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Neuronal organization of olfactory bulb circuits

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Olfactory sensory neurons extend their axons solely to the olfactory bulb, which is dedicated to odor information processing. The olfactory bulb is divided into multiple layers, with different types of neurons found in each of the layers. Therefore, neurons in the olfactory bulb have conventionally been categorized based on the layers in which their cell bodies are found; namely, juxtaglomerular cells in the glomerular layer, tufted cells in the external plexiform layer, mitral cells in the mitral cell layer, and granule cells in the granule cell layer. More recently, numerous studies have revealed the heterogeneous nature of each of these cell types, allowing them to be further divided into subclasses based on differences in morphological, molecular, and electrophysiological properties. In addition, technical developments and advances have resulted in an increasing number of studies regarding cell types other than the conventionally categorized ones described above, including short-axon cells and adult-generated interneurons. Thus, the expanding diversity of cells in the olfactory bulb is now being acknowledged. However, our current understanding of olfactory bulb neuronal circuits is mostly based on the conventional and simplest classification of cell types. Few studies have taken neuronal diversity into account for understanding the function of the neuronal circuits in this region of the brain. This oversight may contribute to the roadblocks in developing more precise and accurate models of olfactory neuronal networks. The purpose of this review is therefore to discuss the expanse of existing work on neuronal diversity in the olfactory bulb up to this point, so as to provide an overall picture of the olfactory bulb circuit.
Basic model of the olfactory bulb network. The illustrated olfactory bulb network is based on the conventional categorization of participating neurons. The axons of olfactory sensory neurons make synapses in the glomerular layer (GL), consisting of spherical structures called glomeruli. Although there are several thousand glomeruli at the surface of the rodent olfactory bulb, olfactory sensory neurons expressing the same type of odorant receptor converge their axons into only a few glomeruli, and thus each glomerulus represents a single odorant receptor. Neurons surrounding glomeruli in the GL are called juxtaglomerular cells (JG cells), consisting of three morphologically distinct cell types: periglomerular (PG) cells, external tufted (ET) cells (not shown), and superficial short-axon (sSA) cells. There are two types of projection neurons, the mitral cells and the tufted cells, which send their axons to the olfactory cortex. The somata of mitral cells are located in the mitral cell layer (MCL), while the tufted cells are scattered throughout the EPL. Both mitral and tufted cells project a single primary dendrite into a single glomerulus, where they receive synaptic inputs from the axons of olfactory sensory neurons and make reciprocal synapses with the dendrites of PG cells. Secondary dendrites of mitral and tufted cells are elongated in the external plexiform layer (EPL), where reciprocal synapses are formed with granule cell dendrites. The internal plexiform layer (IPL), in which axons from mitral cells and axon collaterals of ET cells run, and the granule cell layer (GCL), which is largely composed of granule cells, both lie beneath the MCL. Granule cells are axon-less interneurons extending dendrites apically into the EPL. Abbreviation: ONL, olfactory nerve layer.
