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The Ultrastructure of the Olfactory System in Two Species of Short-Tailed Shrews, Blarina brevicauda and Blarina carolinensis
Lisa Johnson Byrum Old Dominion University
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Recommended Citation Byrum, Lisa J.. "The Ultrastructure of the Olfactory System in Two Species of Short-Tailed Shrews, Blarina brevicauda and Blarina carolinensis" (2004). Doctor of Philosophy (PhD), Dissertation, , Old Dominion University, DOI: 10.25777/5kav-3e84 https://digitalcommons.odu.edu/biomedicalsciences_etds/12
This Dissertation is brought to you for free and open access by the College of Sciences at ODU Digital Commons. It has been accepted for inclusion in Theses and Dissertations in Biomedical Sciences by an authorized administrator of ODU Digital Commons. For more information, please contact [email protected]. THE ULTRASTRUCTURE OF THE OLFACTORY SYSTEM IN
TWO SPECIES OF SHORT-TAILED SHREWS, BLARINA
BREVICAUDA AND BLARINA CAROLINENSIS
by
Lisa Johnson Byrum B.S. December 1986, Old Dominion University M.S. May 1995, Old Dominion University
A Dissertation Submitted to the Faculty of Old Dominion University in Partial Fulfillment of the Requirement for the Degree of
DOCTOR OF PHILOSOPHY
BIOMEDICAL SCIENCES
OLD DOMINION UNIVERSITY August 2004
Approved by:
Keith Carson (Director)
Alan Savitzky (f^Cmber)
Charles Morgan (Memb
David Scott (Member)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT
THE ULTRASTRUCTURE OF THE OLFACTORY SYSTEM IN TWO SPECIES OF SHORT-TAILED SHREWS, BLARINA BREVICAUDA AND BLARINA CAROLINENSIS
Lisa Johnson Byrum Old Dominion University, 2004 Director: Dr. Keith Carson
Several studies of the fine structure of the olfactory system of rodents have been
conducted, but very little research has been done on members of the Insectivora. The
olfactory systems of the northern short-tailed shrew,Blarina brevicauda, and the
southern short-tailed shrew, Blarina carolinensis, were examined by light and electron
microscopy. These shrews were live trapped in the vicinity of Norfolk, Virginia
throughout all months of the year. Olfactory tissues were processed following standard
transmission and scanning electron microscopy protocols. The olfactory system
structures investigated included the olfactory epithelium/mucosa (OEM), main olfactory
bulb (MOB), accessory olfactory bulb (AOB), anterior olfactory nucleus (AON),
olfactory tubercle (OT), and piriform cortex (PC). A new type of supporting cell, the
light supporting cell, was observed in the OEM in Blarina. The MOB and AOB were
laminated structures that were similar to other macrosmatic mammals. The AON was
divided into typical mammalian subdivisions but the AON appeared at a significantly
more rostral level within the MOB compared to other mammals. The AON was
displaced in a dorsolateral direction in shrews. The trilaminar OT and PC had smaller
cells compared to other small mammals. Light and electron microscopic data show that
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the olfactory system inBlarina differed from other small macrosmatic mammals and was
well-developed.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This dissertation is dedicated to my encourager, my comforter, and my dearest friend.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS
I am appreciative for the counsel and direction from my dissertation committee
members, Drs. Alan Savitzky, Charles Morgan, and David Scott whose wisdom was
indispensable in organizing and conducting this research project. My major professor,
Dr. Keith Carson, generously shared his expertise and knowledge throughout this
program. His persistent leadership and instruction were crucial to the successful
completion of this project and I am deeply grateful to him. Mike Adam and Denise Sliter
enthusiastically shared their knowledge of electron microscopy with me, offering many
hours of training and coaching.
I am thankful for the support from my family and friends. From my mother I
learned of the value of education; my father illustrated humbleness that must accompany
an education. I have been blessed with honorable friends whose camaraderie continually
elevated my spirits. Their confidence in me has been an essential factor in my
achievements.
And finally, I must acknowledge my husband, Frank. Helen Keller is attributed
with the words “One can never consent to creep when one feels an impulse to soar.”
Because of Frank, I will never be the same again, for I have learned to soar. He has been
an infinite source of encouragement, inspiration, hope, and optimism for me. Without
him, I scarcely could creep. His faith in me has been and continues to be a priceless
treasure. Frank remains my strongest ally, my loyal advocate, and my steadfast friend.
The years of his commitment and the source of my strength have been my solid anchor.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS
Page
LIST OF TABLES...... ix
LIST OF FIGURES...... x
CHAPTER
I. INTRODUCTION...... 1 1.1 The Order Insectivora...... 1 1.2 The Significant of Sensory Systems...... 2 1.3 The Importance of Olfaction ...... 3 1.4 The Statement of the Problem ...... 4 1.5 Olfactory Epithelium and Mucosa (OEM)...... 4 1.6 Main Olfactory Bulb (MOB)...... 6 1.7 Accessory Olfactory Bulb (AOB)...... 7 1.8 Anterior Olfactory Nucleus (AON)...... 9 1.9 Olfactory Tubercle (OT)...... 9 1.10 Piriform Cortex (PC)...... 11
II. METHODS...... 13 2.1 Introduction...... 13 2.2 Light Microscopy of Epoxy-Embedded Tissues...... 13 2.3 Transmission Electron Microscopy of Epoxy-Embedded Tissues... 14 2.4 Scanning Electron Microscopy of Olfactory Epithelium Tissues.... 15 2.5 Light Microscopic Histochemistry of Frozen Sections...... 15 2.6 Light Microscopic Histochemistry of Paraffin-Embedded Tissues . 16 2.7 Table of Abbreviations...... 16
III. OLFACTORY EPITHELIUM AND MUCOSA...... 18 3.1 Introduction ...... 18 3.2 Results ...... 18 3.3 Discussion ...... 30 3.4 Comparative Studies ...... 34 3.5 Conclusion...... 34
IV. MAIN OLFACTORY BULB ...... 35 4.1 Introduction ...... 35 4.2 Results ...... 36 4.3 Discussion ...... 63 4.4 Comparative Studies ...... 75 4.5 Conclusion...... 77
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. V. ACCESSORY OLFACTORY BULB...... 78 5.1 Introduction...... 78 5.2 Results...... 79 5.3 Discussion...... 92 5.4 Comparative Studies...... 101 5.5 Conclusion...... 102
VI. ANTERIOR OLFACTORY NUCLEUS...... 104 6.1 Introduction...... 104 6.2 Results...... 106 6.3 Discussion...... 123 6.4 Comparative Studies...... 128 6.5 Conclusion...... 129
VII. OLFACTORY TUBERCLE...... 131 7.1 Introduction ...... 131 7.2 Results...... 132 7.3 Discussion ...... 138 7.4 Comparative Studies ...... 141 7.5 Conclusion...... 142
VIII. PIRIFORM CORTEX...... 143 8.1 Introduction...... 143 8.2 Results...... 144 8.3 Discussion...... 149 8.4 Comparative Studies...... 153 8.5 Conclusion...... 154
IX. CONCLUSION...... 155 9.1 Hypothesis...... 155 9.2 Conclusions of Olfactory System Structures...... 155 9.3 Summary of Ultrastructure of the Olfactory System Structures 159
X. LITERATURE CITED...... 160
VITA...... 189
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES
Table Page
1. Abbreviations...... 17
2. Glomerular diameter range ...... 65
3. Synaptic patterns of the glomerular layer...... 66
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LIST OF FIGURES
Figure Page
1. Light micrograph of the olfactory epithelium...... 20
2. Light micrograph of the five cell types of the olfactory epithelium...... 20
3. Light micrograph of the axon bundle of the lamina propria ...... 23
4. TEM micrograph of the receptor cell of the olfactory epithelium...... 23
5. TEM micrograph of the receptor cell dendrite...... 25
6. TEM micrograph of the cilium of the receptor cell...... 25
7. SEM micrograph of the dense carpet of cilia ...... 26
8. TEM micrograph of the microtubule arrangement...... 26
9. TEM micrograph of two types of supporting cells...... 28
10. TEM micrograph of two types of basal cells...... 28
11. TEM micrograph of the axon bundle...... 29
12. TEM micrograph of the Bowman’s glands ...... 29
13. Line drawing of the lateral view of the cortex and cerebellum...... 37
14. Light micrograph of the sagittal plane of the main olfactory bulb...... 38
15. Light micrograph of the rostral main olfactory bulb...... 39
16. Light micrograph of the mid-caudal main olfactory bulb...... 39
17. Light micrograph of the caudal main olfactory bulb...... 40
18. Light micrograph of the main olfactory bulb layers...... 40
19. Light micrograph of the olfactory nerve layer...... 42
20. Light micrograph of the glomerular layer...... 42
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21. Light micrograph of the external tufted cell...... 44
22. Light micrograph of the external plexiform layer...... 44
23. Light micrograph of the mitral cell layer...... 46
24. Light micrograph of the granule cell layer...... 46
25. TEM micrograph of dark ensheathing cells ...... 47
26. TEM micrograph of a light ensheathing cell...... 47
27. TEM micrograph of periglomerular cells ...... 49
28. TEM micrograph of the short-axon cell...... 50
29. TEM micrograph of the external tufted cell...... 51
30. TEM micrograph of axons and dendrites in the glomeruli ...... 52
31. TEM micrograph of a dendro-dendritic synapse ...... 54
32. TEM micrograph of a reciprocal synapse ...... 54
33. TEM micrograph of a axo-dendritic synapse ...... 55
34. TEM micrograph of a dendro-dendritic synapse ...... 55
35. TEM micrograph of dendro-dendritic synapses ...... 56
36. TEM micrograph of pleomorphic vesicles...... 56
37. TEM micrograph of a myelinated neuron ...... 57
38. TEM micrograph of the thin myelin sheath...... 58
39. TEM micrograph of a myelinated dendrite ...... 58
40. TEM micrograph of a mitral cell ...... 60
41. TEM micrograph of myelinated axons...... 61
42. TEM micrograph of the neuropil of the granule cell layer...... 61
43. TEM micrograph of dark and light granule cells ...... 62
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 44. Light micrograph of the rostral accessory olfactory bulb...... 80
45. Light micrograph of caudal accessory olfactory bulb...... 80
46. Light micrograph of the laminar organization of the accessory olfactory bulb...... 81
47. Light micrograph of an overview of the accessory olfactory bulb...... 83
48. Light micrograph of the vomeronasal nerve layer...... 83
49. Light micrograph of the glomerular layer...... 84
50. Light micrograph of the external plexiform/mitral-tufted cell layer...... 84
51. Light micrograph of the granule cell layer...... 86
52. TEM micrograph of vomeronasal nerve layer...... 86
53. TEM micrograph of ensheathing cells of the vomeronasal nerve layer...... 87
54. TEM micrograph of a glomerulus...... 89
55. TEM micrograph of a axo-dendritic synapse...... 89
56. TEM micrograph of a dendro-dendritic synapse ...... 90
57. TEM micrograph of a dendro-dendritic synapse ...... 90
58. TEM micrograph of a periglomerular cell...... 91
59. TEM micrograph of mitral/tufted cells...... 91
60. TEM micrograph of pleomorphic vesicles...... 93
61. TEM micrograph of spherical vesicles...... 93
62. TEM micrograph of the internal plexiform layer...... 94
63. TEM micrograph of the granule cell layer...... 94
64. Light micrograph of the rostral pars lateralis of the anterior olfactory nucleus 108
65. Light micrograph of the rostral pars externa of the anterior olfactory nucleus...... 108
66. Light micrograph of the mid-caudal pars extema...... 109
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67. Light micrograph of the rostral pars dorsalis and pars ventralis...... 109
68. Light micrograph of the rostral pars medialis...... 110
69. Light micrograph of the mid-caudal pars medialis ...... 110
70. Light micrograph of the caudal pars medialis ...... 112
71. Light micrograph of the pars posterior ...... 112
72. Light micrograph of the olfactory ventricle and subventricular zone...... 113
73. Light micrograph of ependymal cells ...... 113
74. Light micrograph of pyramidal cells of the pars lateralis ...... 115
75. Light micrograph of the dorsal limb of the pars externa...... 115
76. Light micrograph of the ventral limb of the pars externa...... 116
77. Light micrograph of pyramidal cells of the pars dorsalis ...... 116
78. Light micrograph of pyramidal cells of the pars medialis...... 118
79. Light micrograph of pyramidal cells of the pars ventralis...... 118
80. Light micrograph of pyramidal cells of the pars posterior...... 119
81. TEM micrograph of pars lateralis pyramidal cell with apical dendrite ...... 119
82. TEM micrograph of synapses in the pars lateralis neuropil...... 121
83. TEM micrograph of pars externa pyramidal cell...... 121
84. TEM micrograph of synapses in the pars externa neuropil...... 122
85. TEM micrograph of pars dorsalis pyramidal cell...... 122
86. Light micrograph of a sagittal plane of the olfactory tubercle...... 133
87. Light micrograph of the rostral olfactory tubercle...... 134
88. Light micrograph of the mid-caudal olfactory tubercle...... 134
89. Light micrograph of the caudal olfactory tubercle...... 135
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90. Light micrograph of the trilaminar olfactory tubercle...... 135
91. Light micrograph of the plexiform layer of the olfactory tubercle...... 137
92. Light micrograph of the pyramidal cell layer of the olfactory tubercle...... 137
93. Light micrograph of the polymorph cell layer of the olfactory tubercle...... 139
94. Light micrograph of granule cells of the Islands of Calleja ...... 139
95. Light micrograph of the rostral piriform cortex...... 145
96. Light micrograph of the mid-caudal piriform cortex...... 145
97. Light micrograph of the caudal piriform cortex...... 146
98. Light micrograph of the trilaminar piriform cortex...... 146
99. Light micrograph of Layer I of the piriform cortex...... 148
100. Light micrograph of Layer II pyramidal cells of the piriform cortex...... 148
101. Light micrograph of pyramidal cell dendrites ...... 150
102. Light micrograph of multipolar neurons of the piriform cortex...... 150
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CHAPTER I
INTRODUCTION
1.1 The Order Insectivora
Shrews make up greater than 70% of the Order Insectivora and are described as having
considerable appetites with a correspondingly high metabolism. As predators, these
small mammals feed mainly upon invertebrates. Shrews share the Order Insectivora with
moles, hedgehogs, and tenrecs. The family Soricidae (shrews) is divided into two
subfamilies: Soricinae and Crocidurinae (Catania et al., 1999). Shrews of the subfamily
Soricinae have a reddish-brown coloration of the teeth due to iron deposits in the enamel
and are known as the red-toothed shrews (Dotsch and Koenigswald, 1978, Larochelle and
Baron, 1989b). Shrews (Soricidae) range from 2.5 grams to over 200 grams in body
weight. Shrews are located on all major landmasses with the exception of Australia, New
Zealand, Antarctica, Greenland, Iceland, and Tasmania. Generally, shrews are terrestrial
mammals occupying woodlands, forests, and grasslands although several species of
aquatic forms thrive in marshy and aquatic habitats (Churchfield, 1990; Nowak, 1999).
As terrestrial insectivores, shrews have numerous morphological specializations that
allow these small mammals to survive in different habitats. The cylindrical body, tail
length, small eyes, and short pelage are examples of various specializations (Stephan et
al., 1991; Baron and Stephan, 1996). Shrews remain active throughout all seasons of the
year. Shrews are among the most primitive placental mammals, first evolving in the late
Eocene/early Oligocene period, approximately 38 million years ago, some time after the
The model journal for this document is the European Journal of Morphology.
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dinosaurs disappeared. Shrews have changed little since their first appearance among the
dinosaurs. Present-day Insectivora have retained many features of the earliest mammals
(Churchfield, 1990). Therefore, the study of their brains may yield significant insight
into the process of mammalian brain evolution.
1.2 The Significance of Sensory Systems
The visual system of the Insectivora is generally poorly developed compared to most
other mammals. Shrews have the smallest eyes of the terrestrial Insectivora, measuring
between 0.7 - 1.5 mm in diameter (Churchfield, 1990; Baron and Stephan, 1996; Stephan
et al., 1991). It appears that vision functions merely to discriminate light intensity
(Merriam, 1884; Branis, 1981). In agreement with previous researchers, Ryzen and
Campbell (1955) reported that shrew visual cortex was thin and poorly developed. The
small size of the optic nerve, the minute lateral geniculate bodies, and the poorly-
developed fiber network are indicative of a greatly reduced visual system. Due to this
reduction in the visual system in the Insectivora, the olfactory sense may be considered to
be a sensory system of compensation (Sigmund and Sedlacek, 1985; Stephan et al., 1991;
Baron and Stephan, 1996).
The auditory sense is of greater importance to shrews than the visual system. As
vocal mammals, shrews perceive and emit a wide range of sounds. The size of the
external ear varies depending upon species and habitat, ranging from the most prominent
ears in ground dwelling shrews to extremely minute structures. In semi-fossorial and
semi-aquatic species of shrews, the external ear is completely lacking with only a narrow
opening covered with a thin fold of skin (Churchfield, 1990). The importance of audition
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to the shrew has not yet been demonstrated, but the brain stem and cortical regions
involved with audition are not well-developed.
Additional sensory information is received via the trigeminal complex which
relays information from the facial vibrissae to higher brain centers (Baron et al., 1990).
The trigeminal complex size index varies significantly among the Insectivora (Baron et
al., 1990). For example, inBlarina, the trigeminal complex has a size index of 140. In
general, the terrestrial Insectivora have an average trigeminal complex size index of 134.
In the Insectivora, the best-developed trigeminal complex with highly-developed
vibrissae is found in the semi-aquatic forms. The average trigeminal complex size index
in the semi-aquatic Insectivora is 283. Variations in the development of the trigeminal
complex and vibrissae may account for olfactory acuity differences in the Insectivora.
1.3 The Importance of Olfaction
The term “macrosmatic” describes a species with a well-developed, well-organized
olfactory system with high sensitivity for odorants. Macrosmatic mammals rely upon
their olfactory system for various behaviors that is essential to their survival. The term
“microsmatic” is typically used to describe a species with a poorly-developed olfactory
system. The term “anosmatic” relates to the impairment or loss of the sense of smell.
Primates are considered to be microsmatic mammals due to the size reduction in
olfactory brain areas and reduced olfactory acuity and olfactory importance (Loo, 1977;
Turner et al., 1978; Laska et al., 2000; Barbado et al., 2001). For the macrosmatic shrew,
the olfactory system typically is highly-developed (Negus, 1958; Wohrmann-
Repenning, 1975; Schmidt and Nadolski, 1979; Sollner and Kraft, 1980; Churchfield,
1990; Stephan et al., 1991) and is thought to be essential for numerous activities critical
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. for survival. These activities include reproductive and social behavior, recognition of
prey and/or predators, aggressive behavior, detection of food, maternal-infant
relationships, and habitat selection (Le Gros Clark, 1932; Allison, 1953b; Johnson, 1957;
Negus, 1958; Wohrmann-Repenning, 1975; Schmidt and Nadolski, 1979; Hutterer, 1985;
Larochelle and Baron, 1986, 1989a, b; Churchfield, 1990; Stephan et al., 1991; Farbman,
1992; Shipley et al., 1995; Shipley and Smith, 1995; Nieuwenhuys et al., 1998).
1.4 Statement of the Problem
Two Soricidae shrews, Blarina brevicauda and B. carolinensis were of particular interest
for this research since both have very small eyes and cochleas and correspondingly small
brain areas involved in visual and auditory sensation (Gould, 1969; Burda, 1979; Branis,
1981; Stephan et al., 1991). However, the olfactory system of shrews appears not only to
be highly developed, but also a system that plays an important part of their sensory
repertoire (Stephan et al., 1991). The present investigation was designed to provide a
detailed ultrastructural analysis of the olfactory system of the two closely related soricid
shrews in order to test the hypothesis that their olfactory systems are well-developed and
differ significantly from those of better known small macrosmatic mammals. The
olfactory system components investigated by light and electron microscopy for this
research included the olfactory epithelium and mucosa, main and accessory olfactory
bulbs, anterior olfactory nucleus, olfactory tubercle, and piriform cortex.
1.5 Olfactory Epithelium and Mucosa (OEM)
The olfactory mucosa consists of the epithelium, a basal lamina, and the underlying
lamina propria. Within the lamina propria, Bowman’s glands, axon bundles, connective
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. tissue, and vasculature are common (Graziadei, 1971; Wohrmann-Repenning and Meinel,
1975; Morrison and Costanzo, 1989,1992; Naguro and Iwashita, 1992; Mendoza, 1993).
The olfactory epithelium is a pseudostratified epithelium consisting of receptor cells,
supporting cells, and basal cells. The receptor cells and supporting cells are produced by
mitosis and differentiation of basal cells throughout life (Graziadei and Graziadei, 1978,
1979; Barber and Raisman, 1979; Doucette et al., 1983; Morrison and Costanzo, 1989;
Farbman and Buchholz, 1992; Oakley and Riddle, 1992; Hirai et al., 1996; Matsuoka et
al., 2002). Receptor cell nuclei are typically located in the lower two-thirds of the
epithelium. Supporting cells are columnar with microvilli on the apical surface with their
nuclei located in the upper one-third of the epithelium (Morrison and Costanzo, 1989).
Basal cells are elongated cells that lie adjacent to the basement membrane (Allison,
1953b; Arstila and Wersall, 1967; Graziadei, 1971). Odor molecules stimulate first-order
neurons, the olfactory bipolar receptor nerves in the olfactory epithelium within the nasal
cavity. The axons of olfactory receptor nerves pass through the lamina propria and
project via the olfactory nerve bundles to the main olfactory bulb which, in turn, projects
to higher olfactory structures in the cerebral hemisphere (Graziadei, 1966, 1971, 1973;
Anholt, 1987; Shipley et al., 1995).
Studies of the mammalian OEM by light and electron microscopy include Arstila
and Wersall (1967), Frisch (1967,1969), Seifert (1970), Graziadei (1966, 1971, 1973),
Adams (1972), Altner and Kolnberger (1975), Cuschieri and Bannister (1976),
Yamamoto (1976), Dodd and Squirrell (1980), Breipohl and Ohyama (1981), and Menco
(1980, 1983). However, few studies have examined the olfactory mucosa of members of
the Order Insectivora and only four of these were ultrastructural studies including
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Graziadei (1966), Meinel and Erhardt (1978), Erhardt and Meinel (1979), and Carson et
al., (1994).
1.6 Main Olfactory Bulb (MOB)
The MOB has been reported to be a phylogenetically old component of the cerebral
cortex (Kosaka and Kosaka, 1999; Gil-Carcedo et al., 2000). The MOB is attached to the
rostral end of the telencephalon by the olfactory peduncle. As a rostral expansion of the
forebrain, the MOB is the site of the first synaptic interactions of the epithelial receptor
cells, contains the second order neurons of the olfactory pathway, and functions as a relay
station for olfactory signals between the olfactory mucosa and higher olfactory centers.
Primary projections from the MOB terminate in the anterior olfactory nucleus, piriform
cortex, and the olfactory tubercle. Projections to the MOB originate in the anterior
olfactory nucleus, piriform cortex, nucleus of the lateral olfactory tract, and the
hypothalamus (Smith, 1896; Young, 1934; Davis et al., 1978; Baron et al., 1983; Carson,
1984; Greer, 1991; Bassett et al., 1992; Eisthen, 1997; Nieuwenhuys et al., 1998; Gil-
Carcedo et al., 2000). The MOB consists of six concentric layers, starting with the outer
most include the olfactory nerve layer, glomerular layer, external plexiform layer, mitral
cell layer, internal plexiform layer, and granule cell layer. There is a general decrease in
size of the MOB from the Insectivora to higher primates with man having a very small
MOB. In some mammals, such as cetaceans, the MOB is absent. In general, a positive
relationship exists between MOB size and the importance of olfaction to a species
(Johnston, 1909; Allison, 1953b; Baron et al., 1983; Farbman, 1992; Shipley et al., 1995).
The majority of MOB research has focused on light/immunohistochemical
microscopic studies (Forbes, 1984; Mouradian and Scott, 1988; Sanides-Kohlrausch and
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Wahle, 1990; Bassett et al., 1992; Crespo et al., 1995; Kasowski et al., 1999; Treloar et
al., 1999; Brinon et al., 2001; Huang and Bittman, 2002). Relatively few studies have
examined the ultrastructure of the MOB (Klinkerfuss, 1964; Andres, 1970; Price and
Powell, 1970c; Switzer and Johnson, 1977; Jackowski et al., 1978; Gomez and Potts,
1981; Hinds and McNelly, 1981; Sturrock, 1981; Doucette, 1984; Lopez-Mascaraque et
al., 1986; Moriizumi et al., 1995; Chuah et al., 1997). Little research has been done on
the MOB of the Insectivora.
1.7 Accessory Olfactory Bulb (AOB)
The main olfactory system and the vomeronasal olfactory system (accessory olfactory
system) are reported to be separate pathways from their point of origin in the nasal
mucosa to their termination site. Therefore, it has been hypothesized that two separate
olfactory systems exist (Scalia and Winans, 1975; Davis et al., 1978; Takami and
Graziadei, 1990; Shipley and Smith, 1995). The main olfactory system plays a major
role in food location and prey detection whereas the vomeronasal olfactory system has
been proposed to function in mammals in reproductive behaviors, mediating pheromonal
communication, pregnancy, puberty, ovulation, and aggression (Coquelin et al., 1984;
Imamura et al., 1985; Wysocki and Meredith, 1991; Takami et al., 1992; Doving and
Trotier, 1998; Jansen et al., 1998; Dudley and Moss, 1999; Wekesa and Anholt, 1999;
Johnston and Peng, 2000; Brennan, 2001; Korsching, 2001; Sahara et al., 2001; Kondo et
al., 2003).
The projection pathways for the AOB are significantly more simple than the
pathways for the MOB. Axons of the AOB mitral/tufted cells form the accessory
olfactory tract which runs with the lateral olfactory tract and projects to the amygdala.
