or)
A nguillidae
(Fam ily)
T eleostei
(Division)
Sea E el
(C onger
m yriaster)
E lop o cep ha la
(Subdivision)
Elopomorpha
(Superorder)
Clup eocep ha la
(Subdivision)
C ypriniform es
J O f d e |^
Eiiiteleostei
(Su b division)
C vpnnidae
O stariophysi
(Superorder)
Z ebrafish*
(Danu?
G oldfish
{Carassuts
an ram s)
Siluriform es
(Ord er)
I eta lurid a e
(Fam ily)
Catfish
(Parasiluriis
asotus)
Figure 17. A phylogenetic tree of the teleost line exhibits the interrelationships of the primary fish
discussed in this study: the zebrafish (*), goldfish, catfish and sea eel. This cladogram was modified
from M. Wullimann (1998).
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In addition, we have injected at various locations in the telencephalon in an attempt to
reconcile the possibility that ruffed cells project to a different location than the other
output neurons. Based on these strict identification criteria, we propose that ruffed
cells make up less than 10% of the population of output neurons.
Edwards and Michel (2002) describe several neuronal phenotypes in the adult
zebrafish olfactory bulb based on labeling with y-aminobutyric acid (GABA),
glutamate and tyrosine hydroxylase (TH). They found that mitral cells labeled with
glutamate, but not with GABA or TH. They describe two populations of
juxtaglomerular cells, both labeled with low levels of glutamate and high levels of
GABA, but only one group labeled with TH. It is possible, based on the distribution
of these cells, that the TH-negative juxtaglomerular cells are mffed cells. Most of the
ruffed cell bodies were located deep in the glomerular layer or the superficial internal
cell layer and their dendrites projected outwardly to the glomerular layer. Also,
studies in our laboratory showed that ruffed cells are TH-negative (unpublished
observation). On the other hand, Edwards and Michel (2002) reported that the THnegative cells were approximately 6.1± 1.2pm in diameter, and the average width of
the ruffed cells in this study measured 9.9± 0.3pm. So, it is unlikely that the THnegative juxtaglomerular cells are ruffed cells. However, differences in tissue
processing and plane of section between the two studies may account for some of the
deviation in the average soma size. Further studies are necessary to resolve this issue.
Ruffed cells, while similar in size and distribution to mitral cells, possess
distinct morphological and physiological properties. Previous studies have suggested
that ruffed cell axonal protrusions synapse with granule cells and that the dendrites
receive no input from mitral cells or olfactory sensory neurons (Kosaka and Hama,
1979b; Kosaka, 1980). Furthermore, physiological studies comparing response
patterns of mitral cells and ruffed cells in goldfish show that during application of a
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stimulus, ruffed cells and mitral cells showed contrasting interactions (Zippel et al.,
1999). For example, when the epithelial activity was blocked, mitral cell responses
were reduced where as ruffed cell activity increased (Zippel et al, 1999). Thus, the
ruffed cell represents a functionally significant class of neuron whose identification in
the adult zebrafish olfactory bulb could prove important in studies of olfactory
coding, olfactory processing, and comparative neurology.
This information was presented in 2005 in a publication entitled, “Ruffed Cells
Identified in the Adult Zebrafish Olfactory Bulb” in Neuroscience Letters.
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CHAPTER V
THE MORPHOLOGY AND DISTRIBUTION OF MITRAL CELLS IN THE
OLFACTORY BULB OF ADULT ZEBRAFISH
Introduction
Mitral cells are the major output neurons and the primary relay in the
olfactory bulb of vertebrates (Allison, 1953; Nieuwenhuys, 1967; Andres, 1970).
Their structure differs significantly among mammals and lower vertebrates (Allison,
1953; Shepherd, 1972). In mammalian systems, the olfactory bulb is a laminar
structure with well-defined cell types in each layer (Shepherd, 1972).
In these
species, mitral cells are mitre-shaped macroneurons with a single primary dendrite
that contacts a single glomerulus (Meisami and Safari, 1981). They have secondary
dendrites that do not form synapses in any glomeruli and have cell bodies ranging in
size from 10pm-20pm in diameter (Meisami and Safari, 1981; Dryer and Graziadei,
1994).
In contrast to the mammalian olfactory bulb, the teleost olfactory bulb has a
diffusely organized laminar structure composed of four principal layers (Oka et al.,
1982; Satou, 1990; Byrd and Brunjes, 1995). These layers include the olfactory nerve
layer (ONL), the glomerular layer (GL), the mitral cell layer (MCL), and the granule
cell layer (GCL). Due to the ill-defined nature of the MCL and GCL, they often are
combined into a single layer termed the internal cell layer (ICL; Byrd, 2000). The
morphology of mitral cells within teleost olfactory bulbs has been characterized
primarily through the use of classic Golgi staining (Sheldon, 1912; Holmgren, 1920;
Kosaka and Hama, 1982; Oka, 1983), although intracellular horseradish peroxidase
methods also have been employed (Fujita et al., 1988). These studies suggest that
mitral cells in teleosts are large neurons of varying soma and dendritic arborization
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characteristics (Oka, 1983). Cell bodies can be triangular or ovoid and, in contrast to
mammalian output neurons, teleost mitral cells reportedly have multiple primary
dendrites with glomerular tufts and no secondary dendrites (Oka, 1983; Alonso et al.,
1988; Dryer and Graziadei, 1994). In goldfish and carp, mitral cells are generally
located 100-200pm from the surface of the bulb and are approximately 10-25 pm in
diameter (Kosaka and Hama, 1982; Oka, 1983). In general, each cell contains several
dendritic projections (2-6pm in diameter) that form tufts at various lengths from the
cell body (Kosaka and Hama, 1982).
Mammals have a single olfactory tract per main olfactory bulb, but fish have
both a lateral and medial olfactory tract where axons from mitral cells bundle and
project to the brain. In zebrafish, these tracts are distinct and can easily be
distinguished as they make their way from the olfactory bulb through the adjacent
telencephalon. It has been suggested that the medial tract processes pheromonal
information and mediates reproductive behavior while the lateral tract conveys
information about food (Satou et al., 1983; Stacey and Kyle, 1983; Demski and
Dulka, 1984; Satou et al., 1984; Kyle et al., 1987; Sorensen et al., 1991; Hamdani,
Alexander and Doving, 2001; Hamdani, Kasumyan and Doving, 2001; Weltzien et
al., 2003). As a result, potential differences between the mitral cells projecting to the
medial or the lateral olfactory tract could provide important information about
olfactory coding in the teleost olfactory bulb.
The zebrafish is an excellent model for studies of the olfactory system (Baier
and Korsching, 1994; Byrd and Brunjes, 1995; Dynes and Ngai, 1998; Whitlock and
Westerfield, 1998; Friedrich and Laurent, 2001; Edwards and Michel, 2002; Tabor et
al., 2004; Miyasaka et al., 2005; Sato et al., 2005) and is becoming an increasingly
popular model in the scientific community. While numerous studies have begun to
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characterize the anatomy of the olfactory system in this species (Baier et al., 1994;
Byrd and Brunjes, 1995; Edwards and Michel, 2002; Sato et al., 2005), there is still
little known about the structure and function of the different cell types within the
olfactory bulb. In order to gain an understanding of the way zebrafish process
information, many researchers have focused on the functional aspects of the olfactory
system, such as odor representation and stimulation (Corotto et al., 1996; Freidrich
and Korsching, 1998; Friedrich and Laurent, 2001; Edwards and Michel, 2002;
Lipschitz and Michel, 2002; Michel et al., 2003; Friedrich et al., 2004; Tabor et al.,
2004; Friedrich and Laurent; 2004; Liy et al., 2005). While some of these studies
have touched upon characteristics of zebrafish mitral cells, a detailed analysis is
lacking. Since the mitral cell is the major output neuron of the olfactory bulb, it is
important to understand its structure, distribution, and interactions with other cells.
This study will analyze mitral cell morphology and distribution in the adult zebrafish
olfactory bulb in order to provide further insight into information processing and
olfactory coding in this important model system.
Materials and Methods
Animals
Adult male and female zebrafish, Danio rerio, at least 2.5cm in length, were
obtained from commercial sources (RJ Ray Distributors, Pontiac, MI). The fish were
maintained in 15-gallon aquaria filled with aerated, conditioned fish water at 28.5oC,
and they were fed commercial flake food (Wardley Corporation) twice daily. All
protocols on animal care and experimental procedures were approved by the
Institutional Animal Care and Use Committee.
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Olfactory Tract Tracing
Fish were anesthetized with 0.03% MS222 (3-amino benzoic acid ethyl ester,
Sigma) and perfused with phosphate buffered saline (PBS). The brains were
dissected, and approximately 0.05pl-0.1pl of a 10000MW dextran (biotinylated
dextran amine, BDA, lOOmg/ml in PBS, Molecular Probes or Texas Red dextran,
5mg/ml in PBS, Molecular Probes) or a 3000MW dextran
(micro-ruby,
tetramethylrhodamine and biotin dextran, 5mg/ml in PBS, Molecular Probes) was
injected either into the medial, lateral, or both olfactory tracts using a PV 800
Pneumatic Picopump (World Precision Instruments). The brains were then placed on
a 3pm polycarbonate filter in a sterilized six-well culture dish (Costar) containing
artificial fish cerebrospinal fluid (lOOmM NaCl, 2.46mM KC1, ImM MgCl2'6H20 ,
0.44mM NaH2P 0 4H20 , 1.13mM CaCl2H20 , 5mM N aHC03) and incubated at 28.5°C
and 1.5% C 0 2 for approximately 4 hours (Tomizawa et al., 2001).
Following fixation in 4% paraformaldehyde for 24 hours, the tissue was
either viewed as whole mounts, or brains were cryostat sectioned in the horizontal or
coronal plane and mounted on positively charged slides. Brains injected with Texas
Red dextran or micro-ruby first were viewed as whole mounts for dendritic analysis
and then were serial sectioned at 20pm-100pm for soma analysis and/or
immunocytochemistry. The tissue was viewed on a Zeiss LSM 510 confocal
microscope using a Texas Red filter. The BDA-treated tissue sections, also taken at
20pm-100pm, were incubated with a fluorescent avidin (Alexafluor 488 avidin, 1:200
in PBS, Molecular Probes) for 1 hour at room temperature, rinsed, coverslipped with
PBS and glycerol containing 0.1% para-phenylenediamine (PPD; Beltz and Burd,
1989) and viewed on a Zeiss LSM 510 confocal microscope using a FITC filter. To
visualize the BDA-labeled cells with light microscopy, avidin-HRP (1:1000 in PBS,
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Molecular Probes BDA Neuronal Tracer Kit, N-7167) treatment was used. The
sectioned tissue was incubated overnight at 4°C, rinsed with PBS for 30 minutes, and
treated with a 0.05% 3,3’-diaminobenzadine solution (Molecular Probes BDA
Neuronal Tracer Kit, N-7167). After rinsing, dehydrating, and coverslipping with
DPX mounting medium (Aldrich Chemical Company), the tissue was viewed on a
Nikon Eclipse E600 microscope.
Golgi-Kopsch Technique
This procedure was modified from Greer (1987) and Riley (1979) and
optimized for zebrafish olfactory tissue. Zebrafish were anesthetized and perfused
with PBS followed by fixative (1% paraformaldehyde, 1% glutaraldehyde in 0.1M
PBS). The brain was dissected, placed in fixative at 4°C for 48 hours, rinsed and
incubated in fresh dichromate solution (1.19M potassium dichromate, 0.44M sucrose,
37% formaldehyde, all from Fisher Scientific), in a light-safe container at room
temperature for 72 hours. Following a rinse in silver nitrate solution (0.04M, Fisher
Scientific), the tissue was stored in this medium, protected from light, at room
temperature for 12 to 24 hours. It was then dehydrated in ethanols followed by a 1:1
solution of ether and ethanol and stored in celloidin (5% parlodion, Mallinckrodt;
50% Ethanol, Fisher Scientific; 45% Ethyl Ether, Fisher Scientific), in a light-safe
container at room temperature overnight. The brains were mounted in celloidin,
solidified in a chloroform chamber and stored in butanol overnight. Sections were
obtained in 100 pm increments on a sliding microtome, dehydrated, mounted on slides
using DPX mounting medium (Aldrich Chemical Company) and viewed on a Nikon
Eclipse E600 microscope using brightfield microscopy. The tissue embedded in this
manner was used to confirm that the morphologies of mitral cells seen with the
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retrograde tract-tracing technique also were present using other methods. These
Golgi-impregnated cells were not used for quantitative analysis.