… 
Subtypes of neurons in the olfactory bulb. (A) Schematic illustration of the subtypes of periglomerular cells (PG cells; purple), superficial short-axon cells (sSA cells; red), and external tufted cells (ET cells; blue). Two subtypes of PG cells are based on their synaptic connections. Type-I PG cells receive synaptic inputs on their dendrites from both olfactory sensory neurons and neurons in the olfactory bulb. Type-II PG cells only receive inputs from neurons in the olfactory bulb. PG cells can be further divided into neurochemical subtypes (not shown in the figure). Classically, sSA cells had an axon and dendrites. The dendrites did not enter a glomerulus. More recently reported types of sSA cells are positive for tyrosine hydroxylase (TH) and connected to a few to tens of glomeruli. Subtypes of ET cells are determined by morphology: those without and those with secondary dendrites. The ET cells with secondary dendrites are more frequently found around the border between the GL and the superficial external plexiform layer (s-EPL). (B) Schematic illustration of subtypes of tufted cells (green) and mitral cells (blue), as well as interneurons (red) in the external plexiform layer (EPL) and the mitral cell layer (MCL). Tufted cells are classified as external, middle, and internal tufted cells based on their soma location. ET cells are discussed in the GL section of the main manuscript and thus are not shown here. The EPL itself can be divided into multiple sub-layers, including the s-EPL, the intermediate EPL (i-EPL), and the deep EPL (d-EPL). The two subtypes of mitral cells are based on the depth of their secondary (basal) dendrites in the EPL sub-layers and are referred to as type-I and type-II mitral cells, respectively. On average, middle tufted cells have smaller cell somata than internal tufted cells, and internal tufted cells have smaller cell somata than mitral cells. Among interneurons, only large short-axon cells have axons. Some of the neurons that had originally been categorized as short-axon cells have an axon-like process (see main text), but they are not shown in this figure to avoid any confusion. Van Gehuchten cells are axonless cells, while inner horizontal and multipolar-type cells are identified by their morphological features and locations. The majority of interneurons have been studied by parvalbumin (PV) labeling. In addition, somatostatin-immunoreactive (SRIF-ir) cells are located in the i-EPL/d-EPL and extend their dendrites specifically to the d-EPL. (C) Schematic illustration of six subtypes of granule cells (GC; black) and three subtypes of deep short-axon cells (dSA cells; red) in the granule cell layer (GCL). The subtypes of granule cells and dSA cells are determined based on the location (depth) of their cell somata in the GCL and the layer or sublayer to which their dendrites extend. These subtypes of GC are referred to as type-I, type-II, type-III, type-IV, type-V, and type-S cells. The subtypes of dSA cells are referred to as GL-dSA, EPL-dSA, and GCL-dSA cells, reflecting the distribution of their axons. Abbreviations: GL, glomerular layer, ONL, olfactory nerve layer; IPL, internal plexiform layer.
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REVIEW ARTICLE
published: 03 September 2014
doi: 10.3389/fncir.2014.00098
Neuronal organization of olfactory bulb circuits
Shin Nagayama
1
*, Ryota Homma
1
and Fumiaki Imamura
2
1
Department of Neurobiology and Anatomy, The University of Texas Medical School at Houston, Houston, TX, USA
2
Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, PA, USA
Edited by:
Benjamin R. Arenkiel, Baylor College
of Medicine, USA
Reviewed by:
Kazushige Touhara, University of
Tokyo, Japan
Veronica Egger,
Ludwig-Maximilians-Universität,
Germany
Kathleen Quast, Baylor College of
Medicine, USA
*Correspondence:
Shin Nagayama, Department of
Neurobiology and Anatomy, The
University of Texas Medical School at
Houston, 6431 Fannin Street,
MSB 7.046, Houston, TX, USA
e-mail: shin.nagayama@uth.tmc.edu
Olfactory sensory neurons extend their axons solely to the olfactory bulb, which is
dedicated to odor information processing.The olfactory bulb is divided into multiple layers,
with different types of neurons found in each of the layers. Therefore, neurons in the
olfactory bulb have conventionally been categorized based on the layers in which their cell
bodies are found; namely, juxtaglomerular cells in the glomerular layer, tufted cells in the
external plexiform layer, mitral cells in the mitral cell layer, and granule cells in the granule
cell layer. More recently, numerous studies have revealed the heterogeneous nature of each
of these cell types, allowing them to be further divided into subclasses based on differences
in morphological, molecular, and electrophysiological properties. In addition, technical
developments and advances have resulted in an increasing number of studies regarding
cell types other than the conventionally categorized ones described above, including short-
axon cells and adult-generated interneurons. Thus, the expanding diversity of cells in the
olfactory bulb is now being acknowledged. However, our current understanding of olfactory
bulb neuronal circuits is mostly based on the conventional and simplest classification of
cell types. Few studies have taken neuronal diversity into account for understanding the
function of the neuronal circuits in this region of the brain. This oversight may contribute
to the roadblocks in developing more precise and accurate models of olfactory neuronal
networks. The purpose of this review is therefore to discuss the expanse of existing work
on neuronal diversity in the olfactory bulb up to this point, so as to provide an overall picture
of the olfactory bulb circuit.