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Other AOB projection sites within the cortex include the bed nucleus of the accessory
olfactory tract, the bed nucleus of the stria terminalis, the supraoptic nucleus, and the
ventromedial hypothalamic nucleus (Scalia and Winans, 1975, 1976; Barber et al., 1978;
Barber and Raisman, 1978; De Olmos et al., 1978; Johns, 1980; Lehman and Winans,
1982; Segovia et al., 1982; Raisman, 1985; Saito and Moltz, 1986; Brennan et al., 1990;
Price, 1991; Wysocki and Meredith, 1991; Shipley et al, 1995; Matsuoka et al., 1997;
Meisami and Bhatnagar, 1998; Dudley and Moss, 1999; Jia et al., 1999; Keveme, 1999;
Sugai et al., 1999; Goldmakher and Moss, 2000; Breer, 2001; Salazar and Brennan, 2001;
Cloutier et al., 2002; Kondo et al., 2003). Centrifugal fibers that originate in the
amygdala project to the AOB (Shipley et al., 1995; Meisami and Bhatnagar, 1998). The
AOB is laminated similar to the MOB but the layers are less distinct and less developed.
Few light and electron microscopy studies have focused on the vomeronasal
organ (Kratzing, 1971; Barber and Raisman, 1979; Breipohl et al., 1981; Vaccarezza et
al., 1981; Fraher, 1982; Adams and Wiekamp, 1984; Mendoza, 1986; Mendoza and
Kuhnel, 1987; Matsuzaki et al., 1993; Doving and Trotier, 1998; Poran, 1998; Smith et
al., 1998; Zuri et al., 1998). Several histochemical and immunohistochemical studies
have focused on the AOB (Carson and Burd, 1980; Salazar et al., 1998; Salazar and
Quinteiro, 1998; Dudley and Moss, 1999; Nakamura et al., 1999; Wekesa and Anholt,
1999; Takigami et al., 2000; Sahara et al., 2001; Salazar and Brennan, 2001; Salazar et
al., 2001; Kondo et al., 2003) whereas very few ultrastructural studies have examined the
AOB (Matsuoka et al., 1997, 1998).
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1.8 Anterior Olfactory Nucleus (AON)
The olfactory peduncle links the MOB to the forebrain and consists of two components,
the anterior olfactory nucleus (AON) and various transition areas between the AON and
the olfactory cortex (Bayer, 1986; Garcia-Ojeda et al., 1998). Herrick (1924) first
introduced the term “nucleus olfactorius anterior” to describe the gray matter just caudal
to the MOB. According to Herrick, the organization of the AON shows significant
variation in different mammal species. Due to the heavy reciprocal connections between
the AON and the MOB, the AON has been proposed to function as a relay for
interhemispheric communication between the two MOBs via the anterior limb of the
anterior commissure (Bennett, 1968; Broadwell, 1975b; Brunjes and Frazier, 1986;
Bassett et al., 1992; Haberly, 2001). Most researchers now agree that the AON is
actually a cortical structure (Shipley et al., 1995). The divisions of the AON are
laminated and consist of an outer plexiform layer and a homogeneous inner layer of AON
pyramidal cells (Davis and Macrides, 1981b; Shipley and Ennis, 1996). This bilayer
form of cortex has been recognized in all mammals examined, including the opossum
(Shammah-Lagnado and Negrao, 1981), hedgehog (Valverde et al., 1989), rabbit
(Broadwell, 1975b), rat (Haberly and Price, 1978b, Bayer, 1986; Garcia-Ojeda et al.,
1998), mouse (Crosby and Humphrey, 1939a), and cat (Ryu, 1980). The mammalian
AON generally is subdivided into six subdivisions including the pars lateralis, ventralis,
medialis, dorsalis, posterior, and extema (Davis and Macrides, 1981b).
1.9 Olfactory Tubercle (OT)
The OT is a trilaminar cortical structure consisting of plexiform, pyramidal, and
polymorph cell layers (Talbot et al., 1988a; Krieger, 1981; Krieger and Scott, 1989). The
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general size of the OT is greatly influenced by the size of other olfactory structures, such
as the MOB (Stephan et al., 1991). In macrosmatic mammals, the OT is well-developed
(Johnson, 1957a; Millhouse and Heimer, 1984; Berezhnaya et al., 1999) whereas in
microsmatic mammals such as humans (Crosby and Humphrey, 1941), the OT is poorly-
developed. The OT receives projections from the MOB via the lateral olfactory tract
(Davis and Macrides, 1981; Meyer, 1981; Krieger, 1981; Fallon, 1983; Bassett et al.,
1992). The OT differs from the AON and the piriform cortex because the OT does not
send reciprocal projections back to the MOB (Shipley et al., 1995). The OT also receives
projections from other regions of the cortex including the PC, AON, and structures within
the limbic system including the hippocampus and amygdala. The OT sends projections
to the globus pallidus, substantia nigra, and thalamus. Due to the extensive projections, it
has been proposed that the OT is not only a part of the olfactory system, but also is a part
of the limbic system (Herrick, 1921; Fallon et al., 1978; Haberly and Price, 1978b; Davis
and Macrides, 1981a; Krieger, 1981; Meyer 1981; Guevara-Aguilar et al., 1982; Luskin
and Price, 1983b; Meyer and Wahle, 1986; Stephan et al., 1991; Kratskin, 1995).
The majority of OT research has focused on histochemical analysis of enzyme
concentrations (Okada et al., 1977; Gilad and Reis, 1979; Gordon and Krieger, 1983;
Talbot et al., 1988b; Krieger and Scott, 1989) or various neurotransmitters (Krieger et al.,
1983). Several studies have examined the morphology of OT by light microscopy
(Fallon et al., 1978; Guevara-Aguilar et al., 1982; Ribak and Fallon, 1982; Fallon, 1983;
Gordon and Krieger, 1983; Krieger et al., 1983; Talbot et al., 1988a; Krieger and Scott,
1989; Berezhnaya et al., 1999) and have focused typically on Golgi-stained sections and
have concluded that the OT of macrosmatic mammals is typically large and well-
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(Krieger, 1981; Ribak and Fallon, 1982; Krieger et al., 1983; Zahm and Heimer, 1985;
Krieger and Scott, 1989; Josephson et al., 1997; Berezhnaya et al., 1999).
1.10 Piriform Cortex (PC)
The piriform cortex is also called the pyriform or prepyriform cortex and is a
phylogenetically old structure of the paleocortex (Haberly, 1985). The PC is located on
the ventrolateral surface of the brain and is divided into three layers including the outer
most superficial Layer I, middle Layer II, and deep Layer III. Layer I contains axons and
terminals of MOB mitral/tufted cells and dendrites of Layer II and III pyramidal cells.
Layer II is a cell layer of tightly packed pyramidal cells that send apical dendrites toward
Layer I. Layer III contains pyramidal cells and multipolar cells. Dendrites of Layer III
multipolar neurons extend in all directions throughout Layer III whereas dendrites of
Layer III pyramidal cells extend toward Layer I (Davis and Macrides, 1981b; Shipley and
Ennis, 1996; Bartolomei and Greer, 1998; Loscher et al., 1998). The PC pyramidal cells
of Layers II and III project to the ipsilateral MOB (Davis and Macrides, 1981b; Shipley
et al., 1995) and provide excitatory input to MOB granule cells (Stripling and Patneau,
1999; Fukushima et al., 2002). The PC also receives projections from the ipsilateral
MOB (David and Macrides, 1981; Bassett et al., 1992; Shipley et al., 1995; Rosin et al.,
1999).
Few studies have focused on the PC and include Haberly and Lewis (1978a, b);
Davis and Macrides (1981); Meyer (1981); Luskin and Price (1983b); Friedman and
Price (1984); Ojima et al., (1984); Curcio et al., 1985; Haberly, (1985); Anders and
Johnson (1990); Bartolomei and Greer, 1998; Loscher et al., (1998); Rosin et al., (1999);
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Johnson et al., 2000; Protopapas and Bower (2000); Datiche et al., (2001); Haberly
(2001); Fukushima et al., (2002); Best and Wilson (2003). Investigation of the
Insectivora PC (Ryzen and Campbell, 1955; Stephan and Andy, 1982) has been
significantly neglected.
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CHAPTER II
METHODS
2.1 Introduction
Fifteen short-tailed shrews, 9 Blarina carolinensis and 6 B. brevicauda, were captured in
the vicinity of Norfolk, Virginia for these ultrastructural studies. Mature shrews of both
sexes were used. Handling of animals was carried out in accordance with our animal care
and use committee guidelines, which are based on National Institutes of Health
guidelines found in Principles o f Animal Care (NIH publication No. 86-23).
2.2 Light Microscopy of Epoxy-Embedded Tissues
For the ultrastructural studies, shrews were lightly etherized and then injected
intraperitoneally with the primary anesthetic, 0.2 ml of 2.5% tribromoethanol. After
waiting about 2 minutes for the shrews to become deeply anesthetized, the chest was
opened and the vascular system was perfused via a 22-gauge needle through the left
ventricle to flush out the blood and deliver the fixative to all body tissues quickly. The
perfusion bottle was suspended approximately 3 feet above the shrews. The right atrium
was cut with small scissors to provide an outlet for the perfusate. The animals were
perfused for about 2 mins with about 10 ml solution of 0.9% sodium chloride, 1% sodium
nitrite, 5 mg/100 ml heparin, and 0.05 M phosphate buffer at pH 7.4, followed by the
primary fixative consisting of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M
cacodylate buffer pH 7.4 for about 30 mins. During the perfusion of fixative, a syringe
was used to deliver additional fixative into each nasal passage. The OEM pieces were
dissected and left overnight in primary fixative at 4° C. For the MOB, AOB, AON, OT,
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and PC, brains were cut on a vibratome at 200 pm and the specific areas cut from slices.
The tissues were then rinsed in cold 0.1 M cacodylate buffer pH 7.4 three times, 10 mins
each. The tissues were then post-fixed for 2 hrs at 4° C in 2% osmium tetroxide in 0.1 M
cacodylate buffer pH 7.4 and then rinsed with cold 0.1 M cacodylate buffer, three times,
10 mins each. The tissues were dehydrated in 10-minute steps with 30%, 50%, 70%,
95%, and 100% (two times) ethanol followed by 100 % acetone (two times). The tissues
were infiltrated with a 1:1 mixture of epoxy resin (Polybed 812, Polysciences) and
acetone for 4 hrs on a rotating mixer followed by 18 hrs in 100% epoxy resin. The OEM
and MOB were embedded in flat molds and polymerized for 48 hrs at 65° C. The AOB,
AON, OT, and PC were embedded in epoxy resin between two Teflon-coated coverslips
and polymerized for 48 hrs at 65° C. These tissues were cut from the coverslip wafer
using a scalpel and were glued onto epoxy blocks. One-micrometer sections of each type
of olfactory system tissue were cut for light microscopy and stained with methylene
blue/azure II stain of Richardson and coworkers (1960). Four slides of each species were
used to count dark and light supporting cells and Bowman’s gland cells. In each slide,
sample areas visible at 400X were randomly selected and cells were counted to arrive at
an estimation of the relative percentages. Identification of light microscopy structures of
the olfactory tubercle and piriform cortex were based upon Millhouse and Heimer (1984)
and Haberly and Feig (1983) respectively.
2.3 Transmission Electron Microscopy of Epoxy-Embedded Tissues
Silver thin sections of all olfactory system tissues were cut with a diamond knife on a
RMC MT2C ultramicrotome (Research and Manufacturing Co., Tucson, AZ) and placed
on naked copper grids. The sections were stained with uranyl acetate for 20 mins and
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lead citrate for 3 mins. The tissues were viewed and photographed using a JEOL 100CX
II transmission electron microscope (TEM). Identification of the fine structure of the
olfactory mucosa was based upon the work of Frisch (1967) and Yamamoto (1976).
Identification of neurons and processes of the main olfactory bulb was based upon the
work of Pinching and Powell (1971 a, b, c). Identification of neurons and processes of
the accessory olfactory bulb was based upon the work of Barber and coworkers (1978).
Structures of the anterior olfactory nucleus were based upon the work of Valverde and
coworkers (1989) and Crosby and Humphrey (1941).
2.4 Scanning Electron Microscopy of Olfactory Epithelium Tissues
The olfactory epithelium selected for SEM was fixed and dehydrated as previously
described. Following dehydration these samples were critical point dried using liquid
carbon dioxide, mounted on aluminum stubs and then coated with a thin layer of
gold/palladium alloy. The samples were then viewed and photographed in a LEO 435VP
SEM.
2.5 Light Microscopic Histochemistry of Frozen Sections
Shrews used for cresyl violet light microscopy studies were anesthetized as described
previously and then perfused with 4% depolymerized paraformaldehyde in 0.1 M
phosphate buffer, pH 7.4. The heads were removed, skinned and fixed for an additional
48 hrs at 4°C. The brain was removed and soaked in the cryoprotectant, 30% sucrose
buffer for 24 hours to prevent ice crystal formation. The brain was placed onto the
sliding/freezing microtome chuck, frozen with dry ice, and sectioned at 40 pm. Sections
were picked up from the microtome blade with a brush and placed in a tray containing
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phosphate buffer rinse. Sections were then mounted on gelatin-coated slides and dried
overnight. The tissue was treated for 4-8 hours with chloroform, then 100% acetone.
Following acetone, the sections were rinsed in 95% ethanol then 70% ethanol. Sections
were stained with 1% cresyl violet for 20 minutes. The sections were washed for several
minutes in water. The tissue was dehydrated through 70%, 95% and 100% ethanol
followed by chloroform for 5 mins. A differentiator of 95% ethanol with glacial acetic
acid, pH 4.1 was used for 5-7 mins to remove any remaining cresyl violet stain. The
sections were then dehydrated with 95% and 100% ethanol followed by xylene and
coverslipped with Permount.
2.6 Light Microscopic Histochemistry of Paraffin-Embedded Tissues
Shrews used for hematoxylin and eosin light microscopy were anesthetized as described
previously and then perfused with 4% depolymerized paraformaldehyde. The heads were
removed, skinned and fixed for an additional 48 hrs at 4°C. The heads were then
decalcified in a solution of formic acid (25%) and sodium citrate (10%) for 24 hours at
room temperature and then rinsed in running water for 4 hours. The heads were then
dehydrated and embedded in paraffin using standard techniques. The heads were
sectioned in the coronal plane at 10 micrometers and the sections were picked up on
slides. Alternate slides were stained for routine histological study with hematoxylin and
eosin, and for mucopolysaccharides with the periodic acid Schiff-alcian blue stain
(Preece, 1972).
2.7 Table of Abbreviations
Table 1 is a list of abbreviations used in the text of this research project.
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Table 1. Abbreviations of olfactory system structures.
AOB accessory olfactory bulb AON anterior olfactory nucleus EPL external plexiform layer EP/M-TCL external plexiform layer/mitral-tufted cell layei ETC external tufted cell GL glomerular layer GrL granule cell layer IPL internal plexiform layer MCL mitral cell layer MOB main olfactory bulb OEM olfactory epithelium and mucosa ONL olfactory nerve layer OT olfactory tubercle PC piriform cortex PD pars dorsalis of the anterior olfactory nucleus PE pars externa of the anterior olfactory nucleus PGC periglomerular cell PL pars lateralis of the anterior olfactory nucleus PM pars medialis of the anterior olfactory nucleus PP pars posterior of the anterior olfactory nucleus PV pars ventralis of the anterior olfactory nucleus RER rough endoplasmic reticulum SAC short-axon cell SEM scanning electron microscopy SER smooth endoplasmic reticulum TEM transmission electron microscopy VNL vomeronasal nerve layer VNO vomeronasal organ
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CHAPTER III
OLFACTORY EPITHELIUM AND MUCOSA
3.1 Introduction
In mammals, the olfactory mucosa consists of the olfactory epithelium and the underlying
lamina propria. The olfactory epithelium is a pseudostratified epithelium that contains
bipolar receptor cells, supporting cells, and basal cells. The lamina propria contains
vasculature, Bowman’s glands, and axon bundles (Morrison and Costanzo, 1992; Naguro
and Iwashita, 1992; Mendoza, 1993). The olfactory epithelium and mucosa have been
studied in numerous macrosmatic mammal species including a mole (Graziadei, 1966),
shrew (Carson et al., 1994), hedgehog (Erhardt and Meinel, 1979), bat (Yamamoto,
1976), guinea pig (Arstila and Wersall, 1967), rabbit (DeLorenzo, 1957; Zinnin, 1964;
Yamamoto, 1976), mouse (Frisch, 1967, 1969; Cuschieri and Bannister, 1975), hamster
(Morrison and Costanzo, 1989), rat (Hinds and McNelly, 1981; Carr et al., 1991; Naguro
and Iwashita, 1992; Weiler and Farbman, 1997), dog (Okano et al., 1967), and cat
(Graziadei, 1964). The olfactory epithelium and mucosa have also been studied in only a
few microsmatic mammals including the rhesus monkey (Shantha and Nakajima, 1970)
and primates including the loris, macaque, and gibbon (Loo, 1977), and humans (Moran
et al., 1982a, b; Morrison and Costanzo, 1990, 1992).
3.2 Results
3.2.1 Light Microscopy Overview
The olfactory mucosa (Fig. 1) varied in thickness from 35 to over 150 pm and consisted
of the overlying olfactory epithelium, a thin basal lamina, and a lamina propria that
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contained Bowman’s glands, bundles of axons, vasculature and connective tissue. The
olfactory epithelium was a pseudostratified columnar epithelium that contained five to
ten layers of cell nuclei. Five cell types (Fig. 2) were common in the olfactory epithelium
including receptor cells, two types of supporting cells, and two types of basal cells.
3.2.2 Light Microscopy of Receptor Ceils
The receptor cell nuclei (Fig. 2) were located within the lower two-thirds of the olfactory
epithelium. The oval, darkly-stained nuclei were more irregular in shape than the other
epithelial nuclei and typically had a distinct nucleolus. Small patches of heterochromatin
were scattered throughout the receptor cell nucleus. The receptor cell nuclei measured up
to 6 pm in diameter. Receptor cells had a single apical dendrite that was topped by a
bulbous olfactory knob that projected above the apical surfaces of the supporting cells
(Fig. 2).
3.2.3 Light Microscopy of Supporting Cells
Supporting cell nuclei were located within the upper one-third of the olfactory
epithelium. Two populations of supporting cells, including dark supporting cells and
light supporting cells were observed. The dark supporting cells (Fig. 2) had an oval
nucleus with scattered patches of heterochromatin. These cells were columnar in shape
with numerous long microvilli on the apical surface. The nuclei measured up to 6 pm in
diameter. The average number of dark supporting cells per random sample area was
approximately 10. The light supporting cell cytoplasm and nucleus were much more
lightly stained than that of the adjacent dark supporting cells. The light supporting cell
nuclei (Fig. 2) measured up to 6 pm in diameter. These light supporting cells averaged
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Fig. 1. Light micrograph of the olfactory epithelium (EPI). Lamina propria consists of
Bowman’s glands (BG), axon bundles (AX) and vasculature (V). Basal lamina (arrow).
1 pm epoxy resin section, Richardson stain. 5 mm = 4 pm.
Fig. 2. Light micrograph of the five cell types of the olfactory epithelium. Receptor cells
(R) topped by a dendritic knob (arrowhead) that extends above the apical surface of the
supporting cells, dark supporting cells (DS), light supporting cells (LS), dark basal cells
(B), and light globose basal cells (arrow). 1 pm epoxy resin section, Richardson stain.
5 mm = 4 pm.
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only 1 cell per sample area and were less numerous than the dark supporting cells,
making up just 8-10% of the supporting cells.
3.2.4 Light Microscopy of Basal Cells
Basal cells were typically flattened and were located in the lower one-third of the
olfactory epithelium just above the basal lamina (Fig. 2). The dark basal cell had darkly-
stained nuclei and contained small patches of heterochromatin. Dark basal cell nuclei
measured up to 3 pm in diameter along the short axis and 6 pm in diameter along the
long axis. Light globose basal cells (Fig. 2) had euchromatic nuclei with small amounts
of heterochromatin. The light basal cell nuclei measured up to 4 pm in diameter along
the short axis and 5 pm in diameter along the long axis.
3.2.5 Light Microscopy of Lamina Propria Axon Bundles
The structures of the lamina propria included olfactory nerve axon bundles with
ensheathing cells, Bowman’s glands, vasculature, and connective tissue. Olfactory nerve
axon bundles (Fig. 3) were oval or spherical in cross-section and measured up to 30 pm
by 60 pm. These bundles were especially large and numerous in areas where the
olfactory epithelium was thickest. Ensheathing cells (Fig. 3) were often located at the
perimeter of axon bundles and had nuclei that measured up to 4 pm in diameter along the
short axis and 5 pm in diameter along the long axis. These cells typically had a
euchromatic nucleus. Occasionally a distinct nucleolus was evident. Some ensheathing
cells had light nuclei and some had darker nuclei. Large axon bundles were often
separated into fascicles by thin septa made up of ensheathing cell processes.
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3.2.6 Light Microscopy of Lamina Propria Bowman’s Glands
Two types of Bowman’s gland cells could be distinguished in the lamina propria. The
more common of these, the mucous cell (Fig. 3), had moderately basophilic cytoplasm
that often contained pale granules. These cells were grouped in an acinar arrangement
around a small lumen. The pale granules were usually more numerous adjacent to the
lumen. Another moderately basophilic gland cell, the serous cell (Fig. 3), was often
observed in the same acinus with the mucous cells. These cells contained small, darkly-
stained secretory granules that were also aggregated near the lumen. Per sample area,
mucous Bowman’s gland cells outnumbered serous cells in a ratio of about five to one.
The nuclei of the Bowman’s gland cells were oval in shape and measured up to 6 pm in
diameter. In PAS-alcian blue stained sections, there was a distinctive staining of the
Bowman’s gland cells. The mucous cells were alcian blue-positive and the serous cells
were PAS-positive.
3.2.7 Light Microscopy of Lamina Propria Vasculature
The lamina propria was highly vascularized by capillaries (Fig. 3) that measured up to 24
pm in diameter. In the spaces between the vessels, Bowman’s glands, and the olfactory
nerve axon bundles, there was a small amount of connective tissue, mostly consisting of
fibroblasts and collagen fibrils.
3.2.8 Transmission Electron Microscopy of Receptor Cells
Receptor cell (Fig. 4) nuclei were spherical with some electron dense patches of
heterochromatin. The perikarya contained numerous mitochondria densely packed in the
apical cytoplasm, lysosomes, and multivesicular bodies. Dispersed strands of rough
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Fig. 3. Light micrograph of the axon bundle (AX) of the lamina propria. Pale staining
ensheathing cells (arrow), vasculature (V), mucous cells (M) and serous cells (curved
arrow) of Bowman’s glands. Bowman’s gland duct (arrowhead) penetrates the basal
lamina and passes up through the epithelium to the surface. 1 pm epoxy resin section,
Richardson stain. 5 mm = 5 pm.
Fig. 4. TEM micrograph of the receptor cell (R) of the olfactory epithelium. A well-
developed Golgi (arrow) is located in the basal cytoplasm. Cilia (arrowhead) extend
from the dendritic knob. 5 mm = 1 pm.
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endoplasmic reticulum (RER) and a well-developed Golgi were located in the apical and
basal cytoplasm. A single dendrite extended to the epithelial surface where it formed a
bulbous knob from which several cilia extended. The dendritic knobs (Fig. 5) measured
up to 3 pm high by 2 pm wide. Ciliary rootlets were often found in dendrites.
Longitudinal sections of cilia showed significant tapering a short distance from the
dendritic knob (Fig. 6). Numerous spherical membranous bodies 0.6-1.1 pm in diameter
were observed among the cilia. In some instances, a cilium was observed to be directly
connected to a spherical body (Fig. 6). Some cilia also appeared to have dilations along
their length. A dense carpet of cilia was observed on the epithelial surface by scanning
electron microscopy (Fig. 7). Cilia were 200-260 nanometers in diameter at their base
and contained the typical 9 + 2 axonemal arrangement of microtubules (Fig. 8). In distal
parts of the cilia, only two microtubules were present.
3.2.9 Transmission Electron Microscopy of Supporting Cells
The dark supporting cell (Fig. 9) had moderately dense cytoplasm and nucleus. The
apical surface of the dark supporting cell had numerous long microvilli up to 2 pm in
length, some of which were branched and irregular in shape. The plasma membrane at
the base of the microvilli often had subsurface cistemae. The apical cytoplasm contained
high amounts of smooth endoplasmic reticulum (SER). In many dark supporting cells,
SER cistemae showed an unusual parallel arrangement. Golgi apparatus was commonly
located in the apical cytoplasm. Light supporting cells (Fig. 9) had a convex apical
surface with fewer microvilli than the dark supporting cells. The light supporting cell
apical cytoplasm contained very small amounts of SER and large numbers of
mitochondria. The light supporting cell nucleus was oval and more electron-lucent than
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Fig. 5. TEM micrograph of the receptor cell dendrite. Mitochondria (M) fill the receptor
cell apical dendrite. The dendritic knob (arrowhead) contains numerous basal bodies of
cilia. Extensive, irregularly shaped microvilli (curved arrow) extend from the apical
surface of the dark supporting cells (DS). 5 mm = 0.4 pm.
Fig. 6. TEM micrograph of the cilium of the receptor cell. Tapering of the cilium
(arrow), swelling at the tip of cilium (arrowhead). 5 mm = 0.6 pm.
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Fig. 7. SEM micrograph of the dense carpet of cilia. Numerous spherical swellings
along the cilia (arrow). 5 mm =1.0 pm.
Fig. 8. TEM micrograph of the microtubule arrangement. Microvillus (arrow), cross-
section of a receptor cell cilium (arrowhead) showing the 9 + 2 axonemal arrangement of
microtubules. 5 mm = 0.05 pm.
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the dark supporting cell nucleus.