Immunocytochemistry
BDA-labeled sections that had already been treated with a fluorescent avidin
were blocked with 3% normal goat serum and 0.4% Triton X-100 in PBS. They were
treated with a mouse monoclonal antibody [anti-HuC/HuD, 1:100 in PBS, Molecular
Probes, CAT#A-21271/LOT# 7201-3, or anti-tyrosine hydroxylase (TH), 1:1000 in
PBS, ImmunoStar, CAT#22941/LOT# 148003] or a rabbit polyclonal antibody [antiFMRFamide,
1:500 in PBS, Chemicon, #AB1917, or anti-keyhole limpet
hemocyanin, KLH, 1:500 in PBS, Sigma, #H0892] for 24 hours at 4°C. HuC/HuD, is
used as a neuronal marker because it recognizes a neuron-specific RNA-binding
protein. The HuC/HuD antibody employed in this study was created against the
peptide sequence QAQRFRLDNLLN (Marusich et al., 1994) and has been shown to
label specifically neurons in zebrafish (Henion et al., 1996; Byrd and Brunjes, 2001;
Zupanc et al., 2005). Tyrosine hydroxylase is the rate-limiting step in catecholamine
synthesis, so the antibody to this enzyme is used commonly to identify dopaminergic
neurons (usually periglomerular cells) in the olfactory bulb. The TH antibody was
made against an epitope in the mid-portion of the molecule (manufacturer’s technical
information) and it did not cross react with other proteins when examined with
Western Blot analysis (data not shown). This antibody produced a pattern of
immunoreactivity in the zebrafish brain that was identical to other studies (Edwards
and Michel, 2002; Fuller, Villanueva, and Byrd, 2005). FMRFamide is a neuroactive
peptide marker shown to label specifically terminal nerve cells in the zebrafish
olfactory bulb (Li and Dowling, 2000; Maaswinkel and Li, 2003). The antibody used
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in this study was made against the 4 amino acid sequence FMRF conjugated to neat
bovine serum albumin (manufacturer’s technical information). The antibody against
KLH labels consistently the olfactory nerve in teleosts (Riddle and Oakley, 1992;
Riddle et al., 1993; Jarrard, 1997; Starcevic and Zielinski, 1997), including this
species (Fuller, Villanueva, and Byrd, 2005). Following a PBS rinse and incubation in
a fluorescently labeled secondary antibody (Alexafluor 594 goat anti-mouse IgG,
1:200 in PBS, Molecular Probes, or Alexafluor 594 goat anti-rabbit IgG, 1:200 in
PBS, Molecular Probes) for 1 hour, the slides were coverslipped with glycerol and
PBS (50:50) and viewed on a Zeiss LSM 510 confocal microscope using a multitrack
overlay with narrow band filters.
Glomerular Tracing
Glomeruli were labeled with DiA (4-(4-(dihexadecylamino) styryl)-Vmethylpyridinium iodide, 5mg/ml in dimethyl sulfoxide, (Fisher Scientific),
Molecular Probes) injections into the olfactory organs according to the protocol
developed by Baier and Korsching (1994). Following this procedure, the animals
were returned to a 15-gallon aquarium for recovery and the tank was kept dark in
order to prevent bleaching of the photosensitive tracer. Twenty-four hours later, fish
were processed for olfactory tract tracing as described above, using BDA (lOOmg/ml
in PBS, Molecular Probes) or Texas Red dextran (5mg/ml in PBS, Molecular Probes)
as a tracer. To optimize visualization of mitral cell dendritic arbors and glomeruli,
80pm cryostat sections and whole mounts were examined on a Zeiss LSM 510
confocal microscope using Z stacks and image projections at multiple angles of
difference. These images were captured using a multitrack overlay with narrow band
pass filters.
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Quantitative Analysis
For a variety of morphological and quantitative analyses, over 1000 cells in 30
adult animals were examined. The morphologies of mitral cells in 10 adult zebrafish,
whole olfactory bulbs were compared for the presence of multiple versus single
primary dendrites. O f these 10 fish, 5 had the medial olfactory tract loaded with tracer
and 5 had the lateral olfactory tract loaded with tracer. A total of 100 cells were
analyzed for dendritic morphology and the percentage of mitral cells with multiple
dendrites versus single dendrites was established for the entire bulb as well as for
cells projecting to the medial olfactory tract and cells projecting to the lateral
olfactory tract. The percentage of mitral cells with single dendrites was determined by
dividing the total number of mitral cells with a single process emanating from the
soma by the number of randomly selected cells (n=100), and the percentage of mitral
cells with multiple dendrites was determined by dividing the total number of mitral
cells with multiple processes emanating from the cell body by the number of
randomly selected cells (n=100). Comparisons between uni-dendritic and multidendritic percentages were made using a one-way ANOVA. For all quantitative
analyses, averages were reported with standard errors of the mean and P values less
than 0.05 were considered significant.
To obtain dendritic reconstructions of these cells, whole mounts were
examined using confocal microscopy. Once a cellular profile was identified, Z-stack
images were used to gather optical sections of the cell at 1pm intervals and the cell
was traced using the LSM510 overlay tool. Different colors represent varying planes
of focus. The image was converted to a projection to visualize the cell in three
dimensions and confirm that all dendritic bifurcations were represented in the z-stack
trace.
66
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The average number of mitral cells in a single olfactory bulb for a single fish
was determined by obtaining counts of profiles retrogradely labeled with a 3000MW
dextran from the right and left olfactory bulbs o f 15 fish. For each fish, the total
number of mitral cells for the right and left bulbs was averaged. These totals were
then combined and divided by the total number of fish evaluated (n=15) to obtain the
average number of mitral cells in a single bulb. Only mitral cells were considered in
these analyses based on specific morphological criteria (see Results).
In order to ascertain if mitral cells possessed a normal distribution with regard
to soma size, cell cross-sectional area measurements from 100 retrogradely labeled
cells in 10 fish were obtained using the LSM 510 confocal microscope overlay and
measurement tools. Once a cellular profile was identified, Z-stack images were used
to gather fine optical sections of the cell. The image was then converted to a
projection to visualize the cell in three dimensions and to determine the largest axis of
the soma. Cell cross-sectional areas were then obtained by tracing the perimeter of the
soma in its largest dimension. The cells were binned based on 10pm areas and the
total number of cells in each category was determined. The Kolmogorov-Smimov test
for normality was used to determine the distribution of all mitral cells as well as TH+
intemeurons. Further, the distribution was examined individually for cells with
multiple dendrites, cells with a single dendrite, cells on the medial side of the bulb,
and cells on the lateral side of the bulb.
Average cross-sectional area comparisons between mitral cells with multiple
dendrites and mitral cells with single dendrites were done using two-sample t-tests, as
the distribution of these populations was normal (p>0.5). Also, cross-sectional area
comparisons were done using the non-parametric Mann-Whitney test to evaluate
potential differences in the soma sizes of mitral cells on the lateral side versus the
67
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medial side of the olfactory bulb because these populations did not exhibit a normal
distribution (p<0.05). Further, a two-way ANOVA was used to compare soma sizes
for all subcategories. For example, the soma sizes of lateral cells with a single
dendrite were compared to the soma sizes of lateral cells with multiple dendrites.
To compare the average sizes of mitral cells to other cellular profiles in the
olfactory bulb, high magnification digital images were obtained from dextran-loaded
or TH-treated olfactory bulbs in similar histological preparations in 20 adult animals.
Diameters of somata were measured using the Spot Advance Software line
measurement tool. Dimensions including soma cross-sectional area, length and width
were used to compare the size of TH-positive intemeurons to mitral cells. THpositive cells on the medial side of the bulb also were compared to TH+ cells on
lateral sides of the olfactory bulb using two-sample t-tests to confirm that the two
populations were not significantly different in size before grouping them to compare
them to mitral cells. Length was defined as the diameter of the cell along the axis
from which the major dendrite(s) projected regardless of the orientation of the cell in
the olfactory bulb, while width was the diameter of the cell perpendicular to that
measure. These measurements were made from a total of 100 presumptive mitral cells
and 75 TH-positive intemeurons.
Hematoxylin and Eosin Staining of Paraffin Sections
Fish were anesthetized and perfused with Bouin’s fixative solution. Two
hours post-fixation, the brains were dissected, dehydrated, and embedded in paraffin.
Following serial sectioning in the horizontal plane at 10pm and mounting on gelatincoated slides, the tissue was dewaxed in xylenes, rehydrated with ethanols, and rinsed
in water. Sections were stained with hematoxylin for 1 minute, rinsed in water, and
68
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incubated in clarifying solution for 1 minute. They were then rinsed with water,
treated with bluing reagent, rinsed, and washed in 95% ethanol. After incubation in
eosin y for 1 minute and three rinses in 100% ethanol, the tissue was rinsed in xylenes
several times and coverslipped with DPX mounting medium (Aldrich Chemical
Company). All hematoxylin and eosin reagents were purchased from Richard Allen,
Inc.
Results
Identification of Mitral Cells
Output cells in the adult zebrafish olfactory bulb were identified using
retrograde tract tracing with various dextrans and a modified Golgi-Kopsch
technique. The labeled profiles were found in the GL and the superficial ICL (Figure
18), and they showed positive immunoreactivity with a neuronal marker, HuC/HuD
antibody (data not shown). Based on the location, neuronal identity, and projection of
the axon to one of the olfactory tracts, we conclude that the majority of these labeled
profiles are mitral cells.
Mitral Cell Morphology and Distribution
The mitral cells exhibited two main types of morphologies with regard to their
dendrites: the uni-dendritic morphology was a single primary dendrite with one or
more tufts, but multi-dendritic mitral cells also were seen. The uni-dendritic mitral
cell somata were generally ovoid or spherical in shape (Figure 19), but there was
variability in that some cells had teardrop, fusiform, pear-shaped or elongated somata.
The typical morphology of this neuron was a cell body ranging in size from 4-13 pm
69
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in diameter with a mean length of 9.3 ± 0.2pm and a mean width of 6.2 ± 0.1 jam
(Table 3). Further, the cell cross-sectional areas at the largest point ranged from 3170pm2 with a mean area of 47.1 ± 1.1pm2 (Table 3). These mitral cells had a single
dendritic trunk, generally 2-5 pm in diameter, with a distinct glomerular tuft.
Although, in some cases a single trunk would branch at some distance from the cell
body and create two or more small tufts. The dendritic tufts generally ranged from
20-50pm in diameter and were typically found within close proximity (under 40pm)
away from the cell body, but they also were seen up to 80pm away from the soma
(Fig. 19). Occasionally, the dendrites projected further; however, this phenomenon
was limited as we have seen only a few instances. When the cell body was located in
the GL the dendrites projected either laterally or deep to reach their synaptic targets,
but when the cell body was located in the superficial ICL the dendrites projected
superficially instead. The axons of these cells, typically l-2pm in diameter, did not
exhibit elaborate protrusions of the membrane such as ruffed cell possess (Fuller and
Byrd, 2005), and they projected to either the medial or the lateral olfactory tract.
A smaller percentage of mitral cells did not exhibit this morphology. Multidendritic mitral cells were similar in shape to the other mitral cells, but they were
significantly larger than mitral cells with a single apical dendrite (P<0.05). They had
a mean length of 11.9 ± 0.4pm, a mean width of 8.0 ± 0.3pm, and their soma cross9
9
sectional areas ranged from 46-96pm with a mean area of 76.8 ± 2.1pm (Table 3).