Keywords: structure of olfactory bulb, cell type, layer formation
INTRODUCTION
Our environment is filled with odorant molecules, and our emo-
tions, moods, and even behaviors can be controlled by olfactory
stimuli. We are predisposed to discriminate between more than
10
12
odors (Bushdid et al., 2014), in part due to the variety of
odorant receptors that bind to different odorants with unique
affinity profiles (Malnic et al., 1999). These odorant receptors are
expressed by olfactory sensory neurons in the olfactory epithe-
lium. Since the first rat odorant receptor was cloned in 1991,
approximately 400 and 1000 different functional odorant recep-
tors have been identified in the human and mouse genome,
respectively (Buck and Axel, 1991; Zhang and Firestein, 2002;
Abbreviations: Brain areas: AON, anterior olfactory nucleus; AONpE, anterior
olfactory nucleus pars externa; SVZ, subventicular zone; Layers: ONL, olfactory
nerve layer; GL, glomerular layer; EPL, external plexiform layer; s-EPL, superfi-
cial EPL; i-EPL, intermediate EPL; d-EPL, deep EPL; MCL, mitral cell layer; IPL,
internal plexiform layer; GCL, granule cell layer; Cells: JG cell, juxtaglomerular
cell; PG cell, periglomerular cell; ET cell, external tufted cell; sSA cell, superficial
short-axon cell; dSA cell, deep short-axon cell; SRIF-ir cell, somatostatinimmunore-
active cell; Molecules: BrdU, 5-bromo-2
-deoxyuridine; CaMKIV, CaM kinase IV;
CB, calbindin; CCK, cholecystokinin; CR, calretinin; CRH, corticotropin-releasing
hormone; DHPG, (RS)-3,5-dihydroxyphenylglycine; GAD, glutamic acid decar-
boxylase; GFP, green fluorescent protein; HCN, hyperpolarization-activated cyclic
nucleotide gated channel; HRP, horseradish peroxidase; Kv, voltage-gated potas-
sium channel; mGluRs, metabotropic glutamate receptors; nNOS, neuronal nitric
oxide synthase; PV, parvalbumin; TH, tyrosine hydroxylase; VGAT, vesicular GABA
transporter; VGLUT, vesicular glutamate transporter; VIP, vasoactive intestinal
polypeptide.
Nei et al., 2008; Adipietro et al., 2012). Because each olfactory
sensory neuron expresses only a single odorant receptor, differ-
ent odorants can activate distinct subsets of olfactory sensory
neurons. Information from activated neurons is first transmit-
ted to the olfactory bulb. Several reviews have summarized the
elaborate neuronal network that extends from the olfactory epithe-
lium to the olfactory bulb (Wilson and Mainen, 2006; Zou et al.,
2009; Sakano, 2010; Mori and Sakano, 2011; Murthy, 2011;
Lodovichi and Belluscio, 2012). In the olfactor y bulb, multi-
ple types of neurons form sophisticated networks to process
information before transmitting it further to the olfactory cor-
tex. Currently, there is a pressing demand for understanding the
numerous neuronal types and networks to elucidate the mech-
anism(s) of olfactory information processing in the olfactory
bulb.
Histologically, the olfactory bulb is divided into multiple lay-
ers. Intensive Golgi analyses in the 1970s succeeded in visualizing
the morphology of neurons in each layer, and showed that the
distinct layers were composed of morphologically distinct cells
(Price and Powell, 1970a,b; Pinching and Powell, 1971a). There-
fore, the neurons in the olfactory bulb have conventionally been
categorized based on the layers in which their cell bodies are found.
According to this categorization, juxtaglomerular (JG) cells, mitral
cells, tufted cells, and granule cells were first defined. JG cells
are now known to include three morphologically distinct cell
types, the periglomerular (PG) cells, external tufted (ET) cells, and
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superficial short-axon (sSA) cells. This categorization provides us
with a basic model of the olfactor y bulb network (Figure 1).