3.2.10 Transmission Electron Microscopy of Basal Cells
Basal cells (Fig. 10) were typically adjacent to the basal lamina. The basal cell
cytoplasm contained numerous polyribosomes and relatively little endoplasmic
reticulum. Dark basal cell nuclei were heterochromatic and more electron-dense than the
light globose basal cell nuclei (Fig. 10). Occasionally, basal cell processes incompletely
surrounded small groups of receptor cell axons. Axons typically were not separated from
each other by any cell processes in the bundles. The basal lamina underlying the
epithelium was not especially prominent or thickened.
3.2.11 Transmission Electron Microscopy of Lamina Propria Axon Bundles
Large bundles of small unmyelinated axons were common throughout areas of olfactory
mucosa (Fig. 11). The axons were 0.2-0.4 pm in diameter. Each bundle of axons was
surrounded by a layer of ensheathing cell processes. In some of the larger axon bundles,
ensheathing cell processes formed septa between groups of axons. The membranes of
adjacent axons were not separated by any visible cellular structures or extracellular
matrix. Ensheathing cells in the lamina propria were surrounded by a thin basal lamina.
3.2.12 Transmission Electron Microscopy of Lamina Propria Bowman’s Glands
Two types of Bowman’s gland cells (Fig. 12) were distinguished. The most common, the
mucous cell, contained electron-lucent secretory granules with fine-grained contents.
The cytoplasm of these cells contained large amounts of SER and small aggregates of
RER. Secretory granules were most numerous in the apical cytoplasm adjacent to the
acinar lumen. The second cell type, the serous cell, contained large areas of RER, and a
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 9. TEM micrograph of two types of supporting cells. Dark supporting cells (DS)
with numerous, irregular microvilli (arrowhead). The apical cytoplasm contains large
amounts of smooth endoplasmic reticulum (SER). Light supporting cells (LS) have a
convex apical surface with microvilli (arrow). 5 mm = 1 pm.
Fig. 10. TEM micrograph of two types of basal cells. Dark basal cells (DB) rest upon
the basal lamina (arrow). Basal cell processes and perikarya incompletely surround
groups of receptor cell axons (A). Light globose basal cell nuclei (LB). 5 mm = 0.7 pm.
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Fig. 11. TEM micrograph of the axon bundle. The ensheathing cell nucleus (ES) lies at
the perimeter of the bundle of unmyelinated axons (A). 5 mm = 1 pm.
Fig. 12. TEM micrograph of the Bowman’s glands. Mucous cells (M) and serous cells
(S) are organized in an acinar arrangement surrounding a lumen (L). 5 mm = 1 pm.
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small amount of SER. Most of the serous granules were smaller than the mucous
granules and were significantly more electron-dense. Secretory granules were not
observed in the cytoplasm of the Bowman’s gland duct cells that passed through the
epithelium to the surface of the mucosa.
3.3 Discussion
The basic organization of the olfactory epithelium and mucosa ofBlarina is well-
developed and is similar to other macrosmatic mammals (Allison, 1953a; Andres, 1966;
Graziadei, 1966; Arstila and Wersall, 1967; Frisch, 1967, 1969; Steinbrecht, 1969;
Seifert, 1970; Cuschieri and Bannister, 1975; Yamamoto, 1976; Loo, 1977; Kratzing,
1978; Moran et al., 1982a, b; Morrison and Costanzo, 1992; Naguro and Iwashita, 1992).
However, several differences were observed inBlarina.
3.3.1 Receptor Cells
The receptor cells in Blarina were comparable to receptor cells in other mammals. These
cells are bipolar neurons with a dendrite at the apical pole and an axon extending from
the basal pole. The dendrite forms a spherical-shaped swelling known as the olfactory
knob, dendritic bulb, or olfactory vesicle. Numerous cilia extend from this knob (Naguro
and Iwashita, 1992; Mendoza, 1993). Approximately 12-18 cilia extended from the
dendritic knob on each receptor cell in Blarina. Two members of the order Insectivora,
the mole, Talpa europaea, is reported to have only 1-5 olfactory cilia per receptor
(Graziadei, 1966) and the hedgehog, Erinaceus europaeus, has about 12 cilia per receptor
cell (Erhardt and Meinel, 1979). Mendoza (1993) reported approximately 11 cilia per
dendritic knob in the rat. In the blind mole rat, Spalax ehrenbergi, (Zuri et al., 1998), an
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average of 59 cilia per dendritic knob was reported. Cilia in Blarina exhibit the typical
9 + 2 microtubule pattern and intermediary vesicles and vesiculated tips, which have been
noted in other species (Frisch, 1969; Graziadei, 1971; Menco, 1983; Burton, 1992). The
mole olfactory receptors lack ciliary rootlets (Graziadei, 1966). However, rootlets were
observed in Blarina. The receptor cell diameter in Blarina measured up to 6 pm which is
comparable to other reports (Graziadei, 1971). The receptor cell axon in Blarina
measured up to 0.4 pm in diameter whereas, Altner and Kolnberger (1975) report axon
diameters to be no larger than 0.2 pm in diameter. Mendoza (1993) reports axon
diameter in the rat to measure up to only 0.2 pm in diameter.
3.3.2 Supporting Cells
Supporting cells are columnar in shape with microvilli extending from the apical surface
(Morrison and Costanzo, 1989; Naguro and Iwashita, 1992). The dark supporting cell of
Blarina was very similar to the most common type of supporting cell reported in other
mammals (Okano et al., 1967; Graziadei, 1971,1973; Yamamoto, 1976). However, the
light supporting cell of Blarina had features not reported for supporting cells in any other
mammal. The apical surface of the light supporting cell of the shrew is similar to the
apical surface of the microvillar cells that have been reported in the dog (Okano et al.,
1967) and human (Moran et al., 1982a, b), although the microvillar cell has been reported
to have an axon and may have a sensory function. The basal process of the light
supporting cell ofBlarina did not appear to be an axon since it is quite wide, contains
rough endoplasmic reticulum, and lacks the microtubules characteristic of axons. The
light supporting cell of Blarina has some similarity to the IA-6 reactive supporting cells
described in the rat olfactory epithelium (Carr et al., 1991), especially with respect to the
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protruding apical surface covered with microvilli. The IA-6 reactive cell is reported to be
non-neuronal and appears to also lack the large amounts of SER found in most olfactory
epithelium supporting cells. Meinel and Erhardt (1978) reported the presence of apical
protuberances on supporting cells in the mole olfactory epithelium. These were not
observed in Blarina. Graziadei (1966) did not report supporting cell apical protuberances
in the mole olfactory epithelium. The function of the light supporting cell in the shrew is
not known. However, the presence of many mitochondria and large numbers of small
vesicles supports the hypothesis that this cell could be involved in transporting molecules
or ions between the lamina propria and the fluid layer surrounding the olfactory receptor
cilia.
The microvilli of these supporting cells inBlarina measured up to 2 pm in length
which is shorter than the microvilli length in the cat which measured up to 5 pm in length
(Graziadei, 1971). According to Graziadei, (1971) no relationship can be established
regarding the number and length of microvilli in various vertebrates.
3.3.3 Basal Cells
Light and dark basal cells are reported to lie near the basal lamina between the basal
processes of supporting cells and contain little cytoplasm around the nucleus (Arstila and
Wersall, 1967; Graziadei, 1971; Naguro and Iwashita, 1992; Mendoza, 1993). In the
mouse (Frisch, 1967) and rat (Mendoza, 1993) basal cells have few distinguishable
features. The light and dark basal cells Blarinain had few distinguishing features and
were comparable to those of other mammal species.
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3.3.4 Lamina Propria
Bowman’s glands are reported to be alveolar glands with ducts that open at the epithelial
surface (Graziadei, 1971; Mendoza, 1993). The Bowman’s glands in the lamina propria
of Blarina olfactory mucosa contained two cell types. The cell with the electron-lucent
granules is similar to mucous cells observed in mammalian salivary glands whereas the
cell type with dense granules is similar to salivary gland cells often classified as serous
(Pinkstaff, 1980). Seifert (1970) reported that Bowman’s glands of several mammalian
species had light and dark secretory cells, each containing secretory granules with
different morphology, one with electron-dense granules and the other with pale granules.
Similar secretory granules have been reported in the mouse (Frisch, 1967). Human
Bowman’s glands are reported to contain only serous cells with electron-dense secretory
granules (Moran et al., 1982a). The observation that Bowman’s glands inBlarina
contained both alcian blue-positive and PAS-positive secretory materials support the idea
that these two cells are producing different secretory products, one of which is probably
an acid mucopolysaccharide and the other a neutral mucopolysaccharide. The presence
of acid and neutral mucopolysaccharides in Bowman’s glands has been previously
reported in mice (Cuschieri and Bannister, 1975), rats (Bojsen-Moller, 1964) and guinea
pigs (Ruseva, 1972). However, in rabbits (Zinnin, 1964), hogs (Herberhold, 1968) and
monkeys (Shantha and Nakajima, 1970) only neutral mucopolysaccharides were found.
Since the basic ultrastructure of the two types of Bowman’s gland cells differed
significantly, especially in the amounts and types of endoplasmic reticulum, it is unlikely
that these cells are the same cell type at different points in the secretory cycle.
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3.4 Comparative Studies
According to studies by Larochelle and Baron (1989a, b), inBlarina, 63% of the nasal
cavity is devoted to the olfactory epithelium.Blarina had the largest surface area of
olfactory epithelium and most developed epithelium of the four shrews studied which
included two terrestrial species, Sorex cinereus and S. fumeus, and one semi-aquatic
species, S. palustris. In Sorex minutus, also a terrestrial shrew, the olfactory epithelium
covered approximately 61.7% of the nasal cavity. The size of the olfactory mucosa is
reduced in the semi-aquatic Insectivora compared to terrestrial forms. Neomys fodiens,
the European watershrew, and S. palustris were reported to have the least-developed
olfactory epithelium of the shrews studied. Only 41% of the nasal cavity ofN. fodiens is
devoted to the olfactory epithelium (Larochelle and Baron, 1989a; Baron and Stephan,
1996). Any differences in the olfactory epithelium and olfactory development are
probably positively related to the ecological niche (Larochelle and Baron, 1989a, b). The
statistical results from these various shrews indicate thatBlarina has a highly-developed
olfactory epithelium.
3.5 Conclusion
The results of this detailed ultrastructural study of the olfactory epithelium and mucosa in
the two species of these Blarina confirm that these small mammals have a very well-
developed olfactory epithelium and mucosa and suggest that these semi-fossorial shrews
rely greatly upon olfaction in their daily activities.
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CHAPTER IV
MAIN OLFACTORY BULB
4.1 Introduction
Main olfactory bulb (MOB) microscopic structure has been studied in numerous
macrosmatic mammalian species including the platypus (Smith, 1895, 1896; Hines,
1929), marsupial shrew opossum (Herrick, 1921); hedgehog (Lopez-Mascaraque et al.,
1986, 1989; Brodmann, 1999; Brinon et al., 2001), mole (Crosby and Humphrey, 1939a;
Johnson, 1957b), opossum (Herrick, 1892; Herrick, 1924; Switzer and Johnson, 1977;
Chuah et al., 1997), rat (Reese and Brightman, 1970; Orona et al., 1983; Orona et al.,
1984; Scheibel and Scheibel, 1975; Mair and Gesteland, 1982; Mair et al., 1982; Struble
and Walters, 1982; Doucette, 1984; Switzer et al., 1985; Moriizumi et al., 1995; Kosaka
et al., 1998), mouse (Hirata, 1964; Burd, 1980; Carson, 1984; Graziadei and Graziadei,
1986; Dellovade et al., 1998), rabbit (Young, 1934, 1936; Allison and Warwick, 1949;
Allison, 1953a; Broadwell, 1975a; Mori et al., 1983; Ojimaet al., 1984; Katoh et al.,
1994; Yilmazer-Hanke et al., 2000), cat (McCotter, 1912; Ryu, 1980; Wahle et al., 1990;
Sztamska and Goetzen, 1997), dog (Read, 1908; McCotter, 1912), and the ferret
(Lockard, 1985). The MOB of the microsmatic mammals has also been studied and
includes the monkey (Alonso et al., 1998; Bassett et al., 1992) and human (Samat and
Netsky, 1981; Bhatnagar et al., 1987; Ohm et al., 1991; Shipley and Reyes, 1991;
Sztamska and Goetzen, 1997).
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4.2 Results
4.2.1 MOB Gross Anatomy
In the lateral view (Fig. 13), the MOB of the short-tailed shrew was relatively large. The
cerebral hemispheres were smooth with few gyri. The olfactory tubercle and piriform
cortex were also large.
4.2.2 Light Microscopy Overview
In the sagittal plane of section (Fig. 14), the large MOB dominated the rostral
telencephalon. The well-developed anterior olfactory nucleus filled the olfactory
peduncle. The rostral-most region of the frontal cortex projected over the caudal parts of
the MOB. The small accessory olfactory bulb (AOB) was positioned at the caudal edge
of the dorsal region of the MOB. In coronal sections, the large MOB was oval and
contained six concentric layers (Fig. 15) including the olfactory nerve layer (ONL),
glomerular layer (GL), external plexiform layer (EPL), mitral cell layer (MCL), internal
plexiform layer (IPL), and granule cell layer (GrL). At rostral levels, the ONL was
thickest on the ventral and medial surfaces. At a more caudal level (Fig. 16), the
olfactory ventricle was deep within the GrL and was partially surrounded by the
subependymal zone (see Fig. 72). At its most caudal level, the crescent-shaped MOB
was found on the ventro-medial surface of the cerebral hemisphere adjacent to the pars
posterior of the AON and the piriform cortex (Fig. 17) and was ventral to the piriform
cortex and rostral lateral ventricle. The laminar organizationof the MOB remained
constant from rostral to caudal levels (Fig. 18).
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CE
CH
MOB
PC OT
13
Fig. 13. Line drawing of the lateral view of the cortex and cerebellum. Large olfactory
structures include the main olfactory bulb (MOB), olfactory tubercle (OT), and piriform
cortex (PC). Smooth cerebral hemisphere (CH), cerebellum (CE).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 14. Light micrograph of the sagittal plane of the main olfactory bulb. Pars dorsalis
(PD) and pars lateralis (PL) of the anterior olfactory nucleus, frontal cortex (FC),
accessory olfactory bulb (arrow), pars externa of the AON (curved arrow), main olfactory
bulb (MOB), olfactory ventricle (arrowhead), nucleus accumbens (NA), caudate-putamen
(C-P). 40 pm frozen section, sagittal plane, cresyl-violet stain. 5 mm = 165 pm.
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Fig. 15. Light micrograph of the rostral main olfactory bulb. The six concentric layers
include the olfactory nerve layer (ONL), glomerular layer (GL), external plexiform layer
(EPL), mitral cell layer (curved arrow), internal plexiform layer (arrowhead) and granule
cell layer (GrL). 40 pm frozen section, coronal plane, cresyl-violet stain.
5 mm = 165 pm.
Fig. 16. Light micrograph of the mid-caudal main olfactory bulb. The open olfactory
ventricle (OV) with adjacent subventricular zone (arrow) and granule cell layer (GrL).
40 pm frozen section, coronal plane, cresyl-violet stain. 5 mm =165 pm.
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Fig. 17. Light micrograph of the caudal main olfactory bulb. The caudal-most MOB is
crescent-shaped. Pars posterior (PP) of the anterior olfactory nucleus and piriform cortex
(PC) are ventral to the frontal cortex (FC). 40 pm frozen section, coronal plane, cresyl-
violet stain. 5 mm = 250 pm.
Fig. 18. Light micrograph of the main olfactory bulb layers. Olfactory nerve layer
(ONL), glomerular layer (GL), external plexiform layer (EPL), mitral cell layer (arrow),
internal plexiform layer (arrowhead) and granule cell layer (GrL). 40 pm frozen section,
coronal plane, cresyl-violet stain. 5 mm = 50 pm.
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4.2.3 Light Microscopy of Olfactory Nerve Layer (ONL)
The ONL (Fig. 19) consisted of olfactory receptor axons and ensheathing cells. Two
types of ensheathing cells, a light and dark cell, were scattered throughout the ONL.
These ensheathing cells contained oval nuclei and a darkly-stained nucleolus was often
present. Ensheathing cell nuclei measured up to 6 pm in diameter along the long axis.
4.2.4 Light Microscopy of Glomerular Layer (GL)
The GL (Fig. 20) consisted of spherical glomeruli and the surrounding periglomerular
region. Neurons in the periglomerular region included periglomerular cells (PGC) and
short-axon cells (SAC). In the ventral regions, glomeruli were stacked two or three thick.
Glomeruli diameters ranged up to 150 pm. Capillaries were common between glomeruli.
In 1 pm epoxy resin sections the glomeruli consisted of darkly-stained olfactory receptor
axons intermingled with pale-staining dendrites. PGCs encircled the glomeruli with
fewer cells on the side facing the ONL. PGCs were most numerous adjacent to the
external plexiform layer often forming a cap-like structure on the glomeruli. These cells
were oval or spherical often with a prominent nucleolus and measured up to 11 pm in
diameter. The second cell type, the SAC was spherical and more lightly stained than the
adjacent periglomerular cells. These cells were less common than the PGCs and
measured up to 13 pm in diameter. A third cell, the external tufted cell (ETC) (Fig. 21)
had a large spherical euchromatic nucleus and measured up to 17 pm. The ETC cell
bodies were located in the superficial EPL. The apical dendrites of the tufted cells
extended into the GL and terminated within the glomeruli.
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Fig. 19. Light micrograph of the olfactory nerve layer (ONL). Unmyelinated axons (A),
dark ensheathing cell (arrow), light ensheathing cell (curved arrow), glomerular layer
(GL). 1 pm epoxy resin section, Richardson stain. 5 mm = 7 pm.
Fig. 20. Light micrograph of the glomerular layer. Glomeruli with dark axons (arrow)
and lighter areas of dendrites (arrowhead). Periglomerular cells (P), short-axons cells
(S). Olfactory nerve layer (ONL) with ensheathing cells. 1 pm epoxy resin section,
Richardson stain. 5 mm = 4 pm.
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4.2.5 Light Microscopy of External Plexiform Layer (EPL)
The EPL (Fig. 22) consisted of tufted cells, numerous capillaries, myelinated axons, and
dendritic profiles. Large tufted cells were scattered throughout the entire EPL and
measured up to 19 pm in diameter. The tufted cells of the EPL were similar in
morphology to the external tufted cells. Pale staining cross-sectional and longitudinal
profiles of tufted cell and mitral cell dendrites were common within the EPL. Some of
these dendrites projected to the GL and some ran horizontally in the EPL. Small
myelinated axons were common.
4.2.6 Light Microscopy of Mitral Cell Layer (MCL)
The MCL consisted of a single row of very large cells with rounded nuclei (Fig. 23). An
occasional granule cell was adjacent to the mitral cells. Mitral cells measured up to 29
pm in diameter. The euchromatic nucleus often had a distinct nucleolus. Abundant pale
cytoplasm surrounded the nucleus. The cell body tapered, forming the apical dendrite
which extended into the EPL. Basal dendrites extended laterally and diagonally into the
EPL. Mitral cell axons extended down through the granule cell layer.
4.2.7 Light Microscopy of Internal Plexiform Layer (IPL)
The IPL (Fig. 23) was located between the MCL and the granule cell layer. This layer
consisted of mitral and tufted cell axons and dendrites of granule cells.
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Fig. 21. Light micrograph of the external tufted cell. External tufted cells (E) are located
deep to the glomerular layer (GL). The external tufted cell apical dendrite (arrowhead)
extends into a glomerulus (arrow). External plexiform layer (EPL). 1 pm epoxy resin
section, Richardson stain. 5 mm = 6 pm.
Fig. 22. Light micrograph of the external plexiform layer. Pale dendritic profiles (D),
tufted cell (T) with apical dendrite (arrowhead), and myelinated axons (arrow) of the
EPL. 1 pm epoxy resin section, Richardson stain. 5 mm = 9 pm.
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4.2.8 Light Microscopy of Granule Cell Layer (GrL)
The GrL (Fig. 24) had elongated clusters of cells oriented parallel to the MCL alternating
with regions of neuropil. Bands of granule cell bodies were typically 3-12 nuclei thick.
Two types of granule cells were present in these bands. One type, a dark granule cell
contained a spherical nucleus with a darkly stained nucleolus. These dark granule cells
were more numerous than the light granule cell. The nuclear diameter measured up to 6
pm. The second type of granule cell had a spherical nucleus with small patches of
heterochromatin that resulted in an overall lighter staining intensity than the dark granule
cells. These light granule cell nuclei measured up to 8 pm in diameter. The surrounding
neuropil in these bands consisted mostly of pale staining dendrites. Between the bands
the neuropil contained small myelinated axons, dendrites, and capillaries.
4.2.9 Transmission Electron Microscopy of ONL
Unmyelinated olfactory receptor axons (Fig. 25) in cross section and longitudinal section
fdled the ONL and contained mitochondria, neurofilaments, and microtubules. The axon
diameters ranged from 0.2-0.4 pm with an average diameter of 0.2 pm. Ensheathing cell
processes formed incomplete septa partially surrounding groups of axons. Dark
ensheathing cells (Fig. 25) had more heterochromatin than the light ensheathing cells
(Fig. 26). Ensheathing cell nuclei were typically oval. Ensheathing cell cytoplasmic
organelles commonly included numerous mitochondria, rough endoplasmic reticulum
(RER), free ribosomes, microtubules, a prominent Golgi apparatus, and intermediate
filaments. Lipofuscin granules were often common in the cytoplasm of both types of
ensheathing cells.
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Fig. 23. Light micrograph of the mitral cell layer. External plexiform layer (EPL) with
numerous pale dendritic profiles (curved arrow), apical dendrite (D) of a tufted cell,
mitral cell (M) of the mitral cell layer, mitral cell apical dendrite (arrowhead), proximal
part of a basal dendrite (arrow), internal plexiform layer (IPL). 1 pm epoxy resin section,
Richardson stain. 5 mm = 10 pm.
Fig. 24. Light micrograph of the granule cell layer. The granule cell layer (GrL) with
distinct bands of light granule cells (arrowhead), dark granule cells (arrow), and
myelinated axons (curved arrow). 1 pm epoxy resin section, Richardson stain.
5 mm = 5 pm.
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Fig. 25. TEM micrograph of dark ensheathing cells. Dark ensheathing cell with
lipofuscin granules (arrowhead). Cross-sections and longitudinal-sections of
unmyelinated olfactory receptor cell axons (A). 5 mm = 0.8 pm.
Fig. 26. TEM micrograph a light ensheathing cell. Light ensheathing cell with lipofuscin
granules (arrowhead). Cross-sections and longitudinal-sections of unmyelinated
olfactory receptor cell axons (A). 5 mm = 0.8 pm.
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4.2.10 Transmission Electron Microscopy of GL
Periglomerular cells were the most common neurons in the periglomerular region of the
GL (Fig. 27). The periglomerular cell nucleus was often indented and had scattered
patches of heterochromatin. The nucleolus was typically eccentric. The thin layer of
cytoplasm contained numerous ribosomes arranged in rosette structures, small amounts
of RER, and Golgi profiles. Lipofuscin granules were often present in periglomerular
neurons. Asymmetrical dendro-somatic synapses between external tufted cell dendrites
and periglomerular cell somas were common (Fig. 27). The second cell type of the
periglomerular region, the short-axon cell was less numerous than the periglomerular cell
and often had an indented nucleus (Fig. 28). The cytoplasm contained a few profiles of
RER and large profiles of Golgi apparatus. Free ribosomes and mitochondria were
scattered throughout the cytoplasm. A third neuronal cell type, the external tufted cell
was identified by its large size, euchromatic nucleus, and abundant electron-lucent
cytoplasm (Fig. 29). These neurons were typically located at the interface between the
EPL and GL. The tufted cell apical dendrite projected into the GL. Long parallel arrays
of RER and Golgi stacks were common in the perinuclear cytoplasm. Rosettes of
ribosomes and mitochondria were uniformly scattered throughout the tufted cell
cytoplasm. These dendrites were smooth in outline and contained mitochondria and
microtubules.
Glomeruli consisted of many profiles of electron-dense receptor cell axons and
terminals with pale dendrites of mitral, tufted, and periglomerular cells (Fig. 30). These
axon terminals were all ultrastructurally similar and contained numerous electron-lucent
vesicles, microtubules and mitochondria. The electron-lucent dendrites from all three
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 27. TEM micrograph of periglomerular cells (PG). Dendritic profiles (D),
myelinated axons (curved arrow), and a dendro-somatic synapse (arrowhead) between the
primary dendrite of an external tufted cell and a periglomerular soma are observed within
the periglomerular region. Nuclear filaments (arrow) are occasionally observed in
periglomerular neurons. 5 mm = 0.6 pm.
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Fig. 28. TEM micrograph of the short-axon cell (SA). The cytoplasm contains numerous
mitochondria (M) and large profiles of Golgi apparatus (G). Nuclear pseudoinclusions
formed by indentations of the nuclear envelope (arrowheads). Glomerulus (GL).
5 mm = 0.6 pm.
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Fig. 29. TEM micrograph of the external tufted cell. The cytoplasm of the external
tufted cell contains a well-developed Golgi apparatus (G). Numerous large dendritic
profiles (D) are common within the periglomerular region. Note the periglomerular cells
(PG) in close proximity to the tufted cell. 5 mm = 1 pm.
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Fig. 30. TEM micrograph of axons and dendrites in the glomeruli. The glomeruli of the
GL typically consist of electron-dense unmyelinated receptor cell axons (A) and
terminals intermingled with electron-lucent dendritic profiles (D). Dendro-dendritic
synapses (arrow) and axo-dendritic synapses (arrowhead) are common. 5 mm = 0.5 pm.
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neuron types were similar and contained scattered mitochondria, microtubules, and
profiles of SER. Dendro-dendritic and axo-dendritic synapses were common in the
glomeruli. Symmetrical and asymmetrical synapses were common. Symmetrical
synapses were characterized by a thin synaptic density between the two membranes
(Figs. 31, 32). Pleomorphic electron-lucent vesicles were grouped near the presynaptic
density. Asymmetrical synapses had a more prominent synaptic density that extended
farther into the postsynaptic structure (Fig. 32). The presynaptic structure was identified
by the presence of predominantly electron-lucent spherical vesicles (Figs. 33,34).