The distinct feature of the multi-dendritic mitral cells was that they possessed
multiple dendritic processes emanating from the cell body (Figure 20). Some of these
dendrites had discrete dendritic tufts while others possessed less distinct projections.
These dendrites occurred at varying lengths from the cell body and in most cases,
regardless of the dendritic morphology, tufts from different dendrites of the same cell
70
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appeared to localize to the same region in the GL (Figure 20A-C). Examination of the
dextran-loaded cells, when the olfactory sensory axons were labeled with an antibody
against KLH (Figure 3D) or traced with DiA (data not shown), showed that even
mitral cells with multiple dendrites can innervate a single glomerulus.
As with the uni-dendritic mitral cells, multi-dendritic mitral cells were
distributed throughout the GL and the superficial ICL, and their dendrites projected
either superficially or deep depending on the location of the soma. Their axons
projected to either the medial olfactory tract (MOT) or the lateral olfactory tract
(LOT) since these morphologies were seen when either the MOT or LOT was
injected with tracer. Analysis of 100 cells in 10 fish revealed that uni-dendritic mitral
cells make up approximately 69% of the total population while multi-dendritic mitral
cells comprise about 31% of the total population.
Mitral Cells and the Olfactory Tracts
Mitral cell distribution was examined based on the projection of the axon to
the medial or the lateral olfactory tract. In most cases, the location of the cell on the
medial or lateral side of the bulb was indicative of the tract to which it would project
(Figure 21). However, it also was apparent that mitral cells in the rostral portion of
the bulb projected primarily to the MOT while mitral cells in the caudal region of the
bulb were more likely to project to the LOT.
There appeared to be no segregation in the morphology of the cells projecting
to the MOT and the LOT, since uni- and multi-dendritic mitral cells were seen on
both the medial and the lateral sides of the olfactory bulb. Uni-dendritic versus multidendritic morphologies can be further analyzed, however, by examining the
percentage of these populations that project to the medial versus the lateral olfactory
71
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tract. Of the MOT-projecting cells analyzed for dendritic morphology (n=50), 42%
were multi-dendritic mitral cells compared to 20% of the LOT-projecting cells
analyzed (n=50). Statistical analysis shows that there are significantly more multidendritic mitral cells on the medial side of the olfactory bulb than on the lateral side
of the olfactory bulb (P<0.05) and there are significantly more uni-dendritic mitral
cells on the lateral side of the olfactory bulb than on the medial side (P<0.05). In
addition, when examining both uni- and multi-dendritic cells on the lateral side of the
olfactory bulb, there are significantly more uni-dendritic cells than multi-dendritic
cells (P<0.05). However, there is no significant difference in the number of unidendritic versus multi-dendritic cells on the medial side of the olfactory bulb
(P>0.05).
The average size of the cell bodies showed significant variation between the
lateral and medial sides of the olfactory bulb (P<0.05 for all comparisons). O f 100
cells examined, the average cross-sectional area of uni-dendritic cells on the lateral
half was 43.9 ± 1.5pm , while the average area of uni-dendritic cells on the medial
half was 51.4 ± 1.3pm2 (Table 3). Further, the average cross-sectional area of multidendritic cells in the lateral half of the bulb was 67.3 ± 4.0pm2, while the average
area of multi-dendritic cells on the medial side of the bulb was 81.3 ± 1.8pm2 (Table
3).
72
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Figure 18. Low magnification (A) and high magnification (B) images of hematoxylin and eosin-stained
horizontal sections o f a zebrafish olfactory bulb are shown. Mitral cells (arrows) were located
primarily in the glomerular layer (GL) and the superficial internal cell layer (ICL) but were not seen in
the olfactory nerve layer (ONL). The axons o f these output neurons projected to either the medial
olfactory tract (MOT) or the lateral olfactory tract (LOT), which are distinct in this fish. For both
panels, rostral is to the top of the image. Scale bar = 50pm (A) or 25pm (B).
Further Examination of Mitral Cells
Area measurements of all mitral cell somata grouped in 10pm increments did
not show a normal distribution (p<0.05). Thus, cells were segregated based on either
their morphologies or their tract projections and the total number o f cells in each
category was reported (Figure 22). Cells segregated based on medial or lateral
olfactory tract projection also did not show a normal distribution (P<0.05), unlike
cells segregated based on uni-dendritic or multi-dendritic morphology (P>0.05).
When the cells are binned according to morphology and tract projection, multidendritic cells and medial cells occupy more of the higher area bins than uni-dendritic
cells and the lateral cells (Figure 22).
73
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Figure 19. Various methods of labeling revealed the typical structure of an adult zebrafish mitral cell: a
soma (*) o f variable-shape, a single primary dendrite (arrow) terminating in fairly discrete dendritic
tufts near the cell body, and a single axon (arrowhead) projecting toward the medial or lateral olfactory
tract. Dextran-labeled cells treated with diaminobenzadine (A) and Alexafluor 488 (B) yielded a
similar morphology to a cell labeled with the fluorescent dextran, micro-ruby, (C) and a Golgi-Kopsch
impregnated cell (D). Dendritic reconstructions of mitral cells showed dendritic processes with a
distinct tufting region in uni-dendritic cells (E, F). Different colors represent varying planes o f focus.
The majority o f the more than 1000 cells examined exhibited this morphology. Scale bar = 10pm (A-
F).
The representative number of mitral cells in a single olfactory bulb was
estimated. Based on calculations from 15 fish, the total number o f labeled mitral cells
in an adult zebrafish olfactory bulb ranged from 188-830 and the average number of
labeled mitral cells seen with our method was 395 ± 53. We did see as many as 830
cells in a single bulb; therefore we suggest that there are over 750 mitral cells per
olfactory bulb in the typical adult zebrafish.
74
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Figure 20. The morphologies o f multi-dendritic mitral cells also were seen using the retrograde tracttracing technique. Mitral cells with multiple dendrites (A, B, D arrows) were less frequent than those
with single primary dendrites. Dendritic reconstructions showed that even cells with multiple dendrites
exhibited dendritic tufts within close proximity to one another (C). Different colors represent varying
planes o f focus. Labeling of olfactory sensory axons with anti-KLH (red) showed that mitral cells with
multiple dendrites can innervate a single glomerulus (D). Scale bar = 20pm (A,B) or 15pm (C,D).
75
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Table 3. Comparisons of Soma Cross-sectional Areas, Lengths, and Widths for All Populations of
Mitral Cells.
All Cells
A11 Medial
Uni
Multi
N
Soma Cross-sectional
Area
(avg. jam2 ± SEM)
Soma Length
(avg. pm ± SEM)
Soma Width
(avg. pm ± SEM)
too
56.3 ± 1.7
10.1 ± 0 . 2
6.8 ± 0 . 1
50
63.9 ± 2.4
11.2 ± 0 . 3
7.0 ± 0.2
29
51.4 ± 1.3
10.2 ± 0 . 3
6.3 ± 0.2
21
81.3 ± 1.8
12.6 ± 0 . 5
8.1± 0.4
50
All Lateral
Uni
Multi
48.6 ± 2 . 0
9.1 ± 0.2
6.5 ± 0.2
40
43.9 ± 1.5
8.7 ± 0.2
6.2 ± 0.2
10
67.3 ± 4.0
9.5 ± 0.9
7.9 ± 0 . 3
All Uni
69
47.1 ± 1.1
9.3 ± 0.2
6.2 ± 0.1
Med
Lat
29
51.4 ± 1.3
10.2 ± 0.3
6.3 ± 0.2
40
43.9 ± 1.5
8.7 ± 0.2
6.2 ± 0.2
All Multi
Med
Lat
31
76.8 ± 2 . 1
11.9 ± 0 . 4
8.0 ± 0 . 3
21
81.3 ± 1 . 8
12.6 ± 0 . 5
8.1 ± 0 . 4
10
67.3 ± 4.0
9.5 ± 0.9
7.9 ± 0 . 3
Comparison of Mitral Cells to Other Cells in the Olfactory Bulb
The retrograde tract-tracing technique also allowed for the identification of
two other types of output neurons: the ruffed cell (Figure 23A) and the terminal nerve
ganglion cells (Figure 23B). These profiles were identified as non-mitral cell by their
unique morphologies. The ruffed cells had an obvious membranous field surrounding
the initial part of the axon (Fuller and Byrd, 2005). The terminal nerve cells were
large, amorphous clusters that often could be found along the axon bundles that made
up the MOT and the LOT or in large groups in the ONL and GL near the most rostral
portion of the bulb, similar to that reported by Li and Dowling (2000).
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Left Bulb
Right Bulb
Dorsal
A a* a * *
Left Bulb
Right Bulb
Figure 21.The distribution of MOT-projecting or LOT-projecting mitral cells was examined in the
horizontal and coronal planes. A horizontal section, viewed with fluorescence, showed that mitral cells
were distributed around the periphery of the bulb when both the MOT and the LOT were labeled (A).
Schematic diagrams shown in B and C illustrate general locations o f MOT-projecting or LOTprojecting mitral cells based on the dye injection site. In the horizontal plane (B), mitral cells
projecting to the MOT (stars) appeared most prominently in the medial and rostral portions o f the bulb,
while mitral cells projecting to the LOT (triangles) were more abundant in the lateral and caudal
regions o f the bulb. A similar distribution pattern was seen in the coronal plane of the olfactory bulb
(C). Scale bar = 50pm
The identity of the latter cell type was subsequently confirmed by co-labeling
with FMRFamide (Figure 23C) and measurements of these output cells established
that mitral cells are not the largest cells in the olfactory bulb. The terminal nerve cells
were much larger. They had an average soma length of 18.8 ± 2.1pm and an average
soma width of 18.3 ± 1.5pm.Thus, even though our tracing technique labeled several
types o f profiles, we were able to identify specifically the mitral cells for this study.
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■ MEDIAL ■ UNI
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Figure 22. Mitral cells were binned according to the cross-sectional area o f their cell bodies at the
largest point. These measurements were further analyzed by grouping them according to tract
projection and/or cellular morphology.
Another neuron in the olfactory bulb, the juxtaglomerular cell, also shared a
common distribution with the mitral cell and sometimes shared a similar morphology
as well (compare Figure 23D and E). This intemeuron, identified using an antibody
against tyrosine hydroxylase (TH), appeared relatively similar in size to mitral cells
(Figure 23F). Given the common distribution pattern and potential for anatomical
resemblance, the sizes of TH-positive intemeurons and mitral cells also were
78
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compared. Analysis of TH+ soma areas showed a normal distribution (P>0.05), and
there was no significant difference between the areas of the cells on the medial side of
the olfactory bulb versus the cells in the later side of the olfactory bulb (P>0.05).
Thus, all TH+ cells were grouped for comparison to mitral cells. The soma length and
width of these two cell types were evaluated and TH+ cells were compared to each
subcategory of mitral cell (Figure 24).
The mitral cell bodies, including all
populations of cells, ranged in size from 6-18pm in length and 4-12pm in width
(Table 3). The TH-positive cells, on the other hand, ranged in size from 5-10pm in
length and 4-10pm in width. The TH-positive cells had a mean length of 7.0 ± 0.1pm
and a mean width of 6.5 ± 0.1pm. The smallest mitral cells, the uni-dendritic cells on
the lateral side of the bulb, had a mean length of 8.7 ± 0.2pm and a mean width of 6.2
± 0.2pm. The largest cells, the multi-dendritic mitral cells on the medial side of the
olfactory bulb, had a mean length of 12.6 ± 0.5pm and a mean width of 8.1 ± 0.4pm.
This shows substantial overlap between TH+ cells and some, but not all, populations
of mitral cells (Figure 24).