4.2.11 Transmission Electron Microscopy of EPL
The EPL consisted of a dense plexus of primary and secondary dendrites of mitral and
tufted cells intermingled with dendrites of granule cells (Figs. 35, 36). The dendrites of
the mitral and tufted cells and granule cells were ultrastructurally indistinguishable from
one another. These dendrites contained many microtubules and mitochondria. Dendro-
dendritic synapses were numerous within the EPL and often three to five synaptic
contacts were common along one dendritic profile (Fig. 35). The dendro-dendritic
synapses were both asymmetrical and symmetrical (Figs. 35, 36). On rare occasions
myelinated tufted cells and dendrites were identified in the EPL. The tufted cell bodies
were partially surrounded by a thin myelin sheath (Figs. 37, 38). In addition, myelinated
dendrites were also noted in the EPL (Fig. 39).
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Fig. 31. TEM micrograph of a dendro-dendritic synapse. This dendro-dendritic
symmetrical synapse in the glomerulus contains electron-lucent pleomorphic vesicles
(arrow) in the presynaptic region. The synaptic density (arrowhead) is thin between the
two dendritic membranes. Numerous cross-sections of microtubules (M) are present in
adjacent dendrites. 5 mm = 0.2 pm.
Fig. 32. TEM micrograph of a reciprocal synapse. Electron-lucent pleomorphic vesicles
in the terminal region (arrowhead), spherical vesicles in the dendrite of a mitral/tufted
cell (arrow) in the glomerulus. 5 mm = 0.2 pm.
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Fig. 33. TEM micrograph of a axo-dendritic synapse. This axo-dendritic asymmetrical
synapse (arrowhead) in the glomerulus contains electron-lucent spherical vesicles
(arrows) in the presynaptic region. 5 mm = 0.2 pm.
Fig. 34. TEM micrograph of a dendro-dendritic synapse. Asymmetrical synapse in the
glomerulus contains electron-lucent spherical vesicles (arrows) in the presynaptic region.
5 mm = 0.2 pm.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 35. TEM micrograph of dendro-dendritic synapses (arrow). Spherical electron-
lucent vesicles are characteristic of these asymmetrical synapses (arrowhead). Darker
structures forming synapses appear to be granule cell dendrites and dendritic spines.
Mitral/tufted cell dendrites (D) in the external plexiform layer. 5 mm = 0.3 pm.
Fig. 36. TEM micrograph of pleomorphic vesicles. Pleomorphic electron-lucent vesicles
(arrowhead) are characteristic of symmetrical dendro-dendritic synapses in the external
plexiform layer. 5 mm = 0.1 pm.
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Fig. 37. TEM micrograph of a myelinated neuron. A tufted cell soma with a thin myelin
sheath (arrow) in the external plexiform layer. 5 mm = 0.4 pm.
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Fig. 38. TEM micrograph of the thin myelin sheath. The sheath typically consists of a
few thin lamellae (arrowhead). 5 mm = 0.1 pm.
Fig. 39. TEM micrograph of a myelinated dendrite (D). Myelinated axons (AX) in the
external plexiform layer. 5 mm = 0.4 pm.
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4.2.12 Transmission Electron Microscopy of MCL
The mitral cells (MC), the largest cells of the MOB, had a euchromatic nucleus
surrounded by abundant cytoplasm (Fig. 40). The nucleus typically contained a large
dense nucleolus. RER was often organized into parallel arrays. Numerous mitochondria,
free ribosomes, and Golgi stacks were common. The initial segments of MC dendrites
typically contained many microtubules and mitochondria.
4.2.13 Transmission Electron Mieroscopy of IPL
The IPL (Fig. 41) contained numerous cross-sections of mitral and tufted cell myelinated
axons. It was not possible to distinguish between the tufted and mitral cell axons or the
afferent fibers, although since the MC were the largest cells, it is likely that the largest
axons were from MC. The axons were densely clustered together forming a thin IPL.
4.2.14 Transmission Electron Microscopy of GrL
The GrL (Figs. 42,43) of the MOB consisted of granule cells and the neuropil of
dendrites of granule cells and numerous unmyelinated axons. Occasional myelinated
axons were also present in the GrL. The dendrites were electron-lucent and contained
microtubules and mitochondria. The axon terminals were typically densely packed
predominantly with small, spherical electron-lucent vesicles. Asymmetric synapses with
spherical vesicles were present common (Fig. 42). The second subdivision contained the
spherical granule cells (Fig. 43). The light and dark granule cells were clustered together.
Both types of granule cells had a spherical or oval nucleus. The dark granule cell was
more numerous than the light granule cell. The lighter granule cell nucleus had less
heterochromatin and appeared more electron-lucent. Both cell types had a narrow band
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mm
Fig. 40. TEM micrograph of a mitral cell. Nucleus (N) with prominent nucleolus. Golgi
apparatus (G), mitochondria (M) and rough endoplasmic reticulum (arrow).
5 mm = 0.8 pm.
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Fig. 41. TEM micrograph of myelinated axons. Myelinated axons (arrow) of mitral and
tufted cells are interspersed with those of afferent fibers in the internal plexiform layer.
5 mm = 0.5 pm.
Fig. 42. TEM micrograph of the neuropil of the granule cell layer. The neuropil consists
of electron-lucent dendrites (D) intermingled with axon terminals with spherical vesicles
(arrow) and asymmetric synapses (arrowhead). 5 mm = 0.1 pm.
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Fig. 43. TEM micrograph of dark and light granule cells. The dark granule cell has
electron-dense patches of heterochromatin (arrowheads) evenly distributed throughout
the nucleus. The lighter granule cell nucleus typically has a more electron-lucent nucleus
with a distinct nucleolus (arrow). 5 mm = 0.5 pm.
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of cytoplasm containing free ribosomes and small amounts of RER.
4.3 Discussion
The typical mammalian MOB organization with the six concentric layers (Young, 1934,
1936; Crosby and Humphrey, 1939a; Jeserich, 1945; Lauer, 1945; Allison, 1953b;
Andres, 1970; MacLeod, 1977; Lockard, 1985; Lopez-Mascaraque et al., 1986; Greer,
1991; McLean and Shipley, 1992; Nickell and Shipley, 1992; Kratskin, 1995; Shipley
and Smith, 1995; Shipley et al., 1995; Butler and Hodos, 1996; Eisthen, 1997; Alonso et
al., 1998; Kosaka et al., 1998) surrounding the olfactory ventricle was also found in
Blarina. Furthermore, the various typical cell types of the MOB (Herrick, 1924; Jeserich,
1945; Johnson, 1957b; Andres, 1970; Brooke and Pinching, 1975; Doucette, 1984; Greer,
1991; Bassett et al., 1992; Dellovade et al., 1998) were also common in the MOB of
Blarina. The synaptic patterns of the mammalian MOB (Hirata, 1964; Andres, 1970;
Price and Powel, 1970a, b, c; Pinching and Powell, 1971a, b, c; Brooke and Pinching,
1975; Shepherd and Greer, 1990; Greer, 1991; Scott and Harrison, 1991; Shipley and
Reyes, 1991; Bassett et al., 1992; Nickell and Shipley, 1992) were also present in
Blarina. One main difference observed in the MOB in Blarina pertained to its caudal
extent. The MOB tissue extended significantly more caudally which caused the shrew
AON to be displaced dorsal and lateral compared to rodents (Bums, 1982). There were
several additional differences in the MOB of Blarina compared to other small
macrosmatic mammals. These differences will be elaborated upon in the following
sections.
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4.3.1 Olfactory Nerve Layer
Olfactory receptor neurons have been reported to continually die throughout the life of
the adult animal and are replaced by mitosis and differentiation of basal cells. Axons of
new receptors grow through the cribriform plate of the ethmoid bone and enter the ONL
on the surface of the MOB. The ONL has been reported to be thickest on the anterior and
ventral surfaces in the rabbit (Young, 1936; Yilmazer-Hanke et al., 2000) and rat
(Doucette, 1984). InBlarina, the ONL was the thickest on the medial and ventral
surfaces of the MOB. Crosby and Humphrey (1939a) reported that the ONL was thickest
on the medial, ventral, and lateral surfaces of the shrew. Within the glomeruli, the
receptor cell axons form synapses with mitral and tufted cell dendrites (Alison, 1953a;
Shepherd and Greer, 1990; Kratskin, 1995; Brinon et al., 2001). In the rabbit, these
axons are reported to have average diameters of 0.6 pm (Yilmazer-Hanke et al., 2000).
In Blarina the axons were slightly smaller than the axon diameter in the rabbit and ranged
from 0.2-0.4 pm in diameter.
According to Doucette (1984), glial cells of the rat ONL include ensheathing cells
and astrocytes. Ensheathing cells have ultrastructural properties of both fibrous
astrocytes and Schwann cells and are found in association with the olfactory nerves
(Barber and Lindsay, 1982; Chuah et al., 2000; Imaizumi et al., 2000; Keyvan-Fouladi et
al., 2002; Wang et al, 2003). The ensheathing cell processes, like Schwann cell
processes, wrap around groups of unmyelinated olfactory receptor axons and are thought
to provide support for the olfactory axon regeneration (Doucette, 1984; Pixley, 1992;
Kafitz and Greer, 1998; Barnett et al., 2000). In the rat, the nuclei of ensheathing cells
and astrocytes are ultrastructurally similar. However, the ensheathing cell cytoplasm
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appears more dense than typical astrocyte cytoplasm. In addition, intermediate filaments
in the ensheathing cells are fewer in number and are scattered throughout the cytoplasm
whereas in the fibrous astrocyte, these intermediate filaments are organized into bundles
and are more distinct. Based upon the morphological observation of scattered
intermediate filaments, absence of filament bundles, and the observed ensheathing of
axonal bundles, the glial cells ofBlarina ONL were identified as ensheathing cells.
Doucette (1984) observed only one type of ensheathing cell in the rat, but inBlarina,
light and dark ensheathing cells were common.
4.3.2 Glomerular Layer
The GL of Blarina was similar to the mammalian MOB GL (Mori et al., 1983; Nickell
and Shipley, 1992; Katoh et al., 1994; Kosaka et al., 1998; Kasowski et al., 1999; Treloar
et al., 1999; Kim and Greer, 2000). Glomerular shape and size varies within and among
mammalian species. Generally, the glomerulus is oval, spherical, or pear-shaped. Table 2
is a summary of glomerular diameter variations among some mammals.
Table 2. Glomerular diameter range.
Mammal species Diameter (pm) Reference Shrew -150 Present study Rat 50-120 Pinching and Powell, 1971b Rat 40-300 Royet et al., 1989
Rat 100-200 MacLeod, 1977 Rabbit 50-200 Katoh et al., 1994 Macaque 70-100 Alonso et al., 1998
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The glomerular diameter in Blarina was similar to glomerular diameter in other
macrosmatic mammals and measured up to 150 pm. The glomerular diameter in Blarina
was significantly larger than the glomerular diameter in the microsmatic macaque
monkey (Alonso et al., 1998).
Two types of synapses have been reported in mammalian MOB glomeruli.
Symmetrical synapses are identified by thin electron-dense membrane thickenings
between the two synaptic membranes with pleomorphic vesicles in the presynaptic
region. Asymmetrical synapses have thicker synaptic densities that typically extend
farther into the postsynaptic region. Spherical vesicles are associated with the
asymmetrical synapses (Price and Powell, 1970a, b, d; Pinching and Powell, 1971a, b, c).
Table 3 is a summary of the common synaptic patterns identified within the rat GL. Cell
types to the left of the arrow are presynaptic. Cells to the right of the arrow are
postsynaptic.
Table 3. Synaptic patterns of the glomerular layer (Pinching and Powell, 1971a, b, c).
Type of synapse Symmetrical Asymmetrical Dendro-dendritic PGC->M/T M/T->PGC PGC->PGC Axo-dendritic SAC^PGC ONA->PGC PGC-»M/T ONA->M/T ETC->PGC CF-»PGC Dendro-somatic M/T->PGC PGC=periglomerular cell; M/T=mitral/tufted cells; SAC=short-axon cell; ONA=olfactory nerve axon; ETC=extemal tufted cell; CF=centrifugal fibers
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The periglomerular cell dendritic processes have been reported to extend into one
or more glomeruli whereas the tufted and mitral cell apical dendrites project to a single
glomerulus (Kratskin, 1995). The axon terminals in the glomeruli originate from various
sources including olfactory receptor cells, SACs, PGCs, and centrifugal fibers (Price,
1968; Pinching and Powell, 1971a, b, c; Getchell and Shepherd, 1975; Kosaka et al.,
1998). The axons of the short-axon cells extend for a short distance throughout the GL,
projecting a distance of only one to three glomeruli; periglomerular cell axons extend
across three to five glomeruli. These axons do not typically extend beyond the
boundaries of the GL, but remain within the GL surrounding the glomeruli. The receptor
cell axons originate from the olfactory epithelial receptor cells and terminate with the
glomeruli. Centrifugal axon fibers approach the MOB via the lateral olfactory tract and
terminate in the glomeruli (Price, 1968; Pinching and Powell, 1971a, b, c; Kratskin,
1995). The synaptic patterns in the MOB glomeruli ofBlarina were comparable to those
reported from other mammals, especially in the rat as described by Pinching and Powell
(1971a, b, c). Symmetrical and asymmetrical dendro-dendritic and axo-dendritic
synapses were observed inBlarina. Spherical, electron-lucent vesicles in the presynaptic
terminals were characteristic of the asymmetrical synapse whereas pleomorphic, electron-
lucent vesicles were associated with the symmetrical synapses. In addition, Pinching and
Powell (1971a, b, c) reported symmetrical somato-dendritic synapses originating on the
periglomerular cells and terminating on mitral and tufted cell dendrites. These synapses
were less frequent than those listed in Table 3 and rarely occurred. This type of synapse
was not observed in Blarina.
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The surrounding periglomerular region of the GL has been defined as the
interstices between individual glomeruli, between the glomeruli and the ONL, and
between the glomeruli and the EPL. This region contains several types of neuronal
somata. Periglomerular cells are the most common neuron of the GL and range in
diameter in the rat from 5-8 pm (Pinching and Powell (1971a, c). Brinon and coworkers
(2001) report similar cellular diameters of 5-9 pm in the hedgehog, a close relative of
Blarina. Periglomerular cells in Blarina were slightly larger than those of the rat and the
hedgehog and measured up to 11 pm in diameter.
A second cell type of the GL is the short-axon cell. These cells range in diameter
from 8-12 pm in the rat and are less numerous than periglomerular and external tufted
cells (Pinching and Powell, 1971a). In Blarina, short-axon cells were comparable to the
rat and measured up to 13 pm. The short-axon cells in Blarina were also less numerous
than the periglomerular cells and the external tufted cells.
A third cell type, the external tufted cell is located within the periglomerular
region or in close proximity to the glomerular region (Pinching and Powell, 1971a;
Haberly and Price, 1977; Mori et al., 1983; Orona et al., 1984). External tufted cells send
a primary dendrite into the GL and several secondary dendrites into the superficial region
of the EPL (Kratskin, 1995). The external tufted cell diameter ranges from 10-15 pm in
the rat (Pinching and Powell, 1971a) and 15-19 pm in the hedgehog (Brinon et al., 2001).
In Blarina, the external tufted cells measured up to 17 pm and were comparable to those
of the rat and hedgehog MOB in their diameter.
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4.3.3 External Plexiform Layer
The EPL is characterized by numerous tufted cells scattered throughout a neuropil
consisting of primary and secondary dendrites from tufted cells and mitral cells and
peripheral dendrites from granule cells (Young, 1934; Shepherd, 1966; Druga, 1980;
Mori et al., 1983; Greer, 1991; Kratskin, 1995). Two of the three types of tufted cells are
located in the EPL. These cells are called middle tufted cells and internal tufted cells.
Middle and internal tufted cells have also been called displaced mitral cells or
outwandered mitral cells in older reports (Young, 1934; Crosby and Humphrey, 1939a).
The middle tufted cells are located within the mid-regions of the EPL whereas the
internal tufted cells are deep within the EPL just above the MCL. The third type of tufted
cell, which has been previously discussed, is the external tufted cell located at the border
between the GL and the EPL. Each type of tufted cell is similar in staining intensity and
morphology (Pinching and Powell, 1970a; Mori et al., 1983; Schneider and Scott, 1983;
Kratskin, 1995; Nakajima et al., 1996; Gil-Carcedo et al., 2000) but may vary in
diameter. According to Haberly and Price (1977), tufted cells become smaller in size as
the distance from the MCL increases. In the hedgehog (Brinon et al., 2001), tufted cells
of the EPL ranged in diameter from 16-24 pm and in the rat (Pinching and Powell,
1971a) tufted cell diameters ranged from 18-25 pm. In Blarina, tufted cells measured
only up to 19 pm in diameter. Additionally, Kosaka and Kosaka (1999) have identified
in the laboratory shrewSuncus murinus a cell type referred to as the tasseled cell that
resembles tufted cells of the EPL. The tasseled cell soma is located within the middle
region of the EPL and its dendrites extend towards the GL. At the border of the GL and
the EPL is a region that contains small glomerular-like structures called nidi. The nidi
resemble the glomeruli of the GL. Surrounding the nidi are specialized cells called
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perinidal cells which are like the periglomerular cells. The dendrites of the tasseled cells
branch into a tuft-like structure and synapse on perinidial cells within the nidi. Kosaka
and Kosaka (1999) have suggested that many of the outwandered mitral cells noted by
Crosby and Humphrey (1939a) should probably be recognized as tasseled cells.
Immunocytochemical studies were used to identify the nidus, perinidal cells, and tasseled
cells. The current project on Blarina focused on the ultrastructure of the olfactory system
by light and transmission electron microscopy so cellular identification based upon
immunocytochemical methods were not within the scope of this research. The presence
or absence of tasseled neurons in Blarina can not be confirmed nor rejected based upon
morphological observations.
Myelinated neuronal somas and dendrites were observed within the EPL of
Blarina. Historically, these types of structures have not been a frequent finding.
However, investigators have discovered these myelinated stmctures in various layers of
the MOB of other mammals. Tigges and Tigges (1980) reported myelinated neuronal
somata within the periglomerular region in the squirrel monkey MOB. These myelinated
somata were identified as external tufted cells based upon their morphology and their
position within the superficial regions of the EPL. In addition, myelinated periglomerular
cells have been observed within the GL. Tigges and Tigges (1980) further reported
occasional myelinated somata of granule cells deep within the GrL. Segments of
myelinated dendrites are also noted within the periglomerular region and the EPL. In the
mouse, Burd (1980) reported myelinated mitral/tufted cell dendrites in the EPL. The
function of these myelinated structures remains unknown. Tigges and Tigges (1980)
hypothesize that myelination may be the result of changes associated with the aging
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process and not due to pathological conditions. In Blarina, based upon position and
ultrastructural characteristics, the myelinated somata appeared to be tufted cells and the
myelinated dendrites appeared to be external tufted cell primary dendrites. The function
of these myelinated structures remains unknown. Their rarity suggests that they do not
play a major role in MOB function.
The synapses within the EPL are relatively simple. Reciprocal dendro-dendritic
synapses between mitral and tufted cell dendrites and granule cell dendrites are common
(Pinching and Powell, 1971a, b, c; Mouradian and Scott, 1988). In dendro-dendritic
reciprocal synapses, it has been suggested that mitral and tufted cell dendrites are
excitatory to the granule cells, whereas the granule cells are inhibitory to the mitral and
tufted cell dendrites. These reciprocal synapses function to inhibit and/or filter incoming
olfactory information from the olfactory mucosa (Brooke and Pinching, 1975; Brunjes et
al., 1982; Kishi et al., 1982; Mori et al., 1983; Mouradian and Scott, 1988; Shipley and
Reyes, 1991). The reciprocal synapse consists of an asymmetrical and a symmetrical
synapse. The asymmetrical synapse originates on mitral and tufted cell secondary
dendrites and terminates on the granule cell dendrites. Electron-lucent spherical vesicles
are reported in the presynaptic dendritic region. The symmetrical synapse originates on
the granule cell dendritic process and terminates on the mitral and tufted cell dendrites.
The vesicles associated with the presynaptic dendrite were reported to be electron-lucent
pleomorphic vesicles (Price and Powel, 1970a, b). In Blarina, asymmetrical synapses
were identified by the presence of predominantly spherical electron-lucent vesicles in
large mitral and tufted cell dendrites. Also, symmetrical synapses were observed
originating from the granule cell dendrite and terminating onto the dendrites of the mitral
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and tufted cells. Small, electron-lucent pleomorphic vesicles were clustered in the
presynaptic dendritic processes of these symmetrical synapses. The asymmetrical
synapses with predominantly spherical vesicles and symmetrical synapses with
pleomorphic vesicles observed inBlarina were similar to the synapses and vesicles
reported in the rat (Price and Powel, 1970a, b; Pinching and Powell, 1971a, b, c).
4.3.4 Mitral Cell Layer
Mitral cells and tufted cells are the efferent neurons of the MOB (Greer, 1991; Cajal,
1995; Kratskin, 1995; Kosaka et al., 1998). These cells share many morphological
characteristics. However, mitral cells are distinguished from tufted cells based on the
position of their somata. In most mammals, mitral cells are confined to a monolayer
whereas tufted cells have been observed scattered within the EPL (Orona et al., 1984).
Both cell types have a single apical dendrite that extends without branching throughout
the EPL into a glomerulus where synaptic contact is made with olfactory nerve axons and
periglomerular cell axons and dendrites. The secondary dendrites of mitral and tufted
cells extend in a tangential pathway, parallel to the MCL, into the EPL and synapse onto
granule cell dendrites. The axons of mitral and tufted cells exit the MOB and form the
lateral olfactory tract (Lohman and Mentink, 1969; Druga, 1980; Orona et al., 1984;
Greer, 1991; Nickell and Shipley, 1992; Kosaka et al, 1998). Blarina, In the MCL
consisted of a monolayer of densely packed mitral cells. This monolayer organizational
feature is not found in the olfactory bulbs of some Monotremes, specifically the platypus
or the echidna. The lack of a monolayer has also been reported in some reptiles, such as
the python (Switzer and Johnson, 1977). In birds, a monolayer was reported in the emu
and kiwi, but was absent in the chicken (Switzer and Johnson, 1977). In the hedgehog,
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the mitral cells are not organized in the typical monolayer but are displaced within the
EPL and IPL (Lopez-Mascaraque et al., 1986). In the macaque monkey (Alonso et al.,
1998), mitral cells are also dispersed throughout the EPL and do not form a monolayer.
It is interesting that a mitral cell monolayer was observed inBlarina but is absent in the
hedgehog. The functional significance of mitral cells organized in a monolayer verses a
generalized displacement remains unclear. The lack of a mitral cell monolayer appears to
be more common in primitive vertebrates including the chameleon Anolis( carolinensis),
copperhead ( Agkistrodon mokaseri), and sparrow {Passer domesticus) (Crosby and
Humphrey, 1939a). In primates, the lack of a monolayer may be part of the regression of
the olfactory system.
4.3.5 Interna] Plexiform Layer
The IPL of Blarina was similar to the typical mammalian IPL and contains relatively few
neuronal somata, but had numerous collaterals of mitral and tufted cell myelinated axons
and peripheral dendrites of granule cells (Allison, 1953a; Shepherd, 1966; Orona et al.,
1984; McLean and Shipley, 1992; Kratskin, 1995). In addition to these efferent fibers,
afferent axons from the forebrain coming into the MOB also pass through the IPL (Scott
and Harrison, 1991).
4.3.6 Granule Cell Layer
Granule cells of Blarina were organized into bands of cells within a neuropil of dendrites
of granule cells and collaterals of axons from mitral and tufted cells. Shipley and Ennis
(1996) also reported that granule cells in other mammals typically form bands of cells.
Afferent projections to the MOB originate from several sources including the ipsilateral
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and contralateral anterior olfactory nucleus and the ipsilateral horizontal limb of the
diagonal band. These afferent fibers make asymmetrical synapses onto the granule cells
(Young, 1934; Cragg, 1962; Price, 1968; Price and Powell, 1970c; Davis et al., 1978;
Macrides et al., 1981; Nickell and Shipley, 1992; Cajal, 1995). InBlarina, afferent axons
also made asymmetrical synapses on granule cell dendritic processes. These synapses
typically occurred within the neuropil surrounding the bands of granule cells. Granule
cells have been reported to lack axons (Pinching and Powell, 1971a; MacLeod, 1977;
Mair et al., 1982). Two types of dendritic processes, the peripheral and deep dendrites
extend from the granule cells. These peripheral processes are relatively thick, terminate
in the EPL and enter into reciprocal synapses with mitral and tufted cell dendrites (Price
and Powell, 1970a, b; Orono et al., 1983). From the deep side of the perikarya, 1-4
dendrites extend toward deeper regions within the GrL, extending away from the MCL.
The diameter of granule cells appears to be relatively consistent among mammals.
In the hedgehog, these cells ranged from 6-8 pm in diameter (Lopez-Mascaraque et al.,
1986; Brinon et al., 2001) and in the rat, granule cells ranged from 6-10 pm in diameter
(Mair et al., 1982). Struble and Walters (1982) have identified two populations of
granule cells in the rat, a dark and a light granule cell. Dark granule cells were 6-11 pm
in diameter, and the light granule cells were 7-13 pm in diameter. Struble and Walters
(1982) concluded that although light granule cells are the larger cell of the two types, the
dark granule cells are more numerous than the light granule cells. These two populations
of granule cells were also identified in Blarina. The dark granule cells in Blarina
measured up to 6 pm in diameter and were more numerous than the light granule cells
which were larger and measured up to 8 pm in diameter. A subset of granule cells,
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termed atypical granule cells has been reported in the cat (Wahle et al., 1990). These
cells, located in the MCL and EPL lack the peripheral dendrite common to typical
granule cells. Rather, two or more dendrites extend in the EPL in an oblique fashion.