Discussion
We have described the morphology and distribution of the major output
neuron, the mitral cell, in the adult zebrafish olfactory bulb. This cell type has been
described in detail in a number of animals including other teleosts (Kosaka and
Hama, 1982; Oka, 1983; Alonso et al., 1988; Fujita et al., 1988; Satou; 1990),
cyclostomes (Johnston, 1902; Heier, 1948; Iwahori et al., 1987), elasmobranchs
(Iwahori et al., 1992; Dryer and Gaziadei, 1993, 1994), amphibians (Scalia et al.,
1991; Jiang and Holley, 1992), reptiles (Garcia-Verdugo et al., 1986; Iwahori et al.,
79
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1989), and mammals (Price and Powell, 1970; Macrides and Schneider, 1982; Mori et
al., 1983; Lopez-Mascaraque et al., 1990; Malun and Brunjes, 1996). While mitral
cell structure varies between the animal groups studied, in general the morphology
remains consistent within the animal groups. For example, in the teleost family mitral
cells in carp, goldfish, and tench appear to be similar (Kosaka and Hama, 1982; Oka,
1983; Alonso et al., 1988; Fujita et al., 1988). In carp, the mitral cell soma is
generally fusiform, elongated, oval, or triangular in shape, has a mean size of 30pm
by 14pm and possesses multiple primary dendrites (Fujita et al., 1988). In goldfish,
the mitral cell usually is round or triangular, ranging in size from 10-25pm in
diameter, and also has multiple dendrites (Kosaka and Hama, 1982; Oka, 1983). The
tench has some variability in that it contains two types of mitral cells. The first type,
which is the most prominent, is generally fusiform or triangular in shape and has two
or more dendritic trunks that have tufts located at a distance from one another
(Alonso et al., 1988). The second type is round or ovoid and possesses one to four
primary dendrites that arborize in a single dendritic field (Alonso et al., 1988). Thus,
the mitral cells of these teleosts exhibit only minor variations in morphological
features such as soma size, which can be accounted for by the difference in the sizes
o f the fishes being examined; the zebrafish is only 2.5cm in length as an adult.
The shapes of zebrafish mitral cell somata are generally similar to that of
other teleosts (ovoid, spherical, fusiform or elongated), although they differ in their
dendritic morphology. Some zebrafish mitral cells have multiple dendrites, but we
have shown that this number is typically less than 35% of the total population. The
typical zebrafish mitral cell has only a single dendritic trunk. Most other teleosts that
have been examined have mitral cells with multiple dendritic trunks (Kosaka and
Hama, 1982; Oka, 1983; Fujita et al., 1988; Satou; 1990).
80
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Figure 23. The morphology and distribution of mitral cells was compared to other neurons in the
olfactory bulb. Ruffed cells (A), which are output neurons that can be identified by a distinct
membranous protrusion on their axon (arrowhead), were even fewer in number than the atypical mitral
cells. These cells exhibited a single primary dendrite extending from the cell body (arrow). Terminal
nerve cells (B) appeared to be the largest neurons in the bulb labeled with our technique, and their cell
bodies (*) were often found in tight clusters near the axon bundles o f the medial olfactory tract and in
large groups in the most rostral portion o f the bulb near the ONL (C, *). In panels B and C, rostral is to
the right o f the image. The identity o f these cells was confirmed using an antibody against
FMRFamide (C, red). While they shared a similar distribution with mitral cells (green) o f the
glomerular layer (GL), their morphology was distinct. Mitral cells (E) also resembled tyrosine
hydroxylase-positive (TH+) cells (D) with regard to size (D-F), morphology (D, E) and distribution
(F). Cells retrogradely-labeled with BDA (green) were found in the GL and the superficial ICL, as
were juxtaglomerular cells labeled with TH (red). Scale bar = 20pm (A,B,D,E) or 15pm (C,F).
81
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14l A Med Uni
12
<
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A
Let Uni
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Lat Multi
+
TH+cells
Jrt
4 fT T
(0
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0)
8
10
12
14
16
18
20
Soma length (microns)
Figure 24. Mitral cells sizes overlapped somewhat with tyrosine hydroxylase-positive (TH+) cells. For
the 75 TH+ cells and 100 mitral cells measured, there was a great deal o f overlap in soma length and
width o f TH+ cells (+) and lateral uni-dendritic mitral cells (triangles) as well as medial uni-dendritic
mitral cells (circles). Multi-dendritic mitral cells on the medial (diamond) and lateral (square) side of
the olfactory bulb were typically much larger in length and width than TH+ cells.
The exception to this rule is found in the tench, where Type II mitral cells can
have from one to four dendrites (Alonso et al., 1988). Even in this case, the Type II
mitral cells are less prominent than the Type I mitral cells that have multiple dendrites
(Alonso et al., 1988). Furthermore, since Type II mitral cells can have from one to
four dendrites, only some of these cells have single primary dendrites. Some teleost
studies, even those that suggest mitral cells have multiple primary dendrites, show
camera lucida drawings that indicate some of these output neurons may have only a
single dendritic trunk. These variations in reporting could result from the fact that
cells in the minority are not described in detail or there are differences in the way
single dendrites are defined. For example, Oka (1983) states that Golgi-impregnated
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mitral cells were identified with very thick dendrites; however, in three of the twenty
schematic drawings, the mitral cells appear to have a single dendrite that extends
from the cell body (see Oka, 1983, Figure 4, Number 12, for example). In these cases,
the dendrite further branches and may innervate different glomeruli, but by our
anatomical classification there is only a single dendrite leaving the cell body. We
classified a cell as having multiple dendrites if more than one dendritic trunk could be
seen leaving the cell body. These minor differences in the definition of a single
dendrite verses multiple dendrites could result in variation in the classification of
cells. Based on our definitions, it appears that goldfish may have mitral cells with a
single dendrite. Regardless, in zebrafish the majority rather than a limited number of
mitral cells appear to have a single primary dendrite.
In contrast to the typical teleost mitral cell, which has multiple dendrites, it is
generally believed that mitral cells in the mammalian olfactory bulb possess only one
primary dendrite (Shepherd, 1972; Meisami and Safari, 1981). However, studies have
shown mammalian mitral cells in the main olfactory bulb can have multiple primary
dendrites, as well (Mori et al., 1983, Puche, personal communication). In rabbits, for
example, about 20% of mitral cells examined had two to three primary dendrites
(Mori et al., 1983). In our study, just over 30% of the mitral cell population in
zebrafish have multiple dendrites. Mitral cells with multiple primary dendrites are
also known to exist in the accessory olfactory bulb of mammals. In mice and rats,
mitral cells of the accessory olfactory bulb, a structure that aids in deciphering
pheromonal cues, each have up to six primary dendrites (Cajal, 1901; Takami and
Graziadei, 1991, 1992; Meisami and Bhatnagar, 1998). In zebrafish, we have shown
that the medial olfactory bulb has a larger number of multi-dendritic mitral cells than
the lateral olfactory bulb. Interestingly, the medial tract in fish has been suggested to
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play a significant role in the processing of pheromonal information while the lateral
tract processes other kinds of odorants (Satou et al., 1983; Stacey and Kyle, 1983;
Demski and Dulka, 1984; Satou et al., 1984; Kyle et al., 1987; Sorensen et al., 1991;
Hamdani, Alexander and Doving, 2001; Hamdani, Kasumyan and Doving, 2001;
Weltzien et al., 2003). This lends further support to the idea that olfactory coding in
this model system may be similar to mammalian coding. Furthermore, our results
suggest that since a large number of mitral cells have only a single primary dendrite
with a discrete dendritic tuft, it is likely that these cells innervate only a single
glomerulus. We also have shown that cells with multiple dendrites appear to tuft
within close proximity to one another and also can innervate a single glomerulus.
Based on these anatomical results, we find that the zebrafish mitral cells may be more
like mammalian mitral cells than previously believed.
One of the most notable differences between the mitral cells of teleosts and
mammals is that teleost mitral cells reportedly lack secondary (basal) dendrites
(Kosaka and Hama, 1982; 1982-1983; Alonso et al., 1988; Dryer and Graziadei,
1994). In mammals, secondary dendrites are horizontal or tangential projections from
the cell body that are confined to the external plexiform layer, meaning they do not
interact with the axons of the olfactory sensory neurons. They are, however, still
active in cellular communication and associate with the dendrites of granule cells
(Shepherd, 1972; Orona et al., 1984). In teleosts, it has been reported that all
dendrites end in a glomerular tuft suggesting that there are no secondary dendrites
(Satou, 1990). In our study, we observed several cells that appeared to have
projections that did not end in tufts (unpublished data). Based on the location of these
projections, extending from the initial portion of the apical dendrite rather than the
cell body, they would not be defined as the morphological equivalent of secondary
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dendrites in mammals, but could be a functional equivalent. It is possible, however,
that these observations were due to incomplete filling of the processes with the
dextran. It also is possible that these branches do not interact with glomeruli and are
just deep to the GL. Since the olfactory bulb of fish is much more diffuse than that of
mammals, it is often difficult to delineate the layers. There is very little segregation
between the GL and the ICL and, as a result, dendrites that do not interact with
glomeruli would be more difficult to identify. Furthermore, many of the studies that
suggest teleosts do not have secondary dendrites examined mitral cells utilizing Golgi
technique. According to one of these Golgi studies, some mitral cells did have thin
branches without terminal arborizations (Kosaka and Hama, 1982). This may have
been a result of partial impregnation and could have resulted in failure to classify
secondary dendrites (Kosaka and Hama, 1982). Subsequently, it does not appear that
zebrafish possess a morphological equivalent of mammalian secondary dendrites, but
it is uncertain whether or not a functional equivalent exists in teleost systems.
Other morphological features, such as soma size, are more easily interpreted.
In this study, we found that mitral cells segregate to two means based on their
morphologies. Uni-dendritic mitral cells have an average length of 9.3 ± 0.2pm, an
average width of 6.2 ± 0.1pm, and an average cross-sectional area of 47.1 ± 1.1pm2
(n=50), while multi-dendritic mitral cells have an average length of 11.9 ± 0.4pm, an
average width of 8.0 ± 0.3pm, and an average cross-sectional area of 76.8 ± 2.1pm2
(n=50). In another zebrafish study, the measurement of 50 cells shows that the
average length of the cells is 14.4 ± 1.8pm, the average width is 10.1 ± 1.7pm, and
the average area is 105.9 ± 13.8pm (Edwards and Michel, 2002). While these mitral
cells were not segregated based on morphologies, the averages were still larger than
those found in this study. It is likely that the variation in sizes between the two studies
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is due to differences in tissue processing and plane of section. Edwards and Michel
(2002) labeled mitral cells with an antibody to glutamate using a three-step
immunocytochemistry procedure. This technique may have added additional apparent
thickness to the cell.
Studies in cyclostomes and reptiles have grouped mitral cells into types,
partially based on soma size (Iwahori et al., 1987, 1989). We binned mitral cells in
lOpm2 increments using cell cross-sectional areas in order to determine if there was a
natural grouping in the size of the cell bodies, for example, one population
segregating to a single mean versus more than one population differing in size and
number. Our results show that the total population of mitral cells does not follow a
normal distribution; rather it appears to be composed of more than one population.
Initial segregation of the cells into medial versus lateral olfactory bulb mitral cells
also yields distributions that are not normal. This suggests that the cells making up
the medial olfactory tract are comprised of two populations o f cells, as are the cells of
the lateral olfactory tract. We then isolated the cells based on uni-dendritic versus
multi-dendritic morphology and found a normal distribution. This suggests that cells
with a single dendrite segregate to one mean that is different from cells with more
than one dendrite. It also suggests that while comparisons can be made between
olfactory bulb mitral cells with specific morphologies regardless of their location, a
group of cells on the medial side of the olfactory bulb cannot be compared to a group
of cells on the lateral side of the olfactory bulb unless their morphologies are first
determined. However, it also is important to note that since the retrograde tracttracing method used in this study does not label all mitral cells, these results were
obtained from a sampling of the total population. Thus, the possibility that mitral cells
follow a different pattern of distribution cannot be eliminated.
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Many of the mitral cell studies that classify these output neurons into groups
use additional morphological features to separate them. In this study, the only major
morphological difference in the cells occurs in the number of dendrites extending
from the cell body. Our results also suggest that cells with multiple dendrites are
significantly larger than those with a single dendrite. In fact, when the results of the
soma cross-sectional groupings and soma length and width measurements are
examined, a clear picture emerges. The largest cells in the olfactory bulb tend to be
cells with multiple dendrites on the medial side and the smallest cells tend to be cells
with a single dendrite on the lateral side. It is uncertain, however whether or not these
morphological variations represent differences in the function of the cells. We know
o f no studies that have begun to elucidate the potential differences in the electrical
properties of mitral cells with single dendrites verses multiple dendrites in zebrafish.
Thus, further physiological studies are required to address the issue of functional
differences between these anatomically variant cells.
We found that mitral cells were most commonly distributed on the side of the
bulb corresponding to the tract to which their axons would project. Cells in the medial
and rostral areas of the bulb most often projected to the MOT, while cells in the
lateral and caudal regions of the bulb most often projected to the LOT. We found that
there is an apparent difference in the mitral cells of the MOT and the mitral cells of
the LOT, with regard to dendritic projections; there are more multi-dendritic mitral
cells on the medial side of the olfactory bulb. This may support the functional
differences seen between the cells of the MOT versus the cells of the LOT. In
teleosts, cellular responses to amino acids and bile components occur in different
regions of the olfactory bulb (Thommesen, 1978; Doving et a l, 1980; Hara and
Zhang, 1998; Nikonov and Caprio, 2001). This suggests that the two classes of
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odorants may be processed independently of one another (Hara and Zhang, 1996;
1998) and that spatial organization of output neurons is extremely important in
olfactory coding (Nikonov and Caprio, 2001). Previous studies also have shown that
the MOT processes information about pheromones while the LOT processes
information dealing with amino acids or food (Satou et al., 1983; Stacey and Kyle,
1983; Demski and Dulka, 1984; Satou et al., 1984; Kyle et al., 1987; Sorensen et al.,
1991; Hamdani, Alexander and Doving, 2001; Hamdani, Kasumyan and Doving,
2001; Weltzien et al., 2003). The variation in the number of uni-dendritic versus
multi-dendritic mitral cells on the medial and lateral sides of the olfactory bulb may
prove significant in understanding the apparent functional differences that exist
between the two olfactory tracts.
While examining the distribution of mitral cells in the adult zebrafish
olfactory bulb, it was noted that there was a great deal of similarity between these
output neurons and TH-positive intemeurons. We found that mitral cells, while larger
than TH-positive cells on average, show substantial size overlap with the
intemeurons. In addition, their shapes can be similar and they have a comparable
location in the bulb. Some studies suggest that mitral cells are easily identified by
their large size and morphology. Our study indicates that size, morphology, and/or
distribution would not be sufficient evidence for mitral cell identification. Indeed, we
have found that mitral cells are not the largest cells in the olfactory bulb; two other
cell types, the ruffed cell and the terminal nerve cell, have larger somata. In a study
examining ruffed cells in the adult zebrafish, ruffed cells were shown to have mean
length of 11.2pm ± 0.6pm and a mean width of 9.9pm ± 0.3pm compared to mitral
cells in the same fish, which had a mean length of 9.0pm ± 0.4pm and a mean width
of 8.3pm ± 0.2pm (Fuller and Byrd, 2005). There also is distributional similarity
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between mitral cells and ruffed cells (Fuller and Byrd, 2005). The current study
shows that terminal nerve cells have larger cell bodies than mitral cells, and
evaluation of their average soma size suggests that they are larger than ruffed cells, as
well. In other fish, retrograde labeling shows the large size of the ganglion cells of the
terminal nerve, approximately 70pm by 80pm in comparison to 30pm by 14pm, the
average size of mitral cells in the same species (Fujita et al., 1985). Based on these
results, mitral cells appear to be the third largest cell type in the olfactory bulb of
zebrafish. It will become increasingly important for those studying this system to use
alternative methods including morphological, pharmacological and/or physiological
evidence to confirm mitral cell identity in the future.
In summary, we have used a variety of techniques for a rigorous analysis of
zebrafish mitral cell morphology and distribution. There are two populations of these
output neurons in this teleost. Uni-dendritic cells have a single dendrite, typically
with a single dendritic tuft, although more than one tuft can be seen when a dendrite
branches at a distance from the cell body. Multi-dendritic cells have multiple
dendritic processes emanating from the cell body with several dendritic tufts
generally located near each other. Both uni-dendritic and multi-dendritic mitral cells
are found throughout the bulb, but the medial side of the olfactory bulb possesses
more multi-dendritic cells than the lateral side of the bulb and the lateral side of the
bulb has more uni-dendritic cells than the medial side of the bulb . In addition, cells on
the medial side of the olfactory bulb tend to project their axons to the MOT while
cells on the lateral side of the bulb tend to project to the LOT, regardless of the
morphology. Further, it does not appear that innervation of multiple glomeruli is a
common feature of zebrafish mitral cells. Because mitral cells are such a vital
component in olfactory coding and processing, studies in our laboratory will continue
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to focus on this area by examining the cellular interactions of these output neurons as
well as activity-dependent cytoarchitecture.
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CHAPTER VI
THE EFFECTS OF DEAFFERENTATION ON MITRAL CELLS: AN
EXAMINATION OF MORPHOLOGY FOLLOWING LOSS OF AFFERENT
INNERVATION
Introduction
Afferent-target interactions continue to be a major focus of study in the
scientific community because of the interest in the natural plasticity of the nervous
system and its ability, or lack thereof, to recover from disease and injury in specific
situations. Many studies have examined the role of afferent innervation on the
development and maintenance of central nervous system structures. For example, in
the visual system it has been shown that removal of innervation modifies molecular
activity in the target (Wong-Riley, 1979; LeVay et al, 1980; Zhang et al, 1995; Pires
et al, 1998; Yan and Ribak, 1998). In the auditory system, cochlear removal leads to
changes in the dendritic profiles of neurons in the medial superior olivary nucleus
(Russell and Moore, 1999) and in the motor-cortical region of birds, afferent lesions
result in increased cell death (Johnson et al., 1997).
Deafferentation also has been well studied in the olfactory system. Removal
of olfactory innervation leads to loss of olfactory bulb volume in both mammals and
teleosts (Matthews and Powell, 1962; Margolis et al., 1974; Harding et al., 1978;
Brunjes, 1985; Poling and Brunjes, 2000). In adult rodents, olfactory deprivation
results in reduced cell number (Henegar and Maruniak, 1991; Meisami and Safari,
1991; Corotto et al., 1994) and decreased neurogenesis (Corotto et al., 1994). Further,
other investigations suggest that loss of innervation results in changes in olfactory
bulb morphology (Wilson and Wood, 1992), cellular morphology (Pinching and
Powell, 1971), enzymatic activity (Nadi et al., 1981; Baker et al., 1984; Baker et al.,
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1993), and mRNA and protein expression (Ehrlich et al., 1990; Stone et al., 1991;
Ferraris et al., 1997; Casabona et al., 1998; Oberto et al., 2001) in the deprived bulb.
Previous studies in our laboratory support these findings showing that removal of
innervation from the nose can have drastic effects on the central nervous system
target. For example, in adult zebrafish loss of olfactory input via cauterization o f the
naris results in reduced bulb volume (Figure 25A; Byrd, 2000), increased apoptosis
(Figure 25B; Vankirk and Byrd, 2003) and reduced immunoreactivity of various
proteins (Figure 25C; Byrd, 2000; Fuller et al., 2005). In this study, we further
examine the role of afferent-target innervation in the maintenance of the adult
zebrafish olfactory system.
The teleost olfactory bulb is a diffuse structure when compared to the
mammalian olfactory bulb. It is organized into four principal layers (Oka et al., 1982;
Satou, 1990; Byrd and Brunjes, 1995) including the olfactory nerve layer (ONL), the
glomerular layer (GL), the mitral cell layer (MCL), and the granule cell layer (GCL).
Based on previous studies in our laboratory, the distribution of mitral cells in
zebrafish does not appear to fall into a single layer as it does in mammals
(unpublished data). Thus, the MCL and the GCL are often are combined into a single
layer termed the internal cell layer (ICL; Byrd, 2000).
Deafferentation studies in adult zebrafish have suggested that ablation of the
olfactory rosette results in significantly reduced volumes of the ONL and the GL, but
not of the MCL and the GCL at 4 weeks post-denervation (Poling and Brunjes, 2000).
The reduction of volume in the ONL is easily explained by the deterioration and
eventual loss of the nerve fibers entering the olfactory bulb. There are other
possibilities that could explain the reduction in laminar volume of the GL. There
could be a reduction in the number of cells in this layer. A previous study in zebrafish
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showed that there is an increase in apoptosis following olfactory denervation,
although, the cells undergoing this process were located primarily in the ONL and the
ICL, not in the GL (Vankirk and Byrd, 2003). However, this study examined the
olfactory bulb at time points varying from 1 hour to 1 week post-deafferentation, so
there could be a greater number of cells undergoing apoptosis after 1 week. Another
study by Baker and colleagues (1993) suggests that even though enzymatic activity of
cells located in the GL of rodents might be reduced following deafferentation, the
cells are still present. This supports the idea that even 2 months after removal of
afferent innervation, a major cell type in the GL does not undergo apoptosis. Another
potential source of reduced GL volume may be loss of the dendritic arborizations of
mitral cells that project to this layer. This study will examine the morphology of these
major output neurons at various time points following deafferentation in an attempt to
address one possible component of volume loss in the deprived bulb.
Materials and Methods
Animals
Adult male and female zebrafish, Danio rerio, at least 2.5cm in length, were
obtained from commercial sources (RJ Ray Distributors, Pontiac, MI). The fish were
maintained in 15-gallon aquaria filled with aerated, conditioned fish water at 28.5°C,
and they were fed commercial flake food (Wardley Corporation) twice daily. All
protocols on animal care and experimental procedures were approved by the
Institutional Animal Care and Use Committee.
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Figure 25. Afferent innervation directly influences the maintenance of the olfactory bulb in adult
zebrafish. A) A hematoxylin and eosin stained section of the adult olfactory bulb shows a reduction in
the volume o f the ipsilateral bulb. B) Twenty-four hours following deafferentation, apotosis increases
in the deafferented bulb (ROB) while there is little cell death in the untreated bulb (LOB). This section
o f the tissue shows that the olfactory epithelium (oe) remains intact in the internal control. Note the
lack of epithelium in the left naris region (*). (C) Tyrosine hydroxylase is markedly reduced in the
denervated bulb following loss o f innervation from the nose.
Deafferentation
Adult zebrafish were anesthetized with tricaine and the right olfactory organ
was ablated using a small-vessel cautery iron. The left olfactory organ remained
intact for use as an internal control for comparison. Fish were returned to aquarium
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water containing the antibiotic Kanacyn (0.01 mg/ml, Aquatronics). For the cellular
morphology experiments, fish were allowed to survive for 3 weeks, 6 weeks, 8
weeks, or 20 weeks. For the laminar volume experiments, fish were allowed to
survive for 1 week, 3 weeks, or 6 weeks. After the appropriate survival period, the
deafferented and control fish were processed as described below.
Olfactory Tract Tracing
Fish were anesthetized with 0.03% MS222 (3-amino benzoic acid ethyl ester,
Sigma) and perfused with phosphate buffered saline (PBS). The brains were
dissected, and approximately 0.05pl-0.1pl of Texas Red dextran (10000MW, 5mg/ml
in PBS, Molecular Probes) was injected either into both olfactory tracts using a PV
800 Pneumatic Picopump (World Precision Instruments). The brains were then placed
on a 3 pm polycarbonate filter in a sterilized six-well culture dish (Costar) containing
artificial fish cerebrospinal fluid (lOOmM NaCl, 2.46mM KC1, ImM MgCl2'6H20 ,
0.44mM NaH2P 0 4H20 , 1.13mM CaCl2H20 , 5mM NaHC03) and incubated at 28.5°C
and 1.5% C 0 2 for approximately 4-6 hours (Tomizawa et al., 2001). Following
fixation in 4% paraformaldehyde (PFA) for 24 hours, the tissue was viewed on a
Zeiss LSM 510 microscope as whole mounts for dendritic analysis.