Other atypical granule cells are located at the border between the GL and EPL. Cells
found within the GL are identified along the surface of the glomeruli themselves, or in
the spaces between the structures. In Blarina, the two populations of dark and light
granule cells were located only within the GrL. In addition, Shepherd and Greer (1990)
noted short axon cells in the GrL of the rat. These cells were not observed in the GrL of
Blarina.
The olfactory ventricle appeared deep within the GrL of Blarina and was similar
to other macrosmatic mammals including the hedgehog (Lopez-Mascaraque et al., 1986,
1989; Valverde et al., 1989) and mole (Johnson, 1957b), marsupial mole (Smith, 1895),
opossum (Herrick, 1892; Herrick, 1924), rabbit (Young, 1934; Broadwell, 1975b; Romfh,
1982), and the cat (Ryu, 1980; Kinney, 1982; Wahle et al., 1990). In some mammals,
such as the mink (Jeserich, 1945), the olfactory ventricle is small. In the platypus (Hines,
1929), rat (Bums, 1982), and mouse (Crosby and Humphrey, 1939a), the ventricle is
occluded. In humans, the ventricle is patent only in fetal life (Samat and Netsky, 1981)
and only in rare cases has an open olfactory ventricle remained within the MOB (Roy et
al., 1987).
4.4 Comparative Studies
According to Stephan and coworkers (1991), the MOB in Blarina makes up
approximately 12% of the telencephalon volume. InNeomys fodiens, a semiaquatic
shrew, the MOB makes up only 9% of the telencephalon. This reduced MOB size is
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expected since olfaction is not thought to be as important in detection of aquatic prey. By
comparison, in the macrosmatic gray wolf, Canus lupus, the MOB is 19% of the total
brain volume and in the red fox, Vulpes vulpes, also a macrosmatic mammal, the MOB is
18% of the total brain volume (Gittleman, 1991). Interestingly, the MOB in
Megachiroptera is well-developed and comparable to the MOB in the Insectivora (Baron
et al., 1996). In Megachiroptera, approximately 8% of the telencephalon is devoted to the
MOB. In Microchiroptera, only 5% of the telencephalon is devoted to the MOB. In
microsmatic mammals, the MOB percentage is significantly less. For example, in
Primates, on average, only 2.4% of the telencephalon is devoted to the MOB. In simians,
the percentage is greatly reduced to only 0.2% (Stephan et al., 1991). The large
telencephalon volume devoted to olfaction inBlarina suggests that the olfactory system
is very well-developed compared to most other Insectivora, as well as other mammals.
Stephan and co workers (1991) plotted the volume of the MOB against body
weight in a double logarithmic scale resulting in a size index. In Blarina, the MOB size
index was reported to be 89. InNeomys fodiens, a semiaquatic shrew, the MOB index
was 59, a significantly smaller index. Other members of the Insectivora were found to be
comparable to Blarina. In Sorex araneus, the common shrew, the MOB size index was
81 and in S. minutus, the pygmy shrew, the MOB index was 72. The MOB size index for
the house shrew, Suncus murinus was 80. In other Insectivora, the MOB size index of
Talpa europa, the European mole, was only 74 whereas in the hedgehog Erinaceus
europaeus, the index measured 93. Thus it appears that the MOB in Blarina is larger
than most Insectivora.
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4.5 Conclusion
Based upon the high percentage of telencephalon volume and the large MOB size index,
it appears that the MOB of Blarina is well-developed. In addition, the morphological
observations of the MOB ofBlarina confirm the hypothesis that this olfactory system
structure differs from that of other small mammals and its high degree of development
suggests that olfaction is of primary importance in these shrews.
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CHAPTER V
ACCESSORY OLFACTORY BULB
5.1 Introduction
The vomeronasal olfactory system, consisting of the vomeronasal organ (VNO),
accessory olfactory bulb (AOB), and the amygdala, has been investigated in many
mammals including the duck-billed platypus (Smith, 1895), shrew (Crosby and
Humphrey, 1939a; Stephan, 1965), mole (Johnson, 1957), opossum (McCotter, 1912;
Halpem et al., 1998a), bat (Frahm, 1981; Acharya et al., 1998; Bhatnagar and Meisami,
1998), mouse (Barber and Raisman, 1978,1979; Weruaga et al., 2001; Taniguchi and
Kaba, 2001), guinea pig (Sugai et al., 1997), hamster (Davis et al., 1978; Meredith,
1980), ferret (Lockard, 1985), mink (Jeserich, 1945; Salazar et al., 1998), rat (Bums,
1982; Rosselli-Austin et al., 1987; Roos et al., 1988; Takami and Graziadei, 1991), rabbit
(Young, 1934; 1936; Romfh, 1982), dog (Adams and Wiekamp, 1984; Salazar et al.,
1994; Nakajima et al., 1998), cat (Wahle et al., 1990; Won et al., 1997), and the human
(Moran et al., 1991; Smith et al., 1998). The AOB has been proposed to function in
mammalian reproductive behaviors (Wysocki and Meredith, 1991; Jansen et al., 1998;
Dudley and Moss, 1999; Brennan, 2001; Korsching, 2001; Kondo et al., 2003).
The location of the AOB varies among mammals. Crosby and Humphrey (1939a,
b) described the location of the AOB as either an “infolding” into the MOB, such as in
the mouse, rat, and red squirrel, or as an “eminence” on the surface of the MOB. In the
guinea pig (McCotter, 1912; Meisami and Bhatnagar, 1998), the extremely large AOB is
described an eminence on the dorsolateral surface of the caudal MOB. In the rabbit
(Young, 1936) and opossum (McCotter, 1912) the AOB also is reported to be an
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eminence on the dorsomedial surface of the caudal MOB. In the mole and shrew, it
appears that the AOB has developed inside the MOB (Crosby and Humphrey, 1939a, b).
In Blarina, the AOB was located on the dorsomedial surface of the caudal MOB and
appeared to develop inside the MOB.
5.2 Results
5.2.1 Light Microscopy Overview
In a parasagittal section, the AOB was located in the dorsal part of the caudal MOB. The
MOB external plexiform layer, mitral cell layer, and granule cell layer were adjacent to
the rostral AOB (see Fig. 14). Brain landmarks found at the most rostral level of the
AOB (Fig. 44) included the pars lateralis and pars extema of the anterior olfactory
nucleus and the olfactory ventricle. At a more caudal level of the AOB (Fig. 45), the
ventral pars lateralis became crescent-shaped and the middle section of the pars extema
disappeared. The layering of the shrew AOB were much less distinct than that of the
MOB and consisted of the vomeronasal nerve layer (VNL), glomerular layer (GL), a
combined external plexiform layer/mitral-tufted cell layer (EP/M-TCL), internal
plexiform layer (IPL), and granule cell layer (GrL) (Fig. 46). The AOB was shaped like
an inverted triangular with the base adjacent to the inferior border of the frontal cortex.
A myelinated fiber tract passed through the IPL of the AOB (Fig. 47).
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Fig. 44. Light micrograph of the rostral accessory olfactory bulb (arrowhead). Frontal
cortex (FC), pars extema (PE) of the anterior olfactory nucleus, olfactory ventricle
(arrow), pars lateralis (PL) of the anterior olfactory nucleus. 40 pm frozen section,
coronal plane, cresyl-violet stain. 5 mm =165 pm.
Fig. 45. Light micrograph of the caudal accessory olfactory bulb (arrowhead). MOB
granule cell layer (GrL), olfactory ventricle (arrow), pars lateralis (PL), pars externa (PE),
frontal cortex (FC). 40 pm frozen section, coronal plane, cresyl-violet stain.
5 mm = 165 pm.
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EPL/MOB
Fig. 46. Light micrograph of the laminar organization of the accessory olfactory bulb.
Layers outlined by dashed lines include the vomeronasal nerve layer (arrow), glomerular
layer (GL), external plexiform/mitral-tufted cell layer (E), internal plexiform layer (IPL),
granule cell layer (G). Landmarks include the frontal cortex (FC), external plexiform
layer of the main olfactory bulb (EPL/MOB), granule cell layer of the main olfactory
bulb (GrL/MOB), pars extema of the anterior olfactory nucleus (PE) and pars lateralis of
the anterior olfactory nucleus (PL). 40 pm frozen section, coronal plane, cresyl-violet
stain. 5 mm = 40 pm.
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5.2.2 Light Microscopy of Vomeronasal Nerve Layer (VNL)
The vomeronasal nerve layer (VNL) consisted of bundles of unmyelinated axons and
ensheathing cells (Fig. 48). The ensheathing cell nuclei were oval and had distinct
nucleoli. Ensheathing cell nuclei measured up to 8 pm in diameter and were interspersed
among the unmyelinated fibers. In the MOB, two types of ensheathing cells, the dark and
light ensheathing cells, were common. In the AOB, only one type of ensheathing cell
was observed. The AOB ensheathing cells were similar to the dark ensheathing cells of
the MOB. Numerous capillaries were scattered throughout the VNL.
5.2.3 Light Microscopy of Glomerular Layer (GL)
The glomerular layer (GL) consisted of acellular glomeruli and a surrounding neuropil
with periglomerular cells (Fig. 49). The glomeruli were not distinct spherical structures
as in the MOB. The AOB glomeruli consisted of darkly stained vomeronasal axons and
pale staining dendrites. Periglomerular cells had an oval nucleus with a distinct nucleolus
and some heterochromatin. Periglomerular cell nuclei measured up to 8 pm in diameter.
Periglomerular cells were scattered throughout the neuropil of the GL and did not form
the cap-like structures seen in the GL of the MOB.
5.2.4 Light Microscopy of External Plexiform /Mitral-Tufted Cell Layer (EP/M-
TCL)
The EP/M-TCL consisted of a neuropil of axons and dendritic profiles, capillaries, and
mitral/tufted cells. The mitral/tufted cells (Fig. 50) were scattered uniformly throughout
the EP/M-TCL. The large euchromatic nuclei measured up to 9 pm and had distinct
nucleoli. Few dendrites were observed extending from these mitral/tufted cell perikarya.
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Fig. 47. Light micrograph of an overview of the accessory olfactory bulb. Vomeronasal
nerve layer (curved arrow), glomerular layer (GL), external plexiform layer/mitral-tufted
cell layer (E), internal plexiform layer/lateral olfactory tract (arrowhead), granule cell
layer (GrL), MOB granule cell layer (G/MOB), MOB external plexiform layer (E/MOB).
1 pm epoxy resin section, Richardson stain. 5 mm = 28 pm.
Fig. 48. Light micrograph of the vomeronasal nerve layer (VNL). Ensheathing cells
(arrow), MOB external plexiform layer (EPL/MOB). 1 pm epoxy resin section,
Richardson stain. 5 mm = 5 pm.
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Fig. 49. Light micrograph of the glomerular layer. Periglomerular cells (arrowhead) are
interspersed between the glomeruli. Glomeruli consist of dark unmyelinated
vomeronasal axons (A) and pale staining dendrites (arrow). AOB glomeruli are not
distinct spherical structures like MOB glomeruli. 1 pm epoxy resin section, Richardson
stain. 5 mm = 5 pm.
Fig. 50. Light micrograph of the external plexiform/mitral-tufted cell layer (EP/M-TCL).
Numerous mitral/tufted cells (arrowheads), glomerular layer (GL). 1 pm epoxy resin
section, Richardson stain. 5 mm = 6 pm.
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AOB mitral/tufted cells appeared to have less cytoplasm and less prominent dendrites
than the MOB mitral or tufted cells.
5.2.5 Light Microscopy of Internal Plexiform Layer (IPL)
Ventral to the EP/M-TCL was a thick fiber tract consisting of densely packed myelinated
axons (Fig. 51) that formed part of the lateral olfactory tract. The IPL separated the
EP/M-TCL from the underlying region that consisted of the AOB granule cell layer and a
subdivision of the anterior olfactory nucleus.
5.2.6 Light Microscopy of Granule Cell Layer (GrL)
The granule cell layer (Fig. 51) consisted of a cluster of granule cells interspersed among
pale staining dendrites and was located ventral to the IPL. Granule cells were not
organized in the banding pattern characteristic of the MOB GrL. The granule cell nuclei
were oval with scarce amounts of heterochromatin and measured up to 8 pm in diameter.
In the MOB, light and dark granule cells were common. The AOB granule cell was
similar to the light granule cell of the MOB. Interspersed between the granule cells were
pale staining dendrites and small myelinated axons. Adjacent to the granule cells were
large euchromatic nuclei of the pars lateralis neurons of the anterior olfactory nucleus,
5.2.7 Transmission Electron Microscopy of VNL
The unmyelinated vomeronasal axons of the VNL (Fig. 52) ranged from 0.2-0.6 pm in
diameter. Ensheathing cells (Fig.53) had an elongated nucleus with densely stained
heterochromatin. A thin layer of perinuclear cytoplasm contained mitochondria, RER,
scattered free ribosomes, and lipofuscin granules. The AOB ensheathing cells were
similar to the dark ensheathing cells of the MOB.
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Fig. 51. Light micrograph of the granule cell layer. Granule cells (arrowhead) of the
AOB granule cell layer (GrL), internal plexiform layer (IPL), pars lateralis neurons of the
anterior olfactory nucleus (arrow). 1 pm epoxy resin section, Richardson stain.
5 mm = 6 pm.
Fig. 52. TEM micrograph of the vomeronasal nerve layer. Unmyelinated fibers of the
vomeronasal nerve layer (VNL), external plexiform layer of the main olfactory bulb
(EPL/MOB). 5 mm = 0.5 pm.
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Fig. 53. TEM micrograph of ensheathing cells of the vomeronasal nerve layer.
Ensheathing cell nuclei contain electron-dense heterochromatin. Lipofuscin granules
(arrow), unmyelinated axons (A). 5 mm = 0.4 pm.
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5.2.8 Transmission Electron Microscopy of GL
The glomeruli of the GL contained vomeronasal receptor cell axon terminals and
electron-lucent periglomerular cell and mitral/tufted cell dendrites. Mitral/tufted cell
dendrites and periglomerular cell dendrites were morphologically identical. The axon
terminals were electron-dense and contained numerous microtubules and mitochondria.
The dendrites were significantly more pale than the axon terminals and contained
microtubules and mitochondria (Fig. 54). In the glomeruli, asymmetrical axo-dendritic
synapses between vomeronasal axons and mitral/tufted cell and periglomerular cell
dendrites contained small spherical vesicles in the axon terminals (Fig. 55). Dendro-
dendritic synapses between mitral/tufted cells and periglomerular cells were also
observed in the glomerular layer. These synapses were identified by the vesicles
contained within the dendrites. Pleomorphic vesicles were common in symmetrical
synapses (Fig. 56) and predominantly spherical vesicles characterized the asymmetrical
synapses (Fig. 57). Few periglomerular cells were observed in the GL. Periglomerular
cell nuclei (Fig. 58) were typically indented and heterochromatic. The thin layer of
cytoplasm contained RER, free ribosomes and numerous mitochondria. These cells were
more commonly found at the border between the GL and the underlying EP/M-TCL. The
AOB periglomerular cells were not located between adjacent glomeruli nor did these
cells form the cap-like structure noted within the GL of the MOB.
5.2.9 Transmission Electron Microscopy of EP/M-TCL
The EP/M-TCL contained numerous mitral/tufted cells surrounded by a neuropil of
axons, synaptic terminals, and dendrites. Mitral/tufted cells (Fig. 59) were identified by
their spherical euchromatic nuclei and abundant cytoplasm. The cytoplasm contained
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Fig. 54. TEM micrograph of a glomerulus. The glomerulus consists of electron-dense
axon terminals (A) and electron-lucent dendrites (D) of mitral/tufted cells and
periglomerular cells. Mitochondria (arrow) are common in the dendrites.
5 mm = 0.3 pm.
Fig. 55. TEM micrograph of a axo-dendritic synapse. Axo-dendritic asymmetrical
(arrows) synapses originate on vomeronasal axons and terminate on dendrites of
mitral/tufted cells and periglomerular cells. 5 mm = 0.1 pm.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 56. TEM micrograph of a dendro-dendritic synapse. Dendro-dendritic symmetrical
synapses in the glomerular layer are identified by the pleomorphic electron-lucent
vesicles (arrowheads). 5 mm = 0.1pm.
Fig. 57. TEM micrograph of a dendro-dendritic synapse. Dendro-dendritic
asymmetrical synapses (arrow) in the glomerular layer are identified by the spherical
electron-lucent vesicles (arrowheads). 5 mm = 0.1 pm.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 58. TEM micrograph of a periglomerular cell. Nuclear indentations (arrow) are
common in the periglomerular cells. The thin layer of perinuclear cytoplasm contains
numerous free ribosomes and mitochondria. 5 mm = 0.8 pm.
Fig. 59. TEM micrograph of mitral/tufted cells. The cytoplasm typically contains RER
(arrow) and numerous mitochondria (arrowhead). 5 mm = 0.9 pm.
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mitochondria, RER profiles, a well-developed Golgi apparatus, and scattered free
ribosomes. Thinly myelinated axons were common adjacent to mitral/tufted cells.
Numerous dendro-dendritic synapses between mitral/tufted cell dendrites and granule cell
dendrites were observed. Symmetrical dendro-dendritic synapses contained electron-
lucent pleomorphic vesicles within the terminal region (Fig. 60) and asymmetrical
dendro-dendritic synapses contained spherical electron-lucent vesicles (Fig. 61).
5.2.10 Transmission Electron Microscopy of IPL
The internal plexiform layer (Fig. 62) was characterized by densely packed thinly
myelinated axons of the LOT. Mitochondria were scattered throughout the axoplasm.
Pale granule cell dendrites were interspersed in the neuropil of the IPL.
5.2.11 Transmission Electron Microscopy of GrL
The GrL was ventral to the IPL and contained few granule cells (Fig. 63). These cells
typically had oval nuclei and distinct electron-dense nucleoli. The thin layer of
perinuclear cytoplasm contained numerous free ribosomes, sparse amounts of RER, and
numerous mitochondria. The AOB granule cells were similar to the light granule cells of
the MOB. The surrounding neuropil consisted of electron-lucent dendrites and
myelinated axons.
5.3 Discussion
Among the vertebrates, the AOB differs in its shape and size. The shape of the AOB
varies from hemispheric (opossum), oval (rat, rabbit) to cone-shaped (hedgehog). In the
mole, the AOB appears wedge-shaped and in the red squirrel, the AOB is crescent
shaped (McCotter, 1912; Crosby and Humphrey, 1939a; Jeserich, 1945; Johnson, 1957;
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Fig. 60. TEM micrograph of pleomorphic vesicles. Dendro-dendritic symmetrical
synapses in the external plexiform/mitral-tufted cell layer contain pleomorphic electron-
lucent vesicles (arrowhead). 5 mm = 0.1 pm.
Fig. 61. TEM micrograph of spherical vesicles. Dendro-dendritic asymmetrical synapses
in the external plexiform/mitral-tufted cell layer are identified by the presence of
spherical electron-lucent vesicles (arrowhead) in the terminal region. 5 mm = 0.1 pm.
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Fig. 62. TEM micrograph of the internal plexiform layer. The layer consists of densely
packed myelinated axons (AX) of the lateral olfactory tract and dendrites of granule cells.
5 mm = 0.5 pm.
Fig. 63. TEM micrograph of the granule cell layer. Granule cell with a prominent
nucleolus (arrow), myelinated axons of the lateral olfactory tract (AX). 5 mm = 0.5 pm.
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Lockard, 1985; Meisami and Bhatnagar, 1998). The shape of the AOB inBlarina was
similar to the mole and was wedge-shaped or triangular-shaped. Additionally, the size
and development of the AOB varies among vertebrates. In some reptiles, including the
garter snake, copperhead, water moccasin, and Gila monster, the AOB appeared to be
highly-developed. However, in other reptiles, such as the green anole Anolis(
carolinensis), homed lizard ( Phrynosoma cornutum), and American alligator (.American
Mississippiensis) no AOB was observed (Crosby and Humphrey, 1939b). In mammals,
the size and presence of the AOB varies significantly. In the primitive monotremes,
including the duck-billed platypus and ant-eaters, the AOB was reported to be small. In
the marsupial kangaroo and opossum, the AOB is well-developed with distinct layers. In
the carnivorous cat and dog, the AOB is relatively small compared to the large, well-
developed MOB. Significant size variations have been reported in rodents. In the guinea
pig, the AOB is extremely large. The AOB is well-developed and relatively large in the
red squirrel, rat, mouse, and hamster. Among bats, the size of the AOB varies greatly. In
some species of bats the AOB is large, yet in other bat species, the AOB is small. In the
Insectivora, the AOB varies in size and development. In shrews and moles, the AOB was
reported to be small and poorly-developed, but in the hedgehog, the AOB appears to be
large with distinct layers. In primates, significant variations have been reported. The
AOB is present in prosimians and appears large and well-developed. Among the simians,
the AOB is well-developed in the New World monkeys, but in the Old World monkeys,
including the macaque, apes, and humans, the AOB is not present (McCotter, 1912,
Crosby and Humphrey, 1939a, b; Frahm, 1980, 1981; Meisami and Bhatnagar, 1998).
According to Meisami and Bhatnagar (1998), there are marked variations in the size and
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development of the AOB among numerous vertebrates and these differences may
represent differences in sensory discrimination.
Typically, a large AOB has a high degree of lamination with well-defined layers
that are named according to the corresponding laminar organization in the MOB.
Matsuoka and coworkers (1998) identified the VNL, GL, mitral/tufted, GrL, and the
olfactory tract layers in the hamster which has a well-developed AOB. In the rat, the
layers are also well-developed and easily identified (Meisami and Bhatnagar, 1998). In
mammals with a small AOB, distinct layers are more difficult to identify (McLean and
Shipley, 1992; Meisami and Bhatnagar, 1998; Salazar et al., 1998). In the dog, only the
GL and EPL with mitral/tufted cells are readily identified (Salazar et al., 1994). In the
mink, a distinct GL, mitral/tufted cell layer, and GrL have been identified (Salazar et al.,
1998). In some species, AOB layers are thin and poorly-differentiated and discrimination
of separate layers is impractical so several layers are often combined. Meisami and
Bhatnagar (1998) reported a well-developed, large AOB in the hedgehog. However, in
other reports (Shipley et al., 1995; Salazar and Quinteiro, 1998; Brinon et al., 2001), the
EPL, MCL, and the IPL of the hedgehog AOB are not distinct so these layers have been
combined into a single EPL/MCL/IPL complex. In Blarina, the small AOB was similar
to other mammals with a small AOB. The poorly-developed layers were not easily
identified and several layers were combined. The layers of the Blarina AOB included the
VNL, GL, external plexiform/mitral-tufted cell layer, IPL, and GrL.
5.3.1 Vomeronasal Nerve Layer
Vomeronasal nerves belong to the peripheral nervous system and are formed by bundles
of unmyelinated axons that originate from sensory receptor cells located within the
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vomeronasal (Jacobson’s organ) epithelium. These nerves pass through the cribriform
plate of the ethmoid bone, bypass the MOB, and spread out over the surface of the AOB
to form a distinct nerve layer (Meisami and Bhatnagar, 1998). Axon bundles are
separated lfom adjacent bundles by ensheathing cells (Barber et al, 1978; Vaccarezza et
al., 1981; Fraher, 1982; Raisman, 1985; Wysocki and Meredith, 1991; Dudley and Moss,
1999). The ensheathing cells of the VNL are similar to the ensheathing cells found in the
ONL of the MOB (Fraher, 1982). The VNL ensheathing cells inBlarina were similar to
the typical mammalian ensheathing cell. In addition, the unmyelinated axons of the VNL
are reported to be approximately 0.2 pm in diameter in the rat (Raisman, 1985). The
vomeronasal axon diameters inBlarina were larger and ranged from 0.3-0.6 pm in
diameter.
5.3.2 Glomerular Layer
The GL in Blarina was less distinct than the GL of the MOB and consisted of
vomeronasal receptor axons and dendrites of mitral/tufted cells and periglomerular cells.
The AOB glomeruli in Blarina were fewer in number compared to the MOB and were
not spherical structures as in the MOB. Therefore, an accurate measurement of
glomerular diameter was not possible. In addition, there were less periglomerular cells in
the AOB. The periglomerular cell in Blarina measured up to 8 pm in diameter and was
significantly smaller than the periglomerular cells in the rat that measure 7-20 pm in
diameter (Takami et al., 1992). Because these cells did not form the cap-like structures
common on the MOB glomeruli, the AOB glomeruli were not well-delineated. Poorly-
defined AOB glomeruli have been reported in other mammals including the hamster
(Matsuoka et al., 1998), guinea-pig (Sugai et al., 1997), mole (Crosby and Humphrey,
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1939a), and in the rat (Takami and Graziadei, 1990,1991). Short-axon cells are common
in the GL of the mammal MOB but these cells are not common in the AOB.
The neuronal circuitry of the AOB is simple in comparison to the MOB (Scalia
and Winans, 1975,1976; Meredith, 1980; Lehman and Winans, 1982; Clancy et al.,
1984; Brennan et al., 1990; Brennan, 2001). The glomerulus is the site of the first
synaptic contact within the vomeronasal system (Halpem et al., 1995; Halpern et al.,
1998b). Direct projections from the VNO sensory axons synapse on the apical dendrites
of mitral/tufted cells and periglomerular cells within multiple glomeruli of the GL
(Crosby and Humphrey, 1939b; Frahm and Bhatnagar, 1980; Imamura et al., 1985;
Wysocki and Meredith, 1991; Jia and Halpem, 1997; Matsuoka et al., 1997, 1998;
Dudley and Moss, 1999; Sugai et al., 1999). In the mouse (Matsuoka et al., 1998) and in
the rat (Jia et al., 1999) axo-dendritic asymmetrical synapses between the vomeronasal
axons and mitral/tufted cell dendrites have been reported. The dendrites of mitral/tufted
cells and periglomerular cell are reported to be morphologically similar. In Blarina,
similar asymmetrical axo-dendritic synapses were observed. The vomeronasal nerve
axon terminal contained numerous electron-lucent spherical vesicles. In addition,
dendro-dendritic asymmetrical and symmetrical synapses were commonly observed
between periglomerular cells and mitral/tufted cells. These asymmetrical and
symmetrical synapses were similar to the synapses in the MOB.
5.3.3 External Plexiform/Mitral-Tufted Cell Layer
In Blarina, the monolayer of mitral cells forming the mitral cell layer commonly found in
the MOB was not present in the AOB. Rather, the mitral/tufted cells appeared scattered
throughout the EPL. Therefore, this layer was referred to as the external
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plexiform/mitral-tufted cell layer. Poorly-defined layers have been reported in other
mammals. In the dog (Salazar et al., 1994), the EPL and MCL are indistinguishable from
each other. In several bat species with a large AOB, the EPL is compressed between a
thickened GL and MCL (Frahm and Bhatnagar, 1980).
Mitral/tufted cells are reported to be the second order output neurons of the AOB
(Takami and Graziadei 1990, 1991; Bonfanti et al., 1997; Jia et al., 1999). Golgi studies
show that the AOB mitral/tufted cells have up to six apical dendrites that project to
several glomeruli whereas in the MOB, a single apical dendrite enters only one
glomerulus (Jia and Halpem, 1997; Meisami and Bhatnagar, 1998; Keveme, 1999;
Brennan, 2001). Mitral/tufted cells of the rat AOB are large and range from 15-24 pm
(Takami and Graziadei, 1990, 1991). InBlarina, the AOB mitral/tufted cells are
significantly smaller than the rat and measured only up to 9 pm in diameter.
Dendro-dendritic reciprocal synapses have often been reported in the EPL.
Asymmetrical dendro-dendritic synapses originate on the mitral/tufted cell basal
dendrites and the symmetrical dendro-dendritic synapses originate on the granule cell
dendrites (Brennan et al., 1990; Matsuoka et al., 1997, 1998; Jia et al., 1999; Taniguchi
and Kaba, 2001). Similar reciprocal dendro-dendritic synapses were also present in
Blarina. Asymmetrical dendro-dendritic synapses were readily identified by the presence
of spherical vesicles and symmetrical dendro-dendritic synapses were identified by
pleomorphic vesicles.
5.3.4 Internal Plexiform Layer/Myelinated Fiber Tract
In the MOB, the IPL is well-developed and consists of mitral/tufted cell axons and
centrifugal afferents. In mammals with a large AOB such as the rat, a distinct IPL is
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common and lies above the lateral olfactory tract which consists of MOB and AOB
mitral/tufted cell myelinated axons (Price, 1991; Meisami and Bhatnagar, 1998). In
mammals with a less-developed AOB, such as some bat species (Frahm and Bhatnagar,
1980), the IPL is thin and not distinct. In the mouse, the thin IPL is enclosed within the
tract of myelinated axons and is not well-differentiated from the tract (Salazar et al.,
2001). In different species, this myelinated fiber tract may run either through the AOB
between the IPL and the GrL or under the GrL of the AOB which separates the AOB
from the MOB (Meisami and Bhatnagar, 1998). According to Meisami and Bhatnagar
(1998), this myelinated fiber tract runs through the AOB in shrew, hedgehog, and mole.
Other mammals with the through pattern include the tenrecs, bushbaby, guinea pig, rat,
and mouse (Switzer et al., 1980). Interestingly, the tract in the opossum runs under the
AOB and deviates from the through pattern seen in other small mammals. The under
pattern appears to be prevalent in numerous carnivores such as the cat, lion, dog, bear,
and weasel. In Blarina, the IPL and myelinated axons were intermixed and ran through
the AOB, a situation similar to that in the hedgehog and mole.
5.3.5 Granule Cell Layer
The GrL is deep to the layer of myelinated fibers (Barber and Raisman, 1978) and has
been difficult to identify in some species including bats (Frahm and Bhatnagar, 1980;
Kinney, 1982) and dogs (Salazar et al., 1994; Meisami and Bhatnagar, 1998). InBlarina,
the GrL was ventral to the IPL/myelinated fiber tract and consisted of a small clustering
of granule cells. In the MOB, granule cells are grouped together in islets (Meisami and
Bhatnagar, 1998), but in the AOB, the granule cells are too few to form islets. In the cat
(Wahle et al., 1990) and in the dog, (Nakajima et al., 1998), AOB granule cells are
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approximately 10 pm in diameter. In the rat, AOB granule cells have been reported to
range from 7-15 pm in diameter (Rosselli-Austin, 1987; Takami et al., 1992). Struble
and Walters (1982) described by light microscopy two populations of AOB granule cells
in rats. One type, the dark granule cell measured 9 pm in diameter and the second type,
the light granule cell measured 10 pm in diameter. The light granule cells are the
predominant granule cell of the rat AOB. In Blarina, only light granule cells were
observed. These cell diameters were smaller than the light granule cells in the rat and
measured only 8 pm in diameter.
5.4 Comparative Studies
To compare the AOB of various mammals, the volume of the AOB is plotted against the
body weight in double logarithmic scales (Stephan et al., 1991). According to Stephan
and coworkers (1991), the AOB has been found in all Insectivora investigated. The size
of the AOB ranges from very small to rudimentary. The AOB size index forBlarina is
71 which is lower than in other shrews. For example, Sorex fumeus, the smoky shrew,
has an index of 93. Other shrews with varying habitats have a significantly higher AOB
index compared to Blarina and include Sorex araneus (157), Suncus etruscus (129), and
Neomys fodiens (144). Two Insectivora are closely related to Blarina, the European
hedgehog, Erinaceus europaeus, and the European mole, Talpa europaea, have AOB
indices of 205 and 143 respectively. Based upon these indices, it appears that no
relationship can be established between AOB size and habitat of the Insectivora. Stephan
and coworkers (1982) reported that the average AOB size index for prosimians was 354
and the average for simians was 56. Stephan and coworkers (1991) also reported a
significantly larger AOB in the prosimians and a much smaller AOB in the simians. This
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gradual decrease results in the absence of the AOB in humans. Since the AOB functions
in sexual behaviors, Stephan and coworkers (1982) suggest that the visual system has
compensated for the reduction of the AOB in those mammals, including humans, with a
small, poorly-developed AOB. There appears to be significant variations in the size and
development of the AOB in the vertebrates. In some reptiles, the AOB is large but in
others, such as the alligator, the AOB is absent. In the various birds investigated by
Crosby and Humphrey (1939a), no AOB was reported. In mammals, the primitive
monotremes have a small AOB. A small AOB has also been reported in various
carnivores. In simians, a large and a small AOB have been reported. In marsupials,
rodents, and prosimians, the AOB appears to be large and well-developed. Although
there are exceptions, it appears that primitive mammals have a small, poorly-developed
AOB and the evolutionary advanced mammals have a large, well-developed AOB.
5.5 Conclusion
The AOB in Blarina is a small, wedge-shaped structure similar to that of the mole AOB.
Compared to the rat which has a well-developed AOB, the cells of the AOB in Blarina
are consistently smaller than the cells of the rat AOB. The layers of theBlarina AOB are
also less-differentiated than the corresponding layers of the MOB. In addition, the
glomeruli of the GL are poorly defined. The low AOB index in Blarina and the
morphological observations by light and electron microscopy indicate that the AOB in
Blarina is a poorly-developed structure that is similar to other Insectivora but differs
significantly from other small mammals such as the rat. Since this accessory olfactory
system inBlarina is poorly developed while the main olfactory system is highly
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developed, these small mammals may be depending upon their main olfactory system and
the trigeminal complex for olfactory acuity and not the accessory system.
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CHAPTER VI
ANTERIOR OLFACTORY NUCLEUS
6.1 Introduction
Herrick (1924) first described the gray matter in the olfactory peduncle, just caudal to the
MOB as a nucleus and termed the structure the “nucleus olfactorius anterior.” This area,
currently known as the anterior olfactory nucleus (AON) is now recognized actually to be
a cortical structure, not a nucleus. This bilayer structure consists of several divisions,
each made up of an outer plexiform layer and an inner layer densely packed with
pyramidal cells (Shipley and Ennis, 1996). The plexiform layer consists of pyramidal
cell dendrites and axons as well as terminals of the lateral olfactory tract axons (LOT).
The AON is a major relay in the olfactory pathway. In addition to input from the MOB
via the LOT, the AON also receives inputs from the ipsilateral and contralateral AON,
the piriform cortex, and olfactory tubercle (Valverde et al., 1989; Shipley and Ennis,
1996). The AON is divided into subdivisions that are named based upon their location
with respect to the anterior commissure and the olfactory ventricle. Exact boundaries
between the subdivisions have not been precisely defined and controversy regarding the
specific names applied to the various subdivisions remains a current problem (Bayer,
1986; Barbado et al., 2001). For example, in the ferret, the AON is divided into the
dorsal, dorsomedial, ventromedial, ventral, lateral, externa, and, the most caudal, the
posterior subdivisions (Lockard, 1985). In the rat, six subdivisions have been identified
which include the externa, lateralis, dorsalis, medialis, ventralis and posterior subdivision
(Bayer, 1986; Garcia-Ojeda et al., 1998). Some researchers have combined the ventralis
and posterior subdivisions in the rat into the ventroposterior subdivision (Reyher et al.,
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1988; Barbado et al., 2001). In the hedgehog, a close relative of the shrew, the AON
subdivisions include the bulbaris, principalis which includes the dorsalis, lateralis,
mediali s, ventralis, the externa, and posterior. The pars bulbaris consists of AON fibers
coursing through the anterior commissure at the level of the olfactory ventricle (Valverde
et al., 1989).
The AON has been investigated in numerous mammalian species including the
platypus (Hines, 1929), opossum (Herrick, 1924; Meyer, 1981; Shammah-Lagnado and
Negrao, 1981), hedgehog (De Carlos et al., 1989; Valverde et al., 1989; Radtke-Schuller
and Kunzle, 2000), mole (Johnson, 1957b), hamster (Ferrer, 1969; Devor, 1976; Davis
and Macrides, 1981; Schoenfeld andMacrides, 1984), guinea pig (Johnson, 1957a), ferret
(Lockard, 1985), rabbit (Young, 1934; Broadwell, 1975b; Mori et al., 1979; Ojima et al.,
1984), rat (Haberly and Price, 1978b; Bennett, 1968; Shafa and Meisami, 1977; De
Olmos et al., 1978; Luskin and Price, 1983a, b; Alheid et al., 1984; Friedman and Price,
1984; Scott et al., 1985; Reyher et al., 1988; Brown and Brunjes, 1990; Garcia-Ojeda et
al., 1998; Barbado et al., 2001), mouse (Creps, 1974), cat (Mascitti and Ortega, 1966;
Ryu, 1980), rhesus monkey (Bassett et al., 1992), and human (Crosby and Humphrey,
1941; Ohm et al., 1991).
According to Crosby and Humphrey (1939a) the most rostral subdivision of the
northern short-tailed shrew {Blarina brevicauda) AON, the pars lateralis, appears as a
mass of densely arranged cells within the inner regions of the granule cell layer of the
MOB, lateral to the olfactory ventricle. The second subdivision to appear is the pars
extema which is separated from the pars lateralis by a fiber mass. Moving caudalward,
the middle region of the pars extema disappears and only the dorsal and ventral limbs of
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the pars extema remain present. At this level, the pars lateralis begins to change shape
forming a crescent-shaped structure with the concave side open to the olfactory ventricle.
The crescent-shaped region of AON is subdivided into the pars dorsalis, pars lateralis,
and pars ventralis. A large portion of MOB remains on the ventromedial surface of each
hemisphere at this caudal level. The tips of the crescent-shaped AON begin to turn
inward toward each other and merge, forming a ring around the olfactory ventricle. The
region where the tips merge forms the pars medialis subdivision. At this level, the rostral
piriform cortex also is present lateral to the AON. As the piriform cortex becomes well-
developed, the pars lateralis disappears, the pars dorsalis merges with the frontal cortex,
and the pars medialis is replaced by the septal area and the nucleus accumbens. The pars
ventralis which is dorsal to the olfactory tubercle is gradually replaced by the caudate.
The AON of the mole (Crosby and Humphrey, 1939a) is comparable to the shrew except
that the pars posterior is the caudal continuation of the pars ventralis and is located
between the caudate nucleus and the polymorph layer of the olfactory tubercle. Crosby
and Humphrey (1939a) found no pars posterior in the short-tailed shrews examined.
The northern short-tailed shrews {Blarina brevicauda) and the southern short
tailed shrews ( B. carolinensis) examined in this project had an AON structure similar to
the mole as reported by Crosby and Humphrey (1939a). The AON subdivisions in
Blarina included the pars lateralis, extema, dorsalis, ventralis, medialis, and posterior.
6.2 Results
6.2.1 Cresyl Violet Rostral to Caudal Series
The pars lateralis (PL), the most rostral subdivision of the AON was found deep within
the granule cell layer in the MOB (Fig. 64) and was rostral to the accessory olfactory
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bulb (AOB). The PL consisted of mass of cells lateral to the olfactory ventricle and
subventricular zone and was separated from the surrounding granule cell layer by a
region of neuropil. Moving in a caudalward direction, the pars extema (PE) subdivision
(Fig. 65) was just ventral to the rostral part of the AOB and dorsolateral to the PL.
Continuing caudalward to the level of the caudal part of the AOB, the mid-region of the
crescent-shaped PE (Fig. 66) disappeared creating dorsal and ventral limbs. The dorsal
limb was immediately ventral to the AOB. The ventral limb was significantly larger than
the dorsal limb and was dorsolateral to the PL. The dorsolateral MOB layers began to
disappear at this more caudal level. The olfactory ventricle remained present and was
surrounded by the subventricular zone. Caudalward, only a small portion of the ventral
pars extema remained and was located just dorsal to the MOB granule cell layer. The
MOB was crescent-shaped at this caudal level and the MOB layers continued to
disappear on the dorsolateral side. The caudal PL was crescent-shaped (Fig. 67) with the
concave side facing the olfactory ventricle. At this level, the PL could be divided into the
pars dorsalis (PD), PL, and pars ventralis (PV). The PL continued to change its shape at
a more caudal level, changing from the crescent shape to a ring-like structure (Fig. 68)
that surrounded the olfactory ventricle. The tips of the crescent merged to become the
pars medialis (PM) subdivision. The PM was dorsolateral to the remaining crescent
shaped caudal MOB. The rostral piriform cortex was lateral to the AON at this level.
The ventral-most remnant of the PE was dorsal to the granule cell layer. At a more
caudal level, the PD began to merge with the frontal cortex (Fig. 69). At this level, the
ring-like structure of the AON completely encircled the olfactory ventricle. The caudal-
most MOB remained crescent-shaped at this level. At the caudal level where the pars
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 64. Light micrograph of the rostral pars lateralis of the anterior olfactory nucleus.
The pars lateralis (PL) begins dorsal and lateral to the olfactory ventricle (arrow), deep
within granule cell layer (GrL) of the MOB. The frontal cortex (FC) is dorsal to the
MOB. The medial surface of the MOB is to the left. 40 pm frozen section, coronal
plane, cresyl-violet stain. 5 mm = 250 pm.
Fig. 65. Light micrograph of the rostral pars externa of the anterior olfactory nucleus.
The pars extema (PE) appears at the level of the accessory olfactory bulb (arrowhead).
Pars lateralis (PL), olfactory ventricle (arrow), frontal cortex (FC). 40 pm frozen section,
coronal plane, cresyl-violet stain. 5 mm = 250 pm.
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FC FC
Fig. 66. Light micrograph of the mid-caudal pars extema. The mid-region of the
crescent-shaped PE disappears and is subdivided into a dorsal (curved arrow) and ventral
limb (PE), pars lateralis (PL), olfactory ventricle (arrow), accessory olfactory bulb
(arrowhead), frontal cortex (FC). 40 pm frozen section, coronal plane, cresyl-violet stain.
5 mm = 250 pm.
Fig. 67. Light micrograph of the rostral pars dorsalis and pars ventralis. Pars lateralis
(PL) is divided into pars dorsalis (PD) and pars ventralis (PV). Subventricular zone
surrounds the olfactory ventricle (arrow), pars extema (PE), frontal cortex (FC). 40 pm
frozen section, coronal plane, cresyl-violet stain. 5 mm = 250 pm.
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v
FC FC
PD PL PL
Fig. 68. Light micrograph of the rostral pars medialis. The caudal AON is subdivided
into the pars dorsalis (PD), pars medialis (PM), pars ventralis (PV), and PL. Frontal
cortex (FC), piriform cortex (PC), ventral pars extema (arrow). 40 pm frozen section,
coronal plane, cresyl-violet stain. 5 mm = 250 pm.
Fig. 69. Light micrograph of the mid-caudal pars medialis. The pars medialis (PM) is
located between the olfactory ventricle and the MOB granule cell layer. The caudal PD
is continuous with the frontal cortex (FC). The piriform cortex (PC) is dorsal to the pars
ventralis (PV). Pars Lateralis (PL). 40 pm frozen section, coronal plane, cresyl-violet
stain. 5 mm = 250 pm.
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dorsalis was continuous with the frontal cortex, the PM and PV (Fig. 70) were
dorsolateral to the thin crescent-shaped MOB. The rostral lateral ventricle was occluded.
The PM and PV merged and formed the pars posterior (PP) (see Fig. 17). This most
caudal AON subdivision was dorsolateral to the caudal-most part of the MOB and ventral
to the lateral ventricle. The trilaminar piriform cortex was well-developed at this level.
Moving caudalward, the PP remained ventral to the lateral ventricle. The piriform cortex
was well-developed and distinct. At the caudal level where the MOB completely
disappeared, the PP (Fig. 71) was ventral to the open lateral ventral and dorsal to the
rostral olfactory tubercle.
6.2.2 Light Microscopy of Pars Lateralis (PL)
The PL (Fig. 72) was located deep within the MOB, dorsolateral to the olfactory ventricle
and its surrounding subventricular zone. The olfactory ventricle was lined with flattened
ependymal cells (Fig. 73). Numerous cilia extended into the open ventricle. A region of
small myelinated axons separated the PL from the olfactory ventricle and subventricular
zone of the MOB. The pyramidal cells (Fig. 74) of the PL had spherical euchromatic
nuclei with distinct nucleoli. These cells measured up to 12 pm in diameter. A
distinctive apical dendrite extended from the pyramidal cells in a dorsolateral direction
toward the PE. Basal dendrites extended radially into the surrounding neuropil of the PL.
The neuropil between the PL and PE consisted of numerous pale dendrites and small
myelinated axons. In the region of the neuropil closest to the PL, the dendrites were
larger in diameter and longer in profile. In the neuropil region closest to the PE, the
dendrites were smaller in diameter and shorter in profile.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 70. Light micrograph of the caudal pars medialis. The caudal pars medialis (PM)
and caudal pars ventralis (PV) remain ventral to the occluded lateral ventricle (arrow).
Piriform cortex (PC), frontal cortex (FC). 40 pm frozen section, coronal plane, cresyl-
violet stain. 5 mm = 250 pm.
Fig. 71. Light micrograph of the pars posterior. The caudal-most pars posterior (PP) is
ventral to the caudate-putamen (curved arrow) and dorsal to the olfactory tubercle (OT).
Piriform cortex (PC), corpus callosum (CC), lateral ventricle (arrow). 40 pm frozen
section, coronal plane, cresyl-violet stain. 5 mm = 250 pm.
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Fig. 72. Light micrograph of the olfactory ventricle and subventricular zone. The pars
lateralis (PL) is dorsolateral to the olfactory ventricle (OV) and subventricular zone
(SVZ). Clusters of myelinated axons (arrow) separate the PL from the ventricular region.
PL dendrites (arrowhead) extend away from the olfactory ventricle. 1 pm epoxy resin
section, Richardson stain. 5 mm = 33 pm.
Fig. 73. Light micrograph of ependymal cells. The olfactory ventricle is lined with
ependymal cells (arrow) with distinct nucleoli. Cilia (arrowhead) extend into the open
lumen. Myelinated axons (curved) in the surrounding neuropil. 1 pm epoxy resin
section, Richardson stain. 5 mm = 5 pm.
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6.2.3 Light Microscopy of Pars Externa (PE)
The PE consisted of neurons and an adjacent neuropil. The neurons of the PE had
euchromatic nuclei with a distinct nucleolus. These cells measured up to 9 pm in
diameter. The PE neurons differed from the pyramidal cells of the PL. The PE neurons
had a thin layer of perinuclear cytoplasm. Dendrites extending from the PE neurons were
not commonly observed. The dorsal limb of the PE (Fig. 75) was ventral to the AOB and
the internal plexiform layer and lateral olfactory tract. The ventral limb was adjacent to
the granule cell layer of the MOB. The neuropil (Fig. 76) of the PE consisted of
myelinated axons, dendrites, capillaries, and several oligodendrocytes.
6.2.4 Light Microscopy of Pars Dorsalis (PD), Pars Medialis (PM), Pars Ventralis
(PV), Pars Posterior (PP)
The PD (Fig. 77) consisted of pale-staining neurons and a surround neuropil. The
neurons had spherical nuclei with a distinct nucleolus. These neurons were similar to the
pyramidal cells of the PL in size and morphology but often lacked apical dendrites.
Occasional basal dendrites extended radially from the PD neurons. The neuropil
contained dendrites and few myelinated axons.
The PM (Fig. 78) contained pyramidal cells with spherical, euchromatic nuclei.
A distinct nucleolus was common. The pyramidal cells of the PM were similar to the
pyramidal cells of the PL. Apical dendrites extended dorsomedial from the olfactory
ventricle towards the granule cell layer o f the MOB. Basal dendrites were radially
oriented. The surrounding neuropil consisted of numerous dendrites and small
myelinated axons.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 74. Light micrograph of pyramidal cells of the pars lateralis. Pyramidal cell apical
dendrites (arrows) extend to the pars externa. Basal dendrites (arrowhead) extend
radially in the neuropil. 1 pm epoxy resin section, Richardson stain. 5 mm = 5 pm.
Fig. 75. Light micrograph of the dorsal limb of the pars externa. The dorsal pars externa
(PE) is ventral to the accessory olfactory bulb (AOB). Internal plexiform layer of the
AOB/lateral olfactory tract (arrowhead). 1 pm epoxy resin section, Richardson stain.
5 mm = 12 pm.
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Fig. 76. Light micrograph of the ventral limb of the pars externa. The ventral pars
externa (PE) is surrounded by a neuropil of dendrites (arrow) and small myelinated axons
(arrowhead). 1 pm epoxy resin section, Richardson stain. 5 mm = 5 pm.
Fig. 77. Light micrograph of pyramidal cells of the pars dorsalis. Basal dendrites
(arrow) extend radially with no specific orientation. The neuropil consists of dendrites
(arrowhead) and myelinated axons (curved arrow). 1 pm epoxy resin section, Richardson
stain. 5 mm = 4 pm.
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The PV (Fig. 79) was comparable to the PM. The pyramidal cells had
euchromatic nucleoli and were similar to the pyramidal cells of the PL. The apical
dendrite extended dorsomedial from the olfactory ventricle towards the granule cell layer
of the MOB. Basal dendrites extended radially from these pyramidal cells. The neuropil
consisted of pale-staining dendrites and small myelinated axons.
The pyramidal cells of the PP (Fig. 80) were similar to the pyramidal cells of the
PL, PM, and PV in size and morphology. The PP pyramidal cells typically had a
euchromatic nucleus with a distinct nucleolus. The apical dendrite extended
ventromedially towards the remaining crescent-shaped granule cell layer of the MOB.
Basal dendrites extended radially and lacked orientation. The neuropil consisted of pale-
staining dendrites and small myelinated axons.
6.2.5 Transmission Electron Microscopy of PL
The pyramidal cells of the PL (Fig. 81) had a spherical, euchromatic nucleus with a
narrow rim of cytoplasm. Ribosomes and mitochondria were common. Apical dendrites
extended from these pyramidal cells. Axo-somatic synapses were observed on the
pyramidal cells. The neuropil of the PL (Fig. 82) consisted of myelinated and
unmyelinated axons, dendrites and synaptic junctions. Axo-dendritic asymmetrical
synapses were commonly observed. The axon terminal was densely packed with small
spherical electron-lucent vesicles. The asymmetrical membrane thickening was
characterized by electron density within the postsynaptic region.
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Fig. 78. Light micrograph of pyramidal cells of the pars medialis. Apical dendrites
(arrows) extend dorsomedially to the granule cell layer of the MOB. Basal dendrite
(arrowhead). Small myelinated axon (curved arrow) in the neuropil. 1 pm epoxy resin
section, Richardson stain. 5 mm = 4 pm.
Fig. 79. Light micrograph of pyramidal cells of the pars ventralis. Apical dendrites
(arrow) extend dorsomedially to the MOB granule cell layer. Basal dendrites
(arrowheads). Small myelinated axons (curved arrow). 1 pm epoxy resin section,
Richardson stain. 5 mm = 5 pm.
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Fig. 80. Light micrograph of pyramidal cells of the pars posterior. Apical dendrites
(arrows) extend dorsomedially to the granule cell layer of the MOB. Basal dendrites
(arrowheads). Small myelinated axons (curved arrow). 1 pm epoxy resin section,
Richardson stain. 5 mm = 5 pm.
Fig. 81. TEM micrograph of pars lateralis pyramidal cell with apical dendrite (arrow).
Small myelinated axons (curved arrow) and dendrites (arrowhead) in the surrounding
neuropil. 5 mm = 1 pm.
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6.2.6 Transmission Electron Microscopy of PE
The PE pyramidal cells (Fig. 83) typically had euchromatic nuclei. A well-developed
Golgi was adjacent to the nucleus. These neurons had just a narrow rim of cytoplasm.
Ribosomes were organized into rosettes and were evenly distributed throughout the
cytoplasm. Numerous mitochondria were common. Axo-somatic synapses were
identified. The surrounding neuropil (Fig. 84) contained myelinated and unmyelinated
axons and electron-lucent dendrites. Asymmetrical axo-dendritic synapses consisted of a
postsynaptic membrane thickening and clusters of spherical vesicles within the
presynaptic axon terminal.