Dendritic Analysis
To obtain dendritic reconstructions of the dextran-labeled cells, whole mounts
were examined using confocal microscopy. Once a cellular profile was identified, zstack images were used to gather fine optical sections of the cell at 1pm intervals.
The image was then converted to a projection to visualize the cell in 3 dimensions
(Figure 26A).
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Figure 26. A stepwise diagram shows the process o f dendritic analysis used to obtain estimates of
dendritic complexity in control and deafferented olfactory bulbs. These steps included obtaining a
three dimensional image o f the mitral cell (A), tracing the dendritic processes (B), and counting the
number of branch tips within the area of the tuft (C).
The cell was traced using the LSM510 overlay tool (Figure 26B), and the area
of the dendritic arbor was measured by connecting the distal tips of the dendritic
branches to form a convex polygon (Figure 26C). The trace was analyzed for
complexity by counting the number of dendritic branch tips in the dendritic arbor and
this number was reported as dendrites per unit area. Dendritic complexities were
compared within survival groups using paired T-tests and between groups using an
ANOVA and Tukey’s test for multiple comparisons. P values less than 0.05 were
considered significant.
Results
Mitral Cell Morphology in Control Animals
In untreated animals, mitral cells exhibited both Type I and Type II
morphologies. The Type I morphology, which was the most prominent, had a single
apical
dendrite
with
a
discrete
dendritic
tuft (Figure
27A-B).
Dendritic
reconstructions of these cells verified the localization of the tuft to a limited area
(Figure 27C). Even cells with a Type II morphology, which was defined by more than
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one dendritic projection, showed dendrites that projected to restricted glomerular
regions and formed a discrete glomerular tuft (Figure 27D).
One of the most
noticeable features of these dendritic arbors was the large number of fine processes
they contained. These morphologies were seen in both the right and left olfactory
bulbs.
Mitral Cell Morphology in Deafferented Animals
At 3 weeks post deafferentation, the treated, or right olfactory bulb appeared
to show no variation from control animals with regard to the fine processes in the
dendritic tufts (Figure 28, 3wk, ROB). Six weeks after ablation, the fine processes
appeared to diminish and the cellular tufts appeared to be less elaborate (Figure 28, 6
wk, ROB). By 8 weeks after denervation the mitral cells appeared to have few fine
processes, yet the large dendritic branches were still apparent (Figure 28, 8wk, ROB).
These changes were not apparent in the internal control bulb (Figure 28, 8wk, LOB).
Quantitative Analysis of Mitral Cell Dendrites
Quantitative analysis of the dendrites showed that there was no significant
difference between the density of the branch tips in the LOB and the ROB at 3 weeks
or 6 weeks following removal o f the olfactory organ (p=0.3 and 0.1, consecutively;
Figure 29). By 8 weeks and 20 weeks, however, there were significantly fewer branch
tips per unit area (p<0.01 and p=0.03, consecutively; Figure 29). When comparing
only right bulb densities between groups, an ANOVA showed that there were
significantly fewer branch tips in the 6week, 8 week, and 20 week deafferented
animals than in the control animals. Additionally, there were significantly fewer
branches in the 8-week and 20-week deafferented animals than in the 3-week and 697
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week deafferented animals (p< 0.01 for all comparisons; Figure 29). Left bulb
densities also were compared between groups and showed no significant difference at
any time point (p= 0.08)
To compare the possibility that primary dendritic processes were more spread
out over time, which might result in a reduction of the number of branches per unit
area, dendritic tuft areas were compared. Results from these findings showed no
significant differences in the tuft areas o f right and left bulbs at any single timepoint
or between groups at any timepoint (P=0.37; Figure 30).
Discussion
The response of cells to loss o f afferent innervation is variable. In some
studies involving motoneurons in adult animals, peripheral nerve injury leads to
decreased size of the dendritic tree (Vanden Noven et al., 1993; O’Hanlon and
Lowrie, 1995). In the auditory system, removal of the cochlea during adulthood
generates reduced dendritic density in the target cells, but these cells do not show a
reduction in area of the dendrite (Russell and Moore, 1999). In one study,
hippocampal lesions result in increased dendritic length and increased dendritic
branching of denervated cells (Pyapali and Turner, 1994). Based on this variability, it
was uncertain how mitral cells would respond to loss of innervation.
Our results indicate that at 3 weeks post-deafferentation, no change is seen in
the dendritic complexity of the mitral cells in the ipsilateral bulb. At 6 weeks after
surgery, there also is no significant difference between the mitral cell dendrites in the
right and left olfactory bulbs, although when compared to control animals the 6-week
time point does show a reduction.
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Figure 27. Retrograde labeling revealed both Type I (A-C) and Type II (D) mitral cell morphologies.
Type I cells had a soma of variable shape (*) ranging from 6-15 pm in diameter, a single primary
dendrite (arrow) terminating in discrete dendritic tufts near the cell body, and a single axon
(arrowhead) projecting toward the medial or lateral olfactory tract. The majority o f more than 1000
cells examined showed this morphology. Fewer cells exhibited the Type II morphology with multiple
dendrites arising from the cell body. Both phenotypes showed significant arborizations and fine
dendritic processes in control animals. Scale bar = 10pm (A-D).
By 8 weeks and 20 weeks after ablation, however, the fine dendritic branches
that are seen in both control animals as well as the internal controls are significantly
reduced. Interestingly, the major dendritic processes still are apparent at this 5-month
timepoint. These findings suggest that mitral cells are highly stable structures that
show little variation in dendritic complexity for extended periods following injury.
The olfactory system is inherently plastic, with constant turnover of cells in the nasal
epithelium and constant renewal of afferent-target interactions (for review see
Farbman, 1992). It is possible that this inherent plasticity results in the highly stable
morphology of the dendritic tuft of mitral cells through 6 weeks post-denervation and
the major processes of those dendrites through 5-months post-deafferentation. It also
is possible that the loss of the fine dendritic processes over time is a result of sensory
neuron loss while the large processes are maintained by the continued presence of
interactions with other cell types in the olfactory bulb such as granule cells.
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LOB
ROB
Figure 28. Mitral cell morphology examined in animals at 3 weeks, 6 weeks, 8 weeks, and 20 weeks
post-deafferentation showed that elaborate dendritic processes still existed in the animals even 2
months after loss o f afferent innervation. The most notable effect was seen at 5 months when the major
dendritic arbors were still present, although fine processes appeared to be greatly diminished. LOB is
shown for comparison as an internal control.
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| LOB
ROB
0.045
0.035
0.025
0.015
0.005
Control
3WK
6 WK
8WK
20 WK
Figure 29. A comparison o f dendritic complexity at 3 to 20 weeks post-deafferentation showed that
mitral cell morphology was affected by loss of afferent input. Dendritic densities were compared
within survival groups using paired T-tests and between groups using an ANOVA and Tukey’s test for
multiple comparisons.
In mammals, for example, the dendritic branches of the apical dendrite interact with
the olfactory sensory axons while the granule cells interact with the secondary
dendrites of the cell, which are more proximal to the cell body (for review see
Farbman, 1992).
In this study, we also show that there is no significant difference in the area of
the dendritic tufts after deafferentation. This suggests that while the fine dendritic
processes that make up the density of the tuft eventually are lost, the cell retains the
major processes that form its structure. This also indicates that the cells may be
capable of re-innervation even after 5 months of olfactory deprivation.
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2500 i
■ LOB
■ ROB
Figure 30. A comparison o f dendritic tuft area at 3 to 20 weeks post-deafferentation showed that mitral
cell tuft areas were not affected by loss o f afferent input. Glomerular tuft areas were compared within
survival groups using paired T-tests and between groups using an ANOVA and Tukey’s test for
multiple comparisons.
Also, as previous work has shown, the size of the olfactory bulb is significantly
reduced at 6 weeks post-deafferentation (Byrd, 2000). This, taken with the stability of
the mitral cell major processes at 6 weeks after surgery, supports the idea that major
reductions in bulb volume after loss of the olfactory nerve layer also occurs in the
internal cell layer. Previous studies have shown that deafferentation results in a
significant loss of granule cells (Henegar and Maruniak, 1991; Corotto et al., 1994)
and reduction in the volume of the granule cell layer (Henegar and Maruniak, 1991),
which is equivalent to the internal cell layer in fish.
Other studies in teleosts suggest that at 4 weeks post-denervation laminar
volume is markedly reduced in the ONL and the GL, but not in the MCL and the
GCL (Poling and Brunjes, 2000). It is possible that our results vary from those of this
study with regard to the M CL and GCL results because of the differences in the
delineations of the olfactory bulb laminae. In the study by Poling and Brunjes (2000),
the investigators use a 4-layer system including a differentiated MCL and GCL. In
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our study, we use a 3-layer system, which groups the MCL and the GCL because
previous work in our laboratory has shown that mitral cells are not uniformly
distributed in a single layer as they are in mammals.
Minor variations in
differentiating between the laminar areas could results in significant differences in
apparent volume.
In general, the results of this study show that mitral cell morphology, is not
grossly affected by loss
of afferent innervation until 8 weeks
following
deafferentation. Even then, these output neurons are highly stable structures, retaining
major dendritic processes and some fine fibers. We will continue to examine the
plasticity of these cells and potential mechanisms by which they remain intact by first
determining what kind of interactions remain between mitral cells and other neurons
in the olfactory bulb following ablation.
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CHAPTER VII
DISCUSSION AND FUTURE DIRECTIONS
Major Contributions of This W ork
In this dissertation, my results show that the morphology of mitral cells in
adult zebrafish is unlike the morphology of mitral cells proposed for all teleosts.
Further, this work describes the presence of two cell types whose morphologies have
not yet been addressed in the zebrafish olfactory bulb. Finally, I have examined the
adult olfactory bulb following removal of olfactory input in order to establish the role
that afferent innervation plays in the maintenance of this central nervous system
structure. I believe that these studies will be of significance to individuals in the fields
of activity-dependence and comparative anatomy, and that they will be a substantial
contribution to sensory systems biologists focusing on olfactory coding and
processing.
General Discussion
The original purpose of this study was to examine the activity-dependent
maintenance of mitral cells in the adult zebrafish olfactory bulb. As I began to
examine the potential methodologies for answering this question, I realized that
almost nothing is known about the structure of the cells in the olfactory bulb o f this
species. This animal has been used in a number of olfactory physiology studies
(Corotto et al, 1996; Freidrich and Korsching, 1998; Friedrich and Laurent, 2001;
Edwards and Michel, 2002; Lipschitz and Michel, 2002; Michel et al, 2003), yet there
is little information about the morphology of the cell types in this system. It seemed
to me that before we could fully understand the physiology of these cells and the
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olfactory coding mechanisms of the entire olfactory bulb, we needed to first establish
the morphology of the cells in zebrafish. For example, in mammalian systems,
olfactory sensory neurons expressing the same odorant receptors project to a discrete
glomerular region, generally a single glomerulus (for review see McClintock, 2000).
Further, mitral cells have a single apical dendrite that innervates a singe glomerulus
(for review see Dryer and Graziadei, 1994). Physiology studies in mammals suggest
that individual mitral cells have the potential to respond to a wide range of related
odorants (Yokoi et al., 1995). If we knew that mitral cells responded multiple
odorants, but were unaware of their morphology, we might conclude that they
innervate multiple glomeruli and thus receive input from multiple types of odorant
receptors. Because we know that they only innervate a single glomerulus based on
their anatomy, our view of olfactory coding in mammals is drastically changed. Thus,
morphology presents a significant contribution to the overall functioning of a system
and should not be overlooked. W ith this in mind, I began examining the morphology
of cells in the adult zebrafish olfactory bulb.