6.2.7 Transmission Electron Microscopy of PD, PM, PV, PP
The pyramidal cells of the PD (Fig. 85) had a euchromatic nucleus surrounded by a thin
rim of cytoplasm. A well-developed Golgi was adjacent to the nucleus. Rosettes of
ribosomes and numerous mitochondria were common. Axo-somatic asymmetrical
synapses were common. The neuropil (Fig. 85) of the PD consisted of myelinated and
unmyelinated axons and dendrites. Axo-dendritic asymmetrical synapses were
commonly observed. The terminal region was densely packed with small electron-lucent
spherical vesicles.
The PM, PV, and PP consisted of pyramidal cells similar to the pyramidal cells of
the PL (see Fig. 81). The spherical, euchromatic nucleus was surrounded by a thin layer
of cytoplasm densely populated with ribosomes and mitochondria. Apical dendrites
extended from these pyramidal cells. Axo-somatic synapses were common. The
neuropil of the PM, PV, and PP was similar to the neuropil of the PL and consisted of
myelinated and unmyelinated axons, dendrites and synaptic junctions. Axo-dendritic
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Fig. 82. TEM micrograph of synapses in the pars lateralis neuropil. Axo-dendritic
asymmetrical synapses (arrowheads). Spherical vesicles (arrow) in the terminal region.
5 mm = 0.4 pm.
Fig. 83. TEM micrograph of pars externa pyramidal cell. Small myelinated axon
(arrow), axo-somatic synapse (curved arrow). 5 mm = 0.6 pm.
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Fig. 84. TEM micrograph of synapses in the pars externa neuropil. Asymmetrical
synapses consist of postsynaptic density (arrows) and clusters of electron-lucent vesicles
in the presynaptic axon terminal (arrowhead). 5 mm = 0.2 pm.
Fig. 85. TEM micrograph of pars dorsalis pyramidal cell. The neuropil consists of small
myelinated axons (curved arrow) and unmyelinated axons and dendrites. Asymmetric
axo-dendritic synapse with spherical vesicles in the terminal region (arrow).
5 mm = 0.8 pm.
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asymmetrical synapses were common. The axon terminals were densely packed with
small spherical vesicles.
6.3 Discussion
The AON of Blarina had comparable subdivisions to the AON of other mammals
(Herrick, 1924; Haberly and Price, 1978b; Valverde et al., 1989) but the rostral-caudal
extent of the AON inBlarina appeared to be significantly larger. The most rostral AON
subdivision ofBlarina was the PL which appeared deep within the granule cell layer of
the MOB, rostral to the AOB. The PL was completely encircled by the well-developed
MOB. In the hedgehog (Valverde et al., 1989), opossum (Herrick, 1924), and mole
(Crosby and Humphrey, 1939a), the rostral PL also appears deep within the MOB rostral
to the AOB. However, in the mouse (Crosby and Humphrey, 1939a) and rat (Bums,
1982), this most rostral AON subdivision first appears more caudally at the level of the
AOB and is only partially surrounded by the MOB since the lateral MOB has
disappeared. The most caudal AON subdivision in Blarina was the PP. The PP appeared
at the level of the rostral olfactory tubercle and mid-caudal piriform cortex. The caudal
AON of the hedgehog (Valverde et al., 1989), opossum (Herrick, 1924), mole (Crosby
and Humphrey, 1939a), mouse (Crosby and Humphrey, 1939a), and rat (Bums, 1982)
also appears at the rostral olfactory tubercle and mid-caudal piriform cortex. In these
Insectivora (Crosby and Humphrey, 1939a; Valverde et al., 1989) and marsupials
(Herrick, 1924), the AON first appears at a significantly more rostral level than in the rat
(Bums, 1982) and the MOB is well-developed from the rostral to caudal levels of the
AON. In Blarina, the rostral appearance of the AON and the well-developed MOB were
similar to the observations of Crosby and Humphrey (1939a) and Valverde and
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coworkers (1989). Furthermore, the general appearance of the AON inBlarina and these
Insectivora and marsupials was rotated counter-clockwise dorsolaterally to accommodate
the caudal MOB. Since the MOB of the rat and mouse disappeared more rostrally than in
these Insectivora and marsupials, rotation of the AON was not observed in the rat and
mouse.
The AON participates in numerous afferent and efferent projection pathways.
The MOB is a major projection source to the AON. These MOB projections travel via
two pathways, the anterior limb of the anterior commissure and the lateral olfactory tract
(Mascitti and Ortega, 1966; Bennett, 1968; Turner et al., 1978; Ojima et al., 1984). All
subdivisions of the AON except the PE receive direct input from the ipsilateral mitral and
tufted cell axons and collaterals of the MOB. All subdivisions of the AON receive
indirect input from the contralateral MOB (Mascitti and Ortega, 1966; Scalia and
Winans, 1975; Davis et al., 1978; De Olmos et al., 1978; Meyer, 1981; Schneider and
Scott, 1983; Ojima et al., 1984; Shipley and Adamek, 1984; Scott et al, 1985; Bayer,
1986; Brown and Brunjes, 1990; Nickell and Shipley, 1992; McLean and Shipley, 1992;
Jansen et al., 1998; Barbado et al., 2001). In additional to the MOB projections, the AON
receives projections from the ipsilateral and contralateral AON, the piriform cortex, and
the olfactory tubercle (Haberly and Price, 1978b; Luskin and Price, 1983b; Schwob and
Price, 1984; Barbado et al., 2001). The MOB receives bilateral AON projections to the
contralateral MOB via the anterior commissure and to the ipsilateral MOB (Broadwell,
1975b; MacLeod, 1977; Shafa and Meisami, 1977; De Olmos et al., 1978; Mori et al.,
1979; Davis and Macrides, 1981; Macrides et al., 1981; Luskin and Price, 1983a; Alheid
et al., 1984; Carson, 1984; Schwob and Price, 1984; Bayer, 1986; McLean and Shipley,
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1992; Reyher et al., 1988; Nickell and Shipley, 1992). The AON does not project to the
AOB (McLean and Shipley, 1992). According to Haberly and Price (1978b), the
extensive projections of the AON serve to coordinate olfactory information on both sides
of the brain.
6.3.1 Pars Lateralis (PL) and Subdivisions
The PL was the most rostral subdivision in Blarina. In other macrosmatic mammals
including the mole (Crosby and Humphrey, 1939a), hedgehog (Valverde et al., 1989),
and rat (Haberly and Price, 1978b; Bums, 1982; Brown and Brunjes, 1990), the PL also
is the most rostral subdivision and consists of a mass of pyramidal neurons located lateral
to the olfactory ventricle. The olfactory ventricle is a rostral extension of the lateral
ventricle is lined with ependymal cells that are ultrastmcturally similar to ependymal
cells lining the ventricles of the brain and the central canal of the spinal cord (Brightman
and Palay, 1963; Allen and Low, 1973; Bruni et al, 1973; Scott et al., 1974; Hannah and
Geber, 1977; Kaplan, 1980; Masuzawa et al., 1981; Bruni et al., 1985; Bruni and Reddy,
1987). In the rat (Garcia-Ojeda et al., 1998), the apical dendrites of the PL pyramidal
cells extend dorsolaterally from the olfactory ventricle towards the PE. InBlarina, the
distinct apical dendrites also extend dorsolaterally from the olfactory ventricle towards
the PE. In other mammals, the PL is not the most rostral subdivision to appear. In the
giant panda (Lauer, 1949) and the cat (Kinney, 1982), the pars dorsalis appears as the
most rostral subdivision. Moving in a caudalward direction, in Blarina, the PL changed
from a nearly linear layer of pyramidal cells into a crescent shape consisting of the PD,
PL, and PV subdivisions followed by the formation of a ring-like structure surrounding
the olfactory ventricle. At this level, the PM was lateral to the olfactory ventricle. This
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pattern of the caudal PL, PD, PV, and PM is also observed in the mole (Crosby and
Humphrey, 1939a), hedgehog (Valverde et al., 1989), opossum (Herrick, 1924) and rat
(Haberly and Price, 1978b). InBlarina, the PD was continuous with the frontal cortex
which is also observed in numerous mammals (Herrick, 1924; Crosby and Humphrey,
1939a; Romfh, 1982; Valverde et al., 1989; Brown and Brunjes, 1990). In the mouse and
mole (Crosby and Humphrey, 1939a), the PM and PV merge to form the most caudal
subdivision, the PP. In Blarina, the PM and PV also merge and form the PP. In the rat
(Brown and Brunjes, 1990) the PP is the caudal continuation of the PV. The pyramidal
cells of the caudal subdivisions PM, PV, and PP extend apical dendrites oriented
dorsomedially towards the tenia tecta. Basal dendrites extend from the cell body without
any specific orientation. The pyramidal cell axons extend towards the olfactory ventricle
and anterior commissure (Herrick, 1924, Haberly and Price, 1978b). InBlarina the
apical dendrites of these caudal subdivisions also extend ventromedially away from the
olfactory ventricle towards the crescent-shaped caudal MOB.
Mascitti and Ortega (1966) report axo-somatic and axo-dendritic synapses on the
neurons of the PL. In Blarina, asymmetrical axo-somatic and axo-dendritic synapses
were also identified. Electron-lucent spherical vesicles in the axon terminal and
increased electron density in the postsynaptic region were commonly observed. These
asymmetrical synapses were similar to the asymmetrical synapses observed in the MOB.
6.3.2 Pars Externa (PE)
The PE, due to its position and neurons, is the only AON subdivision that is noticeably
distinguished from the other AON subdivisions (Broadwell, 1975b; Barbado et al., 2001).
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The PE is ventral to the AOB and is in close proximity to the granule cell layer of the
MOB (Young, 1934; MacLeod, 1977; Davis and Macrides, 1981; Bayer, 1986; Reyher et
al., 1988). InBlarina, the PE was similar in shape and location to the PE of other
mammals. In the rat (Bums, 1982), the mole and shrew (Crosby and Humphrey, 1939a),
and the hedgehog (Valverde et al., 1989) the PE is the second AON subdivision to
appear. In Blarina, the PE also was the second subdivision to appear. In the shrew and
the mole (Crosby and Humphrey, 1939a) and in the opossum (Herrick, 1924), moving in
a caudalward direction, the middle region of the PE disappears, leaving only a dorsal and
ventral limb of the PE. This pattern was followed in Blarina. The dorsal limb of the PE
was located immediately ventral to the AOB and the ventral limb was dorsal to the
granule cell layer of the MOB. In the rat (Haberly and Price, 1978b; Reyher et al., 1988),
the neurons of the PE differ from the neurons of the PL, PD, PM, PV, and PP. The PE
neurons lack basal dendrites. The apical dendrites of these neurons extend away from the
olfactory ventricle. Similarly, in the opossum (Herrick, 1924), the PE dendrites extend
away from the MOB and penetrate the GrL of the MOB. Axons of the PE neurons
extend toward the anterior commissure. In Blarina, by light microscopy, apical dendrites
of the PE neurons were not evident.
The PE is reported to be well-developed in macrosmatic mammals with axo-
somatic and axo-dendritic synapses (Lauer, 1949; Mascitti and Ortega, 1966). In
Blarina, similar axo-somatic and axo-dendritic synapses were commonly identified in the
PE. Both types of synapses were characterized as asymmetrical synapses and contained
predominantly electron-lucent spherical vesicles and increased electron density in the
membrane thickening of the postsynaptic region.
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The PE provides a pathway of communication between the two MOBs within
macrosmatic mammals (Schoenfeld and Macrides, 1984; Reyher, 1988). The PE receives
ipsilateral and contralateral input from MOB mitral and tufted cell axons of the lateral
olfactory tract and contralateral input from most subdivisions of the AON (Schoenfeld
and Macrides, 1984; Barbado et al., 2001). The PE projects extensively to the
contralateral MOB and terminates within the granule cell layer. This subdivision does
not appear to project to any other AON subdivisions or to the piriform cortex. A lesion to
MOB and AON results in contralateral degeneration in the PE (Herrick, 1924; Mascitti
and Ortega, 1966; Shafa and Meisami, 1977; Davis and Macrides, 1981; Luskin and
Price, 1983a; Alheid et al., 1984; Carson, 1984; Schoenfeld and Macrides, 1984; Bayer,
1986; Reyher et al., 1988; De Carlos et al., 1989; Brown and Brunjes, 1990; Nickell and
Shipley, 1992). No PE has been identified in the adult human or in the macaque which
may contribute to a poorly-developed sense of olfaction in these microsmatic mammals
(Crosby and Humphrey, 1941; Lauer, 1945).
6.4 Comparative Studies
To compare the AON of various mammals, two methods have been used-percentages and
size indices. In this second method, the volume of the AON is plotted against the body
weight in double logarithmic scales (Baron et al., 1987; Stephan et al., 1991). Baron and
coworkers (1987) report that the AON (retrobulbar region) index inBlarina brevicauda
had a value o f111 . Sorex fumeus, the smoky shrew, with anAON index o f115 is
comparable to Blarina, Other shrews including Sorex araneus (93), Suncus etruscus
(73), and Neomys fodiens (81) have smaller AON indices thanBlarina. Thus it appears
that based upon the size index, Blarina has a larger AON than most shrews. Two
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insectivores that are closely related toBlarina , the European hedgehog, Erinaceus
europaeus, and the European mole, Talpa europaea, have an index of 116 and 87
respectively. Comparing terrestrial and semiaquatic shrews, the AON index in terrestrial
insectivores is significantly higher (108) than in the semiaquatic insectivores (64). Based
upon these indices, the terrestrial shrews, which include Blarina, have a well-developed
AON that greatly assists these macrosmatic mammals. In addition, the AON index in
Blarina is significantly higher than in microsmatic mammals such as the macaque (8) and
human (18). These low indices appear to indicate a poorly-developed AON. Baron and
coworkers (1987) also report percentages of the olfactory cortex which includes the
retrobulbar region (AON), prepiriform cortex, and olfactory tubercle. In the Insectivora,
the AON makes up approximately 16% of the olfactory cortex. This percentage is higher
than the prosimians, simians, and humans which are reported 12%, 5%, and 4%
respectively. Therefore, based upon percentages, it appears that the Insectivora compared
to the primates have a larger, well-developed AON that would provide more extensive
processing of olfactory inputs.
6.5 Conclusion
The AON in Blarina was well-developed and had comparable subdivisions identified in
other macrosmatic mammals. The AON first appears at a significantly more rostral level
and the MOB continues more caudally in Blarina than in other mammals. This caudal
continuation of the MOB results in a general counter-clockwise dorsolateral rotation of
the AON to accommodate the MOB. This rotation of the AON is not observed in the rat
or mouse. Based upon the rostral-caudal extend of the AON and MOB and the
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morphological observations of the AON, it appears that the AON of Blarina is a well-
developed olfactory system structure that differs from other small mammals.
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CHAPTER VII
OLFACTORY TUBERCLE
7.1 Introduction
The olfactory tubercle (OT) is a trilaminar structure consisting of a superficial plexiform
layer, a middle pyramidal layer, and a deep polymorphic layer which has also been called
the parvicellular layer (Crosby and Humphrey, 1941; Krieger, 1981; Gordon and Krieger,
1983; Talbot et al., 1988a; Krieger and Scott, 1989). Since the deep layer is more
frequently referred to as the polymorphic layer, for the purposes of this paper, the deep
layer will be termed the polymorph cell layer. The OT of macrosmatic mammals is a
prominent swelling located on the ventral surface of the brain caudal to the AON and
medial to the LOT (Krieger, 1981; Millhouse and Heimer, 1984; Shipley et al., 1995).
The OT has been studied in numerous mammals including the opossum (Meyer, 1981),
guinea pig (Johnson, 1957a), shrew opossum (Herrick, 1921), rat (Okada et al., 1977;
Fallon et al., 1978; Ribak and Fallon, 1982; Fallon, 1983; Gordon and Krieger, 1983;
Krieger et al., 1983; Friedman and Price, 1984; Millhouse and Heimer, 1984;), rabbit
(Young, 1934), cat (Meyer and Wahle, 1986; Wahle and Meyer, 1986; Talbot et al.,
1988a, b; Berezhnaya et al., 1999), dog (Berezhnaya et al., 1999), and human (Crosby
and Humphrey, 1941). The OT is not present in anosmatic mammals (Smith, 1909) and
in microsmatic mammals such as humans, the OT is not well-developed.
The northern short-tailed shrew (Blarina brevicauda) and the southern short
tailed shrew (B. carolinensis) are considered to be macrosmatic mammals based upon
MOB, OT, and piriform cortex size, but the OT ofBlarina has yet to be examined in
detail. The purpose of this part of the project was to examine the OT of Blarina by light
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microscopy and, by morphological observations, confirm the hypothesis that the OT of
Blarina is a well-developed olfactory structure that differs from other small mammals
and thus provide further evidence that these primitive placental mammals are
macrosmatic mammals with substantial olfactory systems at all levels.
7.2 Results
7.2.1 Light Microscopy Overview
In a sagittal plane of section (Fig. 86) the OT was ventral to the MOB and appeared at the
level of the AOB, AON and frontal cortex. At the most rostral level (Fig. 87), the
piriform cortex and lateral olfactory tract were dorsolateral to the OT. The corpus
callosum was dorsal to the open lateral ventricle. At a mid-caudal level (Fig. 88), the
islands of Calleja extended into the polymorph cell layer. The lateral ventricle was
occluded at this mid-caudal level. The caudate-putamen and frontal cortex were well-
developed. At the most caudal level (Fig. 89), the third ventricle was lateral to the lateral
ventricle. The well-developed piriform cortex was dorsolateral to the OT. The caudate-
putamen was dorsal to the OT and distinct. The three distinct layers of the OT (Fig. 90)
consisted of the outer plexiform layer, middle pyramidal cell layer, and the deep
polymorph cell layer. Clusters of granule cells formed the islands of Calleja which were
located within the polymorph cell layer. These islands also extended across the
pyramidal cell layer into the plexiform layer. Large multipolar neurons were present in
the polymorph cell layer.
7.2.2 Light Microscopy of Plexiform Layer
The plexiform layer (Fig. 91) consisted of a neuropil of pale-staining dendrites,
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Fig. 86. Light micrograph of a sagittal plane of the olfactory tubercle (OT). The OT is
ventral to the main olfactory bulb (MOB). Frontal cortex (FC), AON pars dorsalis (PD),
AON pars lateralis (PL), nucleus accumbens (NA), caudate-putamen (C-P).
40 pm frozen section, sagittal plane, cresyl-violet stain. 5 mm = 250 pm.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 87. Light micrograph of the rostral olfactory tubercle. The OT is ventral and medial
to the piriform cortex (PC) and lateral olfactory tract (arrow). Pyramidal cell layer
(curved arrow), islands of Calleja (arrowhead), frontal cortex (FC), lateral ventricle (LV).
40 pm frozen section, coronal plane, cresyl-violet stain. 5 mm = 250 pm.
Fig. 88. Light micrograph of the mid-caudal olfactory tubercle. Islands of Calleja
(curved arrow) extend into the polymorph cell layer. Lateral ventricle (LV), frontal
cortex (FC), caudate-putamen (C-P), piriform cortex (PC), lateral olfactory tract (arrow).
40 pm frozen section, coronal plane, cresyl-violet stain. 5 mm = 250 pm.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission...... t-i'- '
£*■' vV --.: 89 OT/^ 90 PL
Fig. 89. Light micrograph of the caudal olfactory tubercle. The caudal OT did not
typically have Islands of Calleja. Lateral ventricle (LV), piriform cortex (PC), lateral
olfactory tract (arrow). 40 pm frozen section, coronal plane, cresyl-violet stain.
5 mm = 250 pm.
Fig. 90. Light micrograph of the trilaminar olfactory tubercle. Multipolar neurons
(arrow) in the polymorph cell layer (PO), Islands of Calleja (IC), pyramidal cell layer
(PY), plexiform layer (PL). 40 pm frozen section, coronal plane, cresyl-violet stain.
5 mm = 45 pm.
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capillaries, and few scattered pyramidal cells. The thickness of the plexiform layer
varied depending upon the location of the pyramidal cell layer. The thinnest regions
measured approximately 115 pm and the thickness regions were approximately 350 pm
deep. The average thickness of the plexiform layer was 175 pm.
7.2.3 Light Microscopy of Pyramidal Cell Layer
The pyramidal layer (Fig. 92) consisted of pyramidal cells and a surrounding neuropil.
Pyramidal cell nuclei were spherical and euchromatic with a distinct, darkly-stained
nucleolus. Pyramidal cell nuclei measured up to 9 pm in diameter. Nissl bodies were
common in thin rim of cytoplasm. Apical dendrites extended towards the plexiform
layer. Basal dendrites projected to the deep polymorph cell layer. The average thickness
of the pyramidal cell layer varied according to the density of pyramidal cells. In some
areas, this layer measured only 35 pm in thickness. In areas of greater pyramidal cell
density, the thickness was 125 pm. The average thickness of the pyramidal cell layer was
approximately 75 pm. The neuropil of the plexiform layer consisted of pale-staining
dendrites, axons and glial cells.
7.2.4 Light Microscopy of Polymorph Cell Layer
The polymorph cell layer (Fig. 93) consisted of three types of cells and a neuropil.
Pyramidal cells were scattered throughout the polymorph cell layer and were comparable
to the pyramidal cells of the pyramidal cell layer. The second cell type was the
multipolar neuron. The multipolar cell nuclei measured up to 19 pm in diameter and had
pale-staining nuclei with a distinct nucleolus. Nissl bodies were common in the abundant
cytoplasm. An apical dendrite extended towards the pyramidal cell layer. Basal
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 91. Light micrograph of the plexiform layer of the olfactory tubercle. The
superficial plexiform layer consisted of pale-staining dendrites (arrow) and scattered
pyramidal cells (arrowhead). 1 pm epoxy resin section, Richardson stain. 5 mm = 7 pm.
Fig. 92. Light micrograph of the pyramidal cell layer of the olfactory tubercle.
Pyramidal cells (arrowhead) are interspersed in a neuropil of pale-staining dendrites
(curved arrow). Apical dendrite (arrow), plexiform layer (PL). 1 pm epoxy resin section,
Richardson stain. 5 mm = 4 pm.
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dendrites extended mostly horizontally and deeper. The third cell type was the granule
cells which were typically grouped in clusters forming the islands of Calleja (Fig. 94).
These islands commonly were located in the polymorph cell layer but also were found
extending into the pyramidal cell and plexiform layers. These granule cells had
heterochromatic nuclei with darkly stained nucleoli. Granule cells were typically
spherical and granule cell nuclei measured up to 7 pm in diameter. The thin layer of
cytoplasm was not visible by light microscopy. On occasion, pyramidal cells were
located within the islands of Calleja. The surrounding neuropil of the polymorph cell
layer consisted of numerous pale-staining dendrites, small myelinated axons, and glial
cells.
7.3 Discussion
Based upon the morphological data in this current study, the trilaminar structure and cell
types of the OT in Blarina were similar to that of other macrosmatic mammals.
However, there were some differences noted in the OT of Blarina when compared to
other macrosmatic mammals.
7.3.1 Plexiform Layer
In several species, such as the guinea pig (Johnson, 1957a) and the rat (Gordon and
Krieger, 1983), the plexiform layer thickness varies in different regions. InBlarina, the
thickness of the plexiform layer also varied. The neuropil inBlarina was consistent with
the typical mammalian plexiform layer neuropil and contained numerous apical
dendrites. These apical dendrites are reported to be the apical dendrites of pyramidal
cells. In addition, MOB axons are reported to terminate on the pyramidal cell dendrites
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 93. Light micrograph of the polymorph cell layer of the olfactory tubercle.
Multipolar neurons with dendrites (arrow) extending toward the pyramidal cell layer.
Pyramidal cells (arrowhead), myelinated axons (curved arrow). 1 pm epoxy resin
section, Richardson stain. 5 mm = 8 pm.
Fig. 94. Light micrograph of granule cells of the Islands of Calleja. Granule cells with
distinct nucleoli (arrow) of the polymorph cell layer. Occasional pyramidal cells
(arrowhead) are located within the islands of Calleja. 1 pm epoxy resin section,
Richardson stain. 5 mm = 4 pm.
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in the plexiform layer (Krieger, 1981; Gordon and Krieger, 1983; Millhouse and Heimer,
1984; Meyer and Wahle, 1986; Shipley and Ennis, 1996).
7.3.2 Pyramidal Layer
The pyramidal layer typically contains a dense population of closely packed pyramidal
cells that are similar to cortical pyramidal cells (Johnson, 1957a; Ribak and Fallon,
1982). The apical dendrite extends into the plexiform layer and the basal dendrites
project toward the polymorph cell layer (Shipley and Ennis, 1996). InBlarina, pyramidal
cell dendrites followed a similar projection pattern. Variations in pyramidal cell
diameters are common. In the rat OT, pyramidal cells measure up to 16 pm (Millhouse
and Heimer, 1984). In the cat OT, pyramidal cells are significantly larger and measure
up to 25 pm (Meyer and Wahle, 1986). InBlarina, pyramidal cells were smaller than
reports of other macrosmatic mammals and measured up to 9 pm in diameter.
7.3.3 Polymorph Cell Layer
The polymorph cell layer is the thickest layer of the trilaminar OT (Johnson, 1957a) and
contains several cell types interspersed among dendrites of pyramidal cells (Meyer and
Wahle, 1986). The largest cells are the polymorph cells, also referred to as multipolar
cells, which measure, in the rat, up to 40 pm in diameter (Krieger 1981; Millhouse and
Heimer, 1984). In Blarina, the multipolar neurons were significantly smaller than the rat
polymorph cells and measured only 19 pm in nuclear diameter. According to Shipley
and Ennis (1996), dendrites of the large neurons do not extend preferentially towards the
pyramidal cell layer. InBlarina, the primary dendrites commonly projected towards the
pyramidal cell layer and basal dendrites extended radially. The second cell type is the
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granule cell which clusters together to form structures called the islands of Calleja.
These islands are typically located within the polymorph cell layer. However,
occasionally the islands of Calleja extend into the plexiform layer (Ribak and Fallon,
1982; Fallon, 1983; Millhouse and Heimer, 1984; Talbot et al., 1988a). Blarina, In the
islands of Calleja were also observed extending into the plexiform layer. Granule cells
measure approximately 8-12 pm in diameter in the rat (Krieger, 1981; Millhouse and
Heimer, 1984). In cats and dogs, Berezhnaya and coworkers (1999) identified three types
of cell in the islands of Calleja. The most granule cell is small and measures 7-10 pm in
diameter. Talbot and coworkers (1988a) also report comparable measurements for small
granule cells. An intermediate size granule cell measuring 25-30 pm in diameter and a
large granule cell measuring 35-50 pm in diameter are also common. According to
Berezhnaya and coworkers (1999), the small granule cells are the most common. The
small granule cells cluster together to form the islands of Calleja whereas the
intermediate and large granule cells typically are located more to the periphery of the
islands. In Blarina, only one population of granule cells was observed. These granule
cells measured 7 pm in diameter and were comparable to the rat granule cells (Krieger,
1981) and small granule cells reported by Berezhnaya and coworkers (1999). In addition,
Berezhnaya and coworkers (1999) identify a narrow cytoplasm with three or four radial
dendrites extending from the granule cells. In Blarina, the thin granule cell cytoplasm
was not visible by light microscopy.
7.4 Comparative Studies
Baron and coworkers (1987) reported OT size indices for various mammals. The OT
index in Blarina brevicauda was 113. Sorex fumeus, the smoky shrew, had an index of
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116 which is comparable to Blarina. Other shrews including Sorex araneus (103),
Suncus etruscus (66), and Neomys fodiens (82) have smaller OT indices thanBlarina.
Thus it appears that based upon the size index, Blarina has a larger OT than most shrews.
Two Insectivores closely related toBlarina, the European hedgehog, Erinaceus
europaeus, and the European mole, Talpa europaea, had a smaller OT index of 77 and
105 respectively. Comparing terrestrial and semiaquatic shrews, the OT index in
terrestrial insectivores is significantly higher (107) than in the semiaquatic insectivores
(69). Based upon these indices, the terrestrial shrews, which include Blarina, have a
well-developed OT that probably provides additional processing capacity for olfactory
input. In addition, the OT index in Blarina is significantly higher than in microsmatic
mammals such as the macaque (45). Based upon statistical data, Blarina has a more
well-developed OT than other macrosmatic Insectivora and microsmatic mammals.
7.5 Conclusion
Light microscopic observations of the OT ofBlarina revealed a well-developed
trilaminar structure with the various cell types commonly reported in other mammals.
Although in Blarina the cells of the OT are smaller than some other macrosmatic
mammals, it appears that the OT is highly-developed for processing olfactory input and
this level of development supports the importance of olfaction in shrews.
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CHAPTER VIII
PIRIFORM CORTEX
8.1 Introduction
The piriform cortex (PC) is a phylogenetically old part of the cerebral cortex (Haberly
and Feig, 1983; Haberly and Bower, 1984; Loscher et al., 1998). It is considered to be
the primary olfactory cortex because it is the largest olfactory area receiving direct
ipsilateral afferent input from the MOB (Stevens, 1969; Davis and Macrides, 1981;
Meyer, 1981; Haberly, 1985; Gracey and Scholfield, 1990; Bassett et al., 1992; Kratskin,
1995; Rosin et al., 1999; Stripling and Patneau, 1999; Johnson et al., 2000; Datiche et al.,
2001; Haberly, 2001; Zinyuk et al., 2001). The PC is a trilaminar structure consisting of
a superficial Layer I, a middle Layer II, and a deep Layer III (DeOlmos et al., 1978;
Zinyuk et al., 2001; Best and Wilson, 2003). The principal neuron of the PC is the
pyramidal cell (Johnson et al., 2000; Truong et al., 2002).
The PC has been studied in numerous mammalian species including the opossum
(Meyer, 1981; Haberly and Feig, 1983; Haberly and Bower, 1984), guinea pig (Gracey
and Scholfield, 1990), rat (Haberly and Price, 1978a, b; Luskin and Price, 1983a, b;
Friedman and Price, 1984; Curcio et al., 1985; Anders and Johnson, 1990; Rosin et al.,
1999; Stripling and Patneau, 1999; Johnson et al., 2000; Datiche et al., 2001; Zinyuk et
al., 2001; Best and Wilson, 2003), mouse (Bartolomei and Greer, 1998), hamster (Davis
and Macrides, 1981); rabbit (Ojima et al., 1984), cat (Stevens, 1969; Willey et al., 1983),
and monkey (Bassett et al., 1992). The purpose of this portion of olfactory system
project was to examine by light microscopy the morphology of the PC ofBlarina and to
test the hypothesis that its PC is a well-developed olfactory system structure that differs
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from other small mammals and thus provide additional evidence that these primitive
placental mammals are macrosmatic mammals with substantial olfactory systems at all
levels.
8.2 Results
8.2.1 Light Microscopy Overview
The most rostral piriform cortex (see Fig. 68) (PC) first appeared at the level of the ring
like structure of the AON that consisted of the pars dorsalis, pars medialis, pars lateralis,
and pars ventralis. The caudal-most region of the MOB was ventromedial to the PC. Just
caudal to the AON (Fig. 95), the rostral lateral ventricle, corpus callosum, and caudate-
putamen were dorsomedial to the PC. The rostral OT was ventral to the PC and the
islands of Calleja of the OT were well developed. At the more caudal level (Fig. 96) the
rostral third ventricle was distinct. At this level, the PC was a prominent structure on the
lateral surface of the brain. At its caudal-most level (Fig. 97), the PC was dorsolateral to
the amygdala and ventrolateral to the hippocampus. The PC was a trilaminar structure
(Fig. 98) and consisted of outer Layer I, middle Layer II, and deep Layer III.
8.2.2 Light Microscopy of Layer I
Layer I (Fig. 99) is typically subdivided into Layer la and lb. Layer la is often further
divided into two subdivisions. The most superficial region of Layer la, Layer la,
consisted of numerous small myelinated axons, few dendrites, capillaries, and glial cells.
The deep region of Layer la was ventral to the layer of myelinated axons. This region
consisted of fewer myelinated axons than the superficial region. Dendrites were common
in this deep region of Layer la. The deepest region of Layer I, Layer lb, consisted of
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Fig. 95. Light micrograph of the rostral piriform cortex. The rostral piriform cortex (PC)
is ventrolateral to the rostral lateral ventricle (arrow) and caudate-putamen (C-P). Islands
of Calleja (arrowhead) are distinct at the rostral olfactory tubercle. Lateral olfactory tract
(curved arrow). 40 pm frozen section, coronal plane, cresyl-violet stain.
5 mm = 250 pm.
Fig. 96. Light micrograph of the mid-caudal piriform cortex. The mid-caudal piriform
cortex (PC) at the level of the rostral third ventricle, caudal lateral ventricle (arrow) and
caudate-putamen (C-P). 40 pm frozen section, coronal plane, cresyl-violet stain.
5 mm = 250 pm.
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AMY
Fig. 97. Light micrograph of the caudal piriform cortex. The most caudal piriform
cortex (PC) is dorsolateral to the amygdala (AMY). 40 pm frozen section, coronal plane,
cresyl-violet stain. 5 mm = 250 pm.
Fig. 98. Light micrograph of the trilaminar piriform cortex. The piriform cortex consists
of an outer Layer I (I), middle Layer II (II), and deep Layer III (III). 40 pm frozen
section, coronal plane, cresyl-violet stain. 5 mm = 20 pm.
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dendrites which were more numerous and larger than dendrites in the other regions of
Layer I . Small myelinated axons were common in Layer lb. The few somata scattered
throughout Layers la and lb appeared to be pyramidal cells. The average thickness of
Layer la and lb varied from rostral to caudal. At the most rostral level, Layer la was
approximately 125 pm thick. Moving caudalward, Layer la was 250 pm thick. At the
most caudal level, Layer la was 140 pm thick. At the most rostral level, Layer lb was
approximately 175 pm thick. Moving caudalward, Layer lb was 200 pm thick and at the
most caudal level, Layer lb measured 125 pm thick.
8.2.3 Light Microscopy of Layer II
Layer II (Fig. 100) consisted of a dense layer of pyramidal cells. These large pale-
staining neurons measured up to 14 pm in diameter with a spherical euchromatic nucleus.
Darkly stained Nissl bodies were common in the cytoplasm. Pyramidal cell apical
dendrites (Fig. 101) extended into Layer lb and basal dendrites extended towards Layer
III. The surrounding neuropil consisted of capillaries, small myelinated axons and
numerous pale-staining dendrites. The average thickness of Layer II varied from rostral
to caudal levels. At the most rostral level, Layer II measured approximately 100 pm in
thickness. Moving caudalward, the thickness increased to 125 pm in thickness. At the
most caudal level, Level II was the thinnest and measured about 90 pm thick.
8.2.4 Light Microscopy of Layer III
Layer III consisted of pale-staining dendrites, myelinated axons, and numerous
capillaries. Several types of cells were common in Layer III. Pyramidal cells
comparable to the pyramidal cells of Layer II were scattered throughout Layer III.
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Fig. 99. Light micrograph of Layer I of the piriform cortex. Layer I is subdivided an
outer region of myelinated axons (Ax) and dendrites, a middle region (la), and a deep
region (lb) with increased concentrations of dendrites. 1 pm epoxy resin section,
Richardson stain. 5 mm = 9 pm.
Fig. 100. Light micrograph of Layer II pyramidal cells of the piriform cortex. Apical
dendrites (arrowhead) extend toward the superficial Layer I (I). 1 pm epoxy resin
section, Richardson stain. 5 mm =10 pm.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The apical dendrites of Layer III pyramidal cells extended toward Layer I and the basal
dendrites extended deep into Layer III. A second cell type, the multipolar neuron (Fig.
102) was common in Layer III. These cells had spherical euchromatic nuclei and
measured up to 25 pm in diameter. Darkly stained nucleoli were common. The abundant
cytoplasm contained Nissl bodies. Multipolar cell dendrites extended radially throughout
Layer III and lacked specific orientation. The surrounding neuropil was densely
populated with pale staining dendrites and numerous small myelinated axons. Darkly-
stained nuclei which appeared to be oligodendrocytes were common. The thickness of
the rostral Layer III measured approximately 400 pm. At caudal levels, the boundaries of
Layer III were not possible to distinguish from the surrounding cortical structures;
therefore, accurate measurements of the caudal regions of thickness of Layer III were not
possible.
8.3 Discussion
The PC is ventral to the rhinal sulcus and extends caudally from the pars lateralis of the
AON to the lateral entorhinal cortex and occupies the majority of the ventral surface of
the forebrain (Davis and Macrides, 1981b; Friedman and Price, 1984). The PC
participates in reciprocal projections with the MOB. Mitral and tufted cell axons of the
MOB project to the ipsilateral PC via the LOT (Stevens, 1969; Meyer, 1981; Luskin and
Price, 1983b; Willey et al., 1983; Ojima et al., 1984; Gracey and Scholfield, 1990;
Shipley and Ennis, 1996; Fukushima et al., 2002).
8.3.1 Layer I
Layer I has been referred to as the molecular layer (Stevens, 1969; Willey et al., 1983) or
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Fig. 101. Light micrograph of pyramidal cell dendrites. Apical dendrites of pyramidal
cells (arrows) extend towards Layer I. Basal dendrites (arrowhead) extend deep towards
Layer III. 1 pm epoxy resin section, Richardson stain. 5 mm = 5 pm.
Fig. 102. Light micrograph of multipolar neurons of the piriform cortex. Multipolar
neurons with abundant cytoplasm (M). Pyramidal cell with basal dendrite (arrowhead).
Myelinated axons (curved arrow) are common in the neuropil. Oligodendrocyte (arrow).
1 pm epoxy resin section, Richardson stain. 5 mm = 5 pm.
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the plexiform layer (Haberly and Feig, 1983; Haberly, 1985; Shipley and Ennis, 1996;
Best and Wilson, 2003). Layer I is subdivided into two subdivisions, a superficial Layer
la and a deep Layer lb (Haberly and Feig, 1983). Layer la is further subdivided into a
superficial region, Layer la which consists of myelinated afferent fibers from the MOB
that make excitatory synaptic contact within Layer la onto the apical dendrites of Layer II
and III pyramidal cells (Mascitti and Ortega, 1966; Stevens, 1969; Davis and Macrides,
1981b; Luskin and Price, 1983b; Willey et al., 1983; Haberly and Bower, 1984; Haberly,
1985; Gracey and Scholfield, 1990; Schwerdtfeger et al., 1990; Bartolomei and Greer,
1998; Loscher et al., 1998; Rosin et al., 1999; Stripling and Patneau, 1999; Best and
Wilson, 2003). Just deep to Layer la is the remaining region of Layer la that contains
dendrites, MOB axons and axon terminals, and glial cells (Friedman and Price, 1984;
Curcio et al., 1985; Anders and Johnson, 1990). The deep region of Layer I, Layer lb
also contains MOB axons and their collaterals and apical dendrites of Layer II and III
pyramidal cells. In addition, fibers from the contralateral PC are common in Layer lb
(Haberly and Price, 1978a; Friedman and Price, 1984; Greer, 1991; Rosin et al., 1999;
Johnson et al,. 2000; Best and Wilson, 2003). In Blarina, Layer I was similar to the
typical mammalian Layer I of the piriform cortex.
8.3.2 Layer II
Layer II consists of a layer of tightly packed pyramidal cells interspersed in a neuropil of
dendritic profiles (Haberly, 1985; Shipley and Ennis, 1996). The apical dendrites of
pyramidal cells extend into Layers la and lb where they receive input from the MOB
(Haberly and Feig, 1983; Haberly and Bower, 1984; Kratskin, 1995; Bartolomei and
Greer, 1998; Truong et al., 2002). Pyramidal cell basal dendrites extend into the deeper
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Layer III. The pyramidal cell axons and collaterals radiate throughout Layer III and
terminate on other pyramidal cells. Axon collaterals are also common in layer lb and
layer II (Haberly, 1985; Shipley and Ennis, 1996). A second cell type, the pyramidal-
type cell has also been reported in Layer II. These cells, referred to as semilunar cells,
are similar to pyramidal cells but lack basal dendrites. Apical dendrites of semilunar
cells extend into Layer I and their axon extends deep into the PC (Haberly, 1985; Shipley
and Ennis, 1996). In Blarina, Layer II also consisted of numerous pyramidal cells that
clustered together forming a distinct cell layer. The pyramidal cells in Blarina did not
appear to have basal dendrites and thus may be semilunar cells. However, by light
microscopy, the presence of these pyramidal-type semilunar cells inBlarina can not be
confirmed.
8.3.3 Layer III
Several cell types are common in the deep Layer III. Pyramidal cells similar to Layer II
pyramidal cells extend a primary dendrite toward Layer I. Basal dendrites extend into
Layer III (Davis and Macrides, 1981; Shipley and Ennis, 1996). InBlarina, pyramidal
cells of Layer III were also similar to pyramidal cells common in Layer II and the
dendrites also extended in a comparable path. A second cell type, the multipolar cell is
uniformly scattered throughout Layer III. These cells have numerous dendrites that
radiate in all directions from the cell body but are confined to Layer III. The multipolar
neurons are reported to receive excitatory synapses from the pyramidal cells and in turn
make excitatory synapses onto the dendrites of other PC pyramidal cells (Shipley and
Ennis, 1996; Stripling and Patneau, 1999). Large multipolar cells in the opossum range
from 15-25 pm in diameter (Haberly and Feig, 1983). In Blarina, these large neurons
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were comparable in size to the opossum multipolar neuron. The multipolar dendrites in
Blarina extended radially and lacked specific orientation. According to Shipley and
Ennis (1996), the typical mammalian neuropil of Layer III contains basal dendrites of
Layer II pyramidal cells and numerous axons from multipolar cells and Layer II
pyramidal cells. These Layer II pyramidal cell axons project in all directions within
Layer III and make axo-dendritic synapses onto Layer III pyramidal cells (Haberly and
Bower, 1984). In addition, multipolar cell dendrites are common in Layer III (Shipley
and Ennis, 1996). The neuropil of Layer III inBlarina was similar to the mammalian
Layer III neuropil reported in the literature.
8.4 Comparative Studies
Baron and coworkers (1987) reported PC size indices for various mammals. The PC
index in Blarina brevicauda was 92. Sorex fumeus, the smoky shrew, has an index of
109 which is slightly larger thanBlarina. Other shrews including Sorex araneus (91),
Suncus etruscus (60), and Neomys fodiens (56) have smaller PC indices than Blarina.
Thus it appears that based upon the size index, Blarina has a larger PC than most shrews.
Two Insectivores closely related toBlarina , the European hedgehog, Erinaceus
europaeus, and the European mole, Talpa europaea, had a smaller PC index of 104 and
83 respectively. Comparing terrestrial and semiaquatic shrews, the PC index in terrestrial
insectivores is significantly higher (109) than in the semiaquatic Insectivores (61). Based
upon these indices, the terrestrial shrews, which include Blarina, have a well-developed
PC. In addition, the PC index in Blarina is significantly higher than in microsmatic
mammals such as the macaque (26) and human (65). The statistical data from Baron and
coworkers (1987) further supports the hypothesis thatBlarina has a well-developed PC
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compared to other shrews and other Insectivores including the mole. Furthermore, the
macrosmatic Blarina has a higher index than various microsmatic mammals.
8.5 Conclusion
Light microscopic observations of the PC ofBlarina revealed a well-developed trilaminar
organization with the various cell types similar to that of other macrosmatic mammals. It
appears that the PC is a highly-developed structure for processing olfactory input.
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CHAPTER IX
CONCLUSION
9.1 Hypothesis
The light microscopic and transmission electron microscopic observations of the
ultrastructure of the olfactory system of the northern short-tailed shrew,Blarina
brevicauda and the southern short-tailed shrew, B. carolinensis confirmed the hypothesis
that the olfactory system inBlarina is well-developed and differs from that of other
macrosmatic mammals.
9.2 Conclusions of Olfactory System Structures
9.2.1 Olfactory Epithelium and Mucosa
In the olfactory epithelium,Blarina has a light supporting cell which differs from the
supporting cells commonly reported in other mammals. The function of this light
supporting cell remains unknown at present. However, the numerous mitochondria and
large numbers of small vesicles in the light supporting cells supports the hypothesis that
this cell could be involved in transporting molecules or ions between the lamina propria
and the fluid layer surrounding the olfactory receptor cilia. Furthermore, the average
number of cilia per olfactory epithelium receptor cells was higher inBlarina than in the
mole and hedgehog. Thus, based upon ultrastructure observations, the olfactory
epithelium and mucosa of Blarina are well-developed.
9.2.2 Main Olfactory Bulb
In the mammalian MOB, a monolayer of mitral cells is located between the external and
internal plexiform layers. Although this monolayer is common in macrosmatic
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mammals, it is not present in all macrosmatic mammals. In the hedgehog, the mitral cells
are more dispersed within the external and internal plexiform layers and do not form a
monolayer. In addition, two primitive Monotremes, the duck-billed platypus
(Ornithorhynchus anatinus) and the echidna ( Tachyglossus aculeatus) also both lack the
monolayer of mitral cells. The absence of a mitral cell monolayer has also been reported
in some reptiles and birds. Since moles, shrews, and hedgehogs are members of the order
Insectivora, significant variations among the olfactory system structures would appear to
be minimal. The absence of a mitral cell layer in the hedgehog represents a significant
variation within an order. The functional significance of mitral cells organized in a
monolayer verses a generalized displacement remains unclear. It is also unclear why the
hedgehog would retain this primitive MOB characteristic and moles and shrews would
not retain this feature. Additionally, the caudal-most extent of the MOB of theBlarina
continued to the level of the piriform cortex. In other macrosmatic mammals such as the
rat and mouse, the MOB does not extend as caudally as in Blarina. Thus it appears that
the MOB of Blarina is proportionately larger and extends over a larger brain area than in
other macrosmatic mammals such as rodents.
Myelinated dendrites and neuronal somas were observed in the MOB of Blarina.
Structures similarly have been reported in the squirrel monkey and in the mouse but these
appear to be rare observations. In Blarina, due to the rarity of these myelinated
structures, it appears that these structures probably play no significant role in the
olfactory sensory system.
An open olfactory ventricle was common in the MOB ofBlarina. This feature
has also been reported in primitive mammals such as the hedgehog, mole, and opossum.
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Open olfactory ventricles have also been reported in several reptiles including the turtle
(Pseudemys elegans), Gila monster (Heloderma suspectum), and the copperhead
(Agkistrodon mokaseri). Occluded olfactory ventricles have been observed in the rat and
mouse. In the human fetus, the olfactory ventricle is patent but only rarely does the
olfactory ventricle remain in the human adult. Thus it appears that the presence of an
open olfactory ventricle is a characteristic of the evolutionary primitive vertebrates.
9.2.3 Accessory Olfactory Bulb
The mammalian AOB is reported to be less developed with poorly-defined layers
compared to the MOB. The AOB of Blarina has a less distinct laminar organization than
the complementary MOB layers. InBlarina , the AOB mitral cells do not form a distinct
monolayer rather the mitral/tufted cells were more dispersed throughout the external
plexiform layer so these layers were combined and resulted in the external
plexiform/mitral-tufted cell layer. The location of the AOB ofBlarina was different
compared to other macrosmatic mammals. In Blarina the AOB appears to develop inside
of the MOB adjacent to the external plexiform and granule cell layer of the MOB, not
infolded into the MOB as in the mouse and rat nor does the AOB appear as an eminence
on the surface of the MOB as in the opossum and rabbit. The Blarina AOB size index is
smaller than other shrews and other Insectivores, including the mole and hedgehog. Due
to the small AOB size index and the poorly-defined layers, it appears that the AOB of
Blarina is less well-developed compared to other macrosmatic mammals.
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9.2.4 Anterior Olfactory Nucleus
The structure and subdivisions of the AON ofBlarina were comparable to other
macrosmatic mammals, but significant variations in the rostral to caudal extent of the
AON in Blarina were noted. In Blarina the AON first appears at a significantly more
rostral level than in the rat and the mouse. In Blarina the AON is first observed rostral to
the AOB and it continues caudally to the level of the rostral OT. In addition, the MOB of
Blarina also continues caudally to the level of the caudal-most AON subdivision, the PP.
To accommodate the caudal continuation of the MOB, the AON is rotated counter
clockwise and displaced dorsolaterally. This rotation is not observed in the rat or mouse.
In Blarina, the shape of the various AON subdivisions was similar to other macrosmatic
mammals including the crescent shape and the ring-like structure. Due to the extensive
caudal continuation and the ultrastructurally distinct cells of the AON, it appears that this
olfactory system structure is well-developed in the macrosmaticBlarina.
9.2.5 Olfactory Tubercle
The OT of Blarina consisted of similar cell types and laminar organization reported in the
mammalian OT. However, several differences were noted in Blarina. The pyramidal
cells and multipolar cells were smaller than those of more highly evolved mammals such
as the rat. Although the layers were thin and the cell types were smaller inBlarina, each
layer was well-organized and the various cells were numerous and distinct. Thus it
appears that the OT in Blarina is highly-developed for processing important olfactory
input for these shrews.
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9.2.6 Piriform Cortex
The PC of Blarina had a distinct trilaminar structure similar to the typical mammalian
PC. However, several differences were identified. In Blarina, the pyramidal cells were
smaller than pyramidal cells of other macrosmatic mammals but the multipolar cells in
the shrew were similar in diameter to other mammals. Semilunar cells are reported in the
PC of other mammals. These cells resemble pyramidal cells but lack basal dendrites. In
Blarina, numerous pyramidal cells with and without basal dendrites were common. By
light microscopy, the presence of the semilunar cell can not be confirmed. Light
microscopy of the PC revealed a trilaminar structure comparable to other macrosmatic
mammals and thus it was concluded that the PC of Blarina was well-developed in these
small mammals.
9.3 Summary of the Ultrastructure of the Olfactory System Structures
The physical environment ofBlarina may have had an evolutionary impact upon its
various sensory systems. The reduction in the visual system appears to have caused the
enhancement of and compensation by the olfactory system. Based upon the ultrastructure
of the olfactory system structures by light and transmission electron microscopy of the
northern short-tailed shrew, Blarina brevicauda and the southern short-tailed shrew, B.
carolinensis, it is concluded that the olfactory system of these small mammals differs
from other macrosmatic mammals. Furthermore, the large amount of brain volume
occupied by the olfactory system and the large surface area of the olfactory epithelium
are both consistent with the importance of the olfactory system in these shrews.
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CHAPTER X
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VITA
LISA JOHNSON BYRUM
Ph.D. Candidate Department of Biological Sciences Old Dominion University Norfolk, Virginia 23529-0276
EDUCATION
2004 Ph.D. Biomedical Sciences, Old Dominion University 1995 M.S. Biological Sciences, Old Dominion University 1986 B.S. Elementary Education, Old Dominion University
RESEARCH INTEREST
Cell ultrastructure, neurobiology, olfactory system
EXPERIENCE
Fall 1996-present Research Assistant, Old Dominion University Fall 1996 Research Assistant, Diabetes Institute, Eastern Virginia Medical School 1994-1995 Teaching Assistant, Old Dominion University Spring/Summer 1996 Instructor, Paul D. Camp Community College
PUBLICATIONS
Byrum LJ, Carson KA, Rose RK (2001) Olfactory Mucosa Ultrastructure in the Short-Tailed Shrews, Blarina brevicauda and Blarina carolinensis. European Journal of Morphology 39: 285-294.
Byrum LJ, Carson KA, Rose RK (2003) The Olfactory System in Two Species of Short-Tailed Shrews, Genus Blarina, and Comparisons with Other Insectivora. Proceedings of the International Colloquium: Biology of the Soricidae II (in press).
PROFESSIONAL SOCIETIES
Sigma Xi, The Scientific Research Society America Society of Mammalogists Virginia Academy of Science
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