A few investigations have examined mitral cell in other teleosts using Golgi
techniques (Kosaka and Hama, 1982; Oka, 1983; Alonso et al., 1988), but this
method is not a reliable indicator of cell type as it appears to label many kinds of
cells. Thus, there are significant issues with using this method as the sole technique in
identifying the morphology of a cell type that has not yet been established in another
manner. Further, few studies have gone beyond this initial series of Golgi
investigations to identify the morphology of mitral cells in their fish. A review of
mitral cells in various animals used these Golgi studies as a basis for defining mitral
cell morphology in all teleosts (Dryer and Graziadei, 1994). This review then
generated a basis for individuals studying the teleost olfactory bulb to define the
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morphology of mitral cells in their respective fish. In turn, few studies have actually
examined the morphology of this cell type in teleosts and yet it is a well-known myth
that all teleost mitral cells have multiple dendrites that innervate multiple glomeruli.
As I have shown in this study, zebrafish mitral cells typically have a single apical
dendrite with a discrete dendritic tuft. This morphology appears to be more similar to
mammals than to other fish, a finding that contributes to the background information
required to make claims regarding olfactory coding and physiological processing in
this commonly used animal model.
The first question I had with regard to this project was how could I confirm
the identification of the cell type? Since the majority of commercial antibodies are
made in mammalian systems, they often fail to produce results in fish. Further, there
have been no studies done to suggest a marker for mitral cells, even in mammals.
Many antibodies label mitral cells in addition to other cells, but none have been
suggested to label mitral cells only. Interestingly, mitral cell identification in
mammals is discerned by the soma location in the mitral cell layer of the olfactory
bulb. Thus, if a cell is located in the mitral cell layer, it is termed a mitral cell. Even
in mammalian studies some authors acknowledge the sometimes-displaced mitral cell
(Mori et al., 1983) or the overlapped distribution of mitral cells and tufted cells
(Pinching and Powell, 1971; Orona et al., 1984). If one were doing a physiological
study where a single neuron response to an odorant was recorded based on its location
in the olfactory bulb, it is quite possible that the cell would be misidentified as a
mitral cell when it could be another cell type with an abnormal distribution. In
zebrafish, especially, this issue is confounded because laminar distribution is not as
defined as in mammalian systems. For example, mitral cells often are intermixed with
juxtaglomerular cells and ruffed cells in the glomerular layer o f the olfactory bulb.
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Because these output neurons share a common distribution, size, and occasional
morphology with other cell types in the bulb, their identification based on any one
factor is not a certainty (see Figure 31).
In one zebrafish study, investigators labeled neurons with Lucifer Yellow
using an intracellular loading technique (Friedrich and Laurent, 2001). In this
particular project, cells were identified by their size and location in the olfactory bulb.
If a cell in this area responded positively to an odorant, it was filled with the dye. The
morphology of the mitral cell was then assumed based on the morphology of the cell
filled with the tracer. First, based on my work, mitral cells cannot accurately be
identified solely based on their size and distribution in the bulb. Thus it is uncertain
that the cells being labeled were mitral cells. The second argument made by the
authors was that the cells responded to an odorant. Another type of cell known as the
juxtaglomerular cell not only overlaps in size and distribution with these output
neurons, but it also receives innervation from olfactory sensory neurons. As a result,
these cells also would respond positively to an odorant. Taken together, this
information implies the possibility that cells other than mitral cells could have been
recorded in this study. In this case, it w asn’t even apparent that the recorded cells
were output neurons. Had the investigators taken the readings simultaneously with
olfactory tract recordings or confirmed the identity of the cells using cellular
backfills, the results would prove more convincing. Thus, it becomes increasingly
important to confirm cellular identity prior to making generalizations about how these
cells respond to odorants and how they interact with other cell types in the olfactory
bulb.
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Cell Type
Distribution
Size (averages)
Mitral Cells
GL/ICL
5-20|im (~9pm)
Granule Cells
ICL
"6pm
Juxtaglomerular Cells
GL
5-10pm ("7 pm)
Short Axon Cells
unknown in zf
unknown in zf
Ruffed Cells
GL/ICL
8-15pm (~11 pm)
Glial Cells
ONL/GL/ICL
variable
Terminal Nerve Cells
clusters in ONL/GL
13-26pm ("18pm)
(Edwards and Michel, 2002}
Figure 31. A chart showing cell types in the olfactory bulb of adult zebrafish describes the features of
these cells including their distribution and size. Images of some o f the more commonly discussed cell
types are shown. Note the substantial overlap in the size and distributions o f some o f these cells.
Another study in zebrafish used multiple labels to identify mitral cells, but
even in this case, the study was pharmacological and morphology was not addressed
because the tissue was processed in thin plastic sections to allow for accurate labeling
with the antibodies (Edwards and Michel, 2002). Based on the uncertainty of
antibody labeling and the likelihood that general morphology questions could not be
addressed using these techniques, I opted to use retrograde tract tracing with either a
biotinylated dextran or a fluorescent dextran. Using this method, I injected a tracer
into the axons of the output neurons and allowed the tracer to be transported
retrogradely into the olfactory bulb. By using this approach, I was able to label output
neurons in the olfactory bulb of adult animals. Since the amount of tracer injected
directly correlated with the number of cells labeled, it was important to establish the
question being addressed prior to loading the tracts. For example, when counting the
number of presumptive mitral cells in the olfactory bulb, the maximum amount of
dextran was injected into both the medial and the lateral olfactory tracts. When
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addressing the issue of cellular morphology, it was important to be able to discern the
dendritic processes of one cell from another cell. In these cases, a lesser amount of
tracer was inserted into a single tract.
Following this method, I was able to establish the presence of two other cell
types whose axons projected to the olfactory tracts: the ruffed cells and ganglion cells
of the terminal nerve. The presence of ruffed cells has been described in other teleosts
(Kosaka and Hama, 1979a; 1979b; Kosaka, 1980), but it has not been identified in
zebrafish. As a result, the work done for this dissertation modifies previous studies
that did not account for this cell type when considering olfactory coding and
processing in this animal. Another cell type, the ganglion cells of the terminal nerve,
have been greatly overlooked in zebrafish olfactory bulb studies. One study addresses
the presence of this cell type in the zebrafish olfactory bulb, but the primary concern
of this study was the association of the terminal nerve cells to the olfactoretinal
centrifugal pathway (Li and Dowling, 2000). Another study discusses the expression
of a neuropeptide gene in neurons of the terminal nerve, but again, morphology was
not a primary component of this investigation (Oehlmann et al., 2002). Few studies
have acknowledged the presence of these cells in the bulb of this species. Because of
the close association of these output cells with the axons of the medial olfactory tract
and the olfactory nerve, their function needs to be established as it may act to
substantially modify olfactory output. Again, anatomy plays a significant underlying
role in the physiology of various central nervous system structures.
Once the morphology of the cells in the olfactory bulb of adult zebrafish had
been established, I was able to return to the initial question of activity-dependent
maintenance of mitral cells. Using the same technique, I loaded the output neurons at
various time points following deafferentation. My results showed that even at 2
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months post-ablation, the morphology of the mitral cells was grossly intact. By 5
months after surgery, the fine dendritic fibers of the tuft had been reduced, but the
overall structure of the cell with its major processes was present. These findings show
that mitral cells are highly stable stmctures. It is likely that since the olfactory system
is inherently plastic by nature, that these cells were simply “waiting” to be re­
innervated. It also is possible that the existing connections between mitral cells and
intemeurons of the olfactory bulb helped to retain the general structure of the neurons
even though their major source of innervation was lost. Further studies are required to
examine this possibility.
Future Directions
Morphology Studies
Based on the significant variation seen between some species of teleosts and
the zebrafish, it might be interesting to do a follow-up study on the morphology of
mitral cells in other teleosts. Since the majority of previous studies have used Golgi
technique to identify these cells, it would be interesting to examine several of these
species using the retrograde tracing technique employed for this dissertation. This
series of experiments might further our understanding of output neurons in teleost
systems. If, for example, the mitral cells were drastically different than reported by
Golgi studies, it might speak to accuracy of cell identification using the Golgi
method. If, however, the cells looked inherently similar to the descriptions given by
other investigators, it would speak to the variation that exists in the teleost order and
would lend more support to the idea that more caution should be taken when making
broad generalizations.
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Development Studies
Another question that should be addressed involves the development of mitral
cells in this system. Several studies suggest that mitral cells in mammals undergo a
period where they have multiple dendrites and that during their development these
dendrites are retracted (Malun and Brunjes, 1996). It would be interesting to see if the
Type I and Type II mitral cells described in this study are two different types of adult
cells or if they are simply at different developmental stages. At this point, it is
uncertain if two developmental stages of mitral cells exist in adult animals. In fact,
there is little information about the development of output neurons in teleost olfactory
bulbs when comparing it to existing knowledge of mitral cell development in
mammals. Further studies may shed light on this question. On the other hand, the
retrograde tracing technique in juvenile zebrafish may prove difficult to master since
they have less developed brains. Other methods for labeling mitral cells in this
situation may need to be discovered.
Neurogenesis Studies
Another question that needs to be addressed involves the identification of
progenitor cells in the olfactory bulb. Previous studies have shown that adult
zebrafish undergo continuous neurogenesis and that the cells being formed migrate to
several areas of the olfactory bulb including the internal cell layer and the glomerular
layer
(Byrd and Brunjes, 2001). Retrograde labeling in combination with Brdu
treatment, which labels cell in the S-phase of mitosis, might suggest if any of these
new cells are mitral cells.
Ill
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Molecular Studies
One investigation that would have significant impact on a number of
individuals examining this cell type would be the creation of an antibody to identify
it. It might be possible to use the retrograde tracing technique combined with cell
culture methods to selectively identify output neurons in the olfactory bulb and then
examine these cells for potential protein markers. The fact that there is no specific
antibody for mitral cells at present suggests that this task may be quite difficult.
Deafferentation Studies
One potential question for future studies is what interactions remain between
mitral cells and other cells types in the deafferented olfactory bulb? One suggestion
for the maintenance of the cell structure following loss of innervation is that the cell
remains in contact with a number of other cell types including juxtaglomerular cells.
Further, centrifugal fibers carrying efferent input may influence the morphology of
these output neurons. This study could be accomplished several ways. First,
retrograde labeling could be employed to identify mitral cells. Once these cells are
labeled, an antibody against tyrosine hydroxylase could be used to identify the
juxtaglomerular cells and examine their morphology following loss of innervation. If
their projections are still intact, it is possible that they are helping to maintain the
mitral cell dendritic structure. Another possibility would be to use electron
microscopy to examine the potential interactions between mitral cells and other
neurons in the olfactory bulb to determine if synaptic contact is retained. It also might
be interesting to see if the number of synapses on the major processes is significantly
reduced in the deafferented animals. This experiment might suggest the degree to
which synaptic input still exists and shapes the cell.
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Another possible question for further examination involves the activity of
mitral cells in the deafferented olfactory bulb. Are the cells in this bulb spontaneously
active? If so, is there a reduction in the amount of spontaneous activity between
mitral cells in control olfactory bulbs versus mitral cells in deafferented olfactory
bulbs? Do cells that lack afferent innervation from the ipsilateral bulb respond to
odorants that are applied to the contralateral bulb? These questions might begin to
address the level at which cellular activity is retained in mitral cells following
ablation of the nose. This might help us begin to understand their plasticity following
injury.
One investigation that should be addressed involves apoptosis following
deafferentation. While previous studies have shown that the majority of cells
undergoing apoptosis following deafferentation are non-neuronal, some are neurons.
Future studies should combine retrograde tracing techniques with antibody labeling
against apoptosis to determine if any of the cells undergoing programmed cell death
are mitral cells. Further, this study should be completed through the 8-week postdeafferentation timepoint, which corresponds to the 8-week deafferentation timepoint
examined in this dissertation.
Final Thoughts
This dissertation begins to answer some of the questions about olfactory bulb
morphology and circuitry in the adult zebrafish, findings that seem to contradict much
of what is discussed in literature involving teleosts. Since this model system is being
more frequently employed for studies involving olfactory coding and physiology, the
anatomical foundation for these investigations becomes increasingly important.
Further, this dissertation shows the incredible stability of output neurons in the
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olfactory bulb following loss of afferent innervation, a finding that supports the
inherent plasticity of this system. I have learned a great deal in the process of writing
this manuscript; primarily that one of the largest problems in the scientific community
involves making unfounded generalizations. Too often researchers take for granted
the foundations upon which their scientific claims are made. In turn, conclusions
based on these initial generalizations can lead to further setbacks in the field when the
commonly held belief must be refuted. As scientists, it is our duty to do what it takes
to answer the question. Sometimes this means forgetting everything you’ve learned
and starting at the very beginning.
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Zippel HP, Reschke CH, Korff V. 1999. Simultaneous Recordings From Two
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Cells, in the Olfactory Bulb of Goldfish. Cell Mol Biol. 45: 327-337.
135
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APPENDIX A
Results from Additional Examinations of Mitral Cell and Olfactory
Sensory Neuron Interactions
136
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B
LB
MB
MB
LB
C
Figure 1. Images of olfactory axons labeled with DiA show that both distinct
glomeruli (A) and diffuse glomeruli (B) are present in the olfactory bulb of zebrafish.
The olfactory nerve forms two distinct bundles, a lateral bundle (LB) and a medial
bundle (MB). The LB appears to be thicker as it makes its way into the olfactory
bulb. Interestingly, the lateral olfactory tract, which is comprised of mitral cell axons
that exit the bulb, is much more diffuse and thinner than the medial olfactory tract. A
high magnification image o f a single glomerulus (C) supports the idea that mitral cellglomerular interactions can be examined in this species.
137
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A
B
"k
c
138
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Figure 2. An image of axons traced with the anterograde tracer, DiA, shows the
presence of distinct glomeruli (A, arrows) in the olfactory bulb. Mitral cells (B, *)
labeled with the retrograde tracer, microruby, also can be seen using fluorescence
microscopy. Note the presence of the discrete dendritic tuft associated with the mitral
cell, as well as the distinct tuft presumably associated with another unidentified cell
(arrows). An overlay image of the two tracers (C) shows that the dendritic tuft
associated with the large mitral cell occupies the entire area of the small glomerulus.
These images were obtained in conjunction with Ms. Holly Yettaw, an undergraduate
researcher from the Biological Sciences department at Western Michigan University.
139
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APPENDIX B
Results from Mitral Cell Studies in Other Teleosts
140
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Figure 1. Mitral cells were identified in the olfactory bulb of the bullhead catfish,
Ictalurus punctatus, using the retrograde tracing method employed for this
dissertation. While mitral cells have been described by Golgi methods in other catfish
species, they have not been described in this species. The cells were variable in size
and shape and typically had multiple dendrites although some cells with single apical
dendrites were seen. In this series of experiments, the dendritic tufts appeared much
more diffuse than in zebrafish; however, since the olfactory bulbs of this species are
significantly larger than those of zebrafish, it is possible that these cells still projected
to a localized glomerular area. Further studies are required to address this issue.
142
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GOLDFISH
CATFISH
ZEBRAFISH
Figure 2. Ruffed cells also were identified in the goldfish and the catfish using the
retrograde tracing method. Studies have previously identified goldfish ruffed cells,
but as an additional measure of control I wanted to confirm that our method would
label these cells. In bullhead catfish, ruffed cells had not yet been identified. These
finding show that retrograde tracing is an adequate method for identifying ruffed cells
in the olfactory bulbs of these species. Notice that the ruffed cells in the goldfish and
the catfish olfactory bulbs are quite similar in morphology to the ruffed cell identified
in zebrafish.
These studies were done in collaboration with Dr. John Caprio at Louisiana State
University.
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APPENDIX C
Animal Care and Use Committee Approval Form
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R E C E I V E D
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Name: Cynthia DeYoung
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E lectronic M ail A ddress: [email protected]
1.
Professor
The research, as approved by the IACUC, is com pleted:
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To:
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The
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Y o u m a y n o w b e g in to im p le m e n t th e re s e a rc h as d e s c rib e d in th e
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W alw o o d H a ll. K a la m a /o o , M l 4 9 0 0 8 - S-156
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WESTERN MICHIGAN UNIVERSITY
Institutional Animal Care and Use Committee
A N N U A L R E V I E W O F V E R T E B R A T E A N IM A L U S E
p r o j e c t o r c o u r s e t i t l e : Cel! Genesis In The Olfactory Bulb Of Adult Zebrafish
IA CU C Protocol N um ber: 03-05-01
D ate o f R eview R equest: 05/17/04
D ate o f L ast A pproval: 6-11-03
Purpose o f project (select one): | [Teaching
^ R esearch
F lO th e r (specify):
P R IN C IP A L IN V E S T IG A T O R O R A D V IS O R
Name: Christine Byrd
T itle: A ssoc/A ssist. P rofessor
D epartm ent: BIOS
E lectronic M ail A ddress: byrd@ w m ichedu
C O -P R IN C IP A L O R S T U D E N T IN V E S T IG A T O R
Name: Cynthia Fuller
T itle: Student
D epartm ent: BIOS
E lectronic M ail A ddress: cynthia.deyoung@ wmich.edu
1.
T he research, as approved by the LACUC, is com pleted:
Q ]Y e s (C ontinue w ith item s 4-5 below .)
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2. Have th ere been any changes in Principal o r C o-Principal Investigators? ^ ^ Y e s
| INq
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tX lN o
C um ulative n u m b er o f m ortalities:
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A nim al usage:
/I /
N um ber o f anim als used during
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Date
APPRO V A L
tjf^ relevant inform ation regarding this protocol, the IACUC approval for this project has
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A N N U A L R E V I E W O F V E R T E B R A T E A N IM A L U S E
p r o j e c t o r c o u r s e t i t l e : Cell Genesis In The Olfactory Bulb Of Adult Zebrafish
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D ate o f R eview Request: 05/03/05
D ate o f Last A pproval: 06/01/04
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^R ese arch
I [Other (specify):
P R IN C IP A L IN V E S T IG A T O R O R A D V IS O R
N ame: Christine Byrd
T itle: Assoc/A ssist. Professor
D epartm ent: BIOS
E lectro n ic M ail A ddress: [email protected]
C O -P R IN C IP A L O R S T U D E N T IN V E S T IG A T O R
N am e: Cynthia Fuller
T itle: Student
D epartm ent: BIOS
E lectro n ic M ail A ddress: cynthta.deyoung@ wmich.edu
1.
T he research, as approved by the IA C U C , is com pleted:
I |Y es (C ontinue w ith item s 4-5 below .)
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explanation on an a tta c h e d sh eet o f paper. Include details o f any m odifications m ade to the p rotocol
b a sed on new fin d in g s o r publications, ad verse events o r m ortalities.
2. H ave th ere been any changes in Principal or C o-Principal Investigators?
I |Y es
[XjNo
3. H ave th ere been any new findings o r publications relative to this research? | |Y es
(XjNo
D escribe the so u rces used to determ ine the availability o f new findings or publications:
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[ ICm rcnl Research Inform ation Service (CRIS)
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A re there any adverse ev en ts, in term s o f anim al well being, or m ortalities to report as a result o f this
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Q V es
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C um ulative n u m b er o f m ortalities:
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/
N um ber o f anim als used d uring
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Cum ulative num ber of
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U pon reviet*' flff tKe r e l i a n t inform ation regarding this protocol, the IACUC approval for this project has
been exte/m efl riyronefyear fro m the date o f this signature.
/I I
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IA C U C C h air Signature
Dale
R e v ise d 10/04
W M U IA C U C
All o th e r c o p ie s ob so lete.
150
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W e s t e r n . M i c h i g a n ..Un i v e r s i t y ,
In s titu tio n a l Animal C are an d Use C om m ittee
0 0 ) 200) C e l e 'o r jt ,o n
Date
June 15,2004
To:
Christine Byrd, Principal Investjgatoi
From: Robert Eversole, Chair
Re:
IACUC Protocol No. 04-06-01
Your protocol entitled “Physical and Chemical Deafferentiation o f the Olfactory Bulb o f
Adult Zebrafish” has received approval from the Institutional Animal Care and Use
Committee. The conditions and duration o f this approval are specified in the Policies o f
Western Michigan University. You may now begin to implement the research as
described in the application.
The Board wishes you success in the pursuit o f your research goals.
Approval Termination:
June 15, 2005
W afw o od H a ll, K a la m a z o o Ml 49008-5456
phone- ( 616 ) 387-8293
fax: (616 ) 387-8276
151
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WESTERN MICHIGAN UNIVERSITY
Institutional Animal Care and Use Committee
A N N U A L R E V I E W O F V E R T E B R A T E A N IM A L U S E
p r o j e c t o r c o u r s e t i t l e : Physical And Chemical Deafferentation Of The Olfactory
Bulb Of Adult Zebrafish
IA C U C Protocol N um ber: 04-06-01
D ate o f R eview R equest: 05/03/05
Date o f Last A pproval: 06/15/04
Purpose o f project (select one): I {Teaching
^ R ese arch
Q O t h e r (specify):
P R IN C IP A L IN V E ST IG A T O R O R A D V ISO R
N a m e : Christine Byrd
D e p a rtm e n t:
BIOS
T itle :
E le c tr o n ic M a i! A d d r e s s :
A ssoc/A ssist. P rofessor
christine.byrd@ wmidi.edu
C O -P R IN C IP A L O R S T U D E N T IN V E ST IG A T O R
/
,
Nam e: Cynthia Fuller
T itle: Student / (rzwt S vx
D epartm ent: BIOS
E lectronic M ail A ddress: [email protected]
/
£>K)S
,
1.
A- ,,
....
hlle
.\
T he research, as approved by th e IA C U C , is com pleted:
'
I |Y es (C ontinue w ith item s 4-5 below .)
[X jN o (C ontinue w ith item s 2-5 below .)
I f the a n sw er to a n y o f the fo llo w in g questions (item s 2-4) is " Y e s ,” please p ro vid e a d eta iled
explanation on an a tta c h e d sh eet o f paper. Include d e tails o f a n y m odifications m ade to the p rotocol
b a sed on new fin d in g s o r p u b lica tio n s, adverse events o r m ortalities.
2. H ave there been any changes in Principal or C o-Principal Investigators?
CZlYes
{XjNo
3. Have there been any new findings o r publications relative to this research? | }Yes
(XjNo
D escribe the sources used to determ ine the availability o f new findings o r publications:
Q N o search conducted (P lease provide a ju stification on an attached sheet.)
I {Animal W elfare Inform ation C en ter (A W IC )
I {Search o f literature d atabases (select all applicable)
□ A G R IC O L A
Q C u rre n t R esearch Inform ation Service (C R IS)
C jB io lo g ic al A bstracts
IXlM edline
I lO ther (p lease specify):
D ate o f search: <05 j o i ( 0 S
Years covered by the search: 190c
K ey w ords.
f
{ ^ A d d itio n a l search strategy narrative:
4. A re there any adverse events, in term s o f anim al well being, or m ortalities to report as a result o f this
research?
C ]Y e s
[XjNo
C u m u lativ e num ber o f m o rtalities: 0
5.
A nim al usage
N um ber o f anim als used during
th is q u arter (T m o n th s): 30
Principal Investigator
C um ulative num ber of
anim als used to date: 100
A dvisor Signature
S 'A ru A b 'tA 'A -
C o-Principal o r §jo d en t Investigator S ignature
IA C U C R E V IE W A N j^ A P P R O V A L
U pon re v ie y o y lty t relevant inform ation regarding this protocol^the IA C U C approval for this project has
been e x te ^ f e ^ f ^ f o tr f y ear from the d a te o f this signature. ,— /
f
> ( s (a
IA C U C C h air Signature
Revised 10/01
Date
W M U IACUC
A ll o th e r c o p ie s o b s o le te
152
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