However, we are far away from forming a comprehensive
model of the olfactory bulb network. An increasing number of
studies suggest that conventionally categorized neurons in the
brain comprise heterogeneous populations, and that the neuronal
types in the olfactory bulb are among the most diverse (Shipley
and Ennis, 1996). For example, PG cells are molecularly hetero-
geneous (Kosaka et al., 1995; Kosaka and Kosaka, 2005, 2007a;
Panzanelli et al., 2007; Parrish-Aungst et al., 2007), and subgroups
of mitral cells with different morphological and/or electrophysio-
logical properties have been identified (Padmanabhan and Urban,
2010; Angelo et al., 2012). Furthermore, granule cells can be sep-
arated into morphologically distinct subgroups (Mori et al., 1983;
Orona et al., 1983), and divergent properties between granule
cells generated during developmental and adult stages are noted
(Lemasson et al., 2005). In addition, some olfactory bulb neurons
have not been typically included in conventional categories due to
their relatively small numbers [e.g., short-axon cells in the External
plexiform layer (EPL) and the granule cell layer (GCL)]. Nonethe-
less, recent technological advances now make it possible to target
and analyze even these miniscule cell populations. Moreover, new
neuronal types and connections in the olfactory bulb continue to
be discovered (Merkle et al., 2014).
Nevertheless, new neuronal types and connections are often
not taken into account in building a model of the olfactory bulb
network. We are concerned with this trend of omission as it may
hinder future progress in research. The major problem is that novel
findings are scattered across many literary references, and recent
reviews have not effectively summarized the neuronal diversity
in the olfactory bulb. Here, we gather the scattered results and
summarize the discoveries regarding the new neuronal types and
connections in each layer. Since understanding the inputs and
outputs of neurons is fundamental to building a network model,
we focus mainly on somata locations, axon/dendrite extension
patterns, neurotransmitters, and/or the physiological properties of
neurons. We believe that this review greatly adds to our knowledge
of the gener a l model of the olfactory bulb network, and brings the
information about this brain region to another level. In this article,
we focus on neurons rather than on glia and the rodent main
olfactory bulb rather than accessory olfactory bulb. The authors
apologize to those whose work was not included here due to space
limitations.
GLOMERULAR LAYER
NEURONS IN THE GLOMERULAR LAYER AND THEIR MORPHOLOGY
Neurons in the glomerular layer (GL) are morphologically
heterogeneous and are of three identified types, PG cells, sSA cells,
FIGURE 1
|
Basic model of the olfactory bulb network. The illustrated
olfactory bulb network is based on the conventional categorization of
participating neurons. The axons of olfactory sensory neurons make synapses
in the glomerular layer (GL), consisting of spherical structures called
glomeruli. Although there are several thousand glomeruli at the surface of the
rodent olfactory bulb, olfactory sensory neurons expressing the same type of
odorant receptor converge their axons into only a few glomeruli, and thus
each glomerulus represents a single odorant receptor. Neurons surrounding
glomeruli in the GL are called juxtaglomerular cells (JG cells), consisting of
three morphologically distinct cell types: periglomerular (PG) cells, external
tufted (ET) cells (not shown), and superficial short-axon (sSA) cells. There are
two types of projection neurons, the mitral cells and the tufted cells, which
send their axons to the olfactory cortex. The somata of mitral cells are located
in the mitral cell layer (MCL), while the tufted cells are scattered throughout
the EPL. Both mitral and tufted cells project a single primary dendrite into a
single glomerulus, where they receive synaptic inputs from the axons of
olfactory sensory neurons and make reciprocal synapses with the dendrites
of PG cells. Secondary dendrites of mitral and tufted cells are elongated in the
external plexiform layer (EPL), where reciprocal synapses are formed with
granule cell dendrites. The internal plexiform layer (IPL), in which axons from
mitral cells and axon collaterals of ET cells run, and the granule cell layer
(GCL), which is largely composed of granule cells, both lie beneath the MCL.
Granule cells are axon-less interneurons extending dendrites apically into the
EPL. Abbreviation: ONL, olfactory nerve layer.
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and ET cells (Pinching and Powell, 1971a). Generically, they may
also be referred to as JG cells when their morphological type is not
specified. Most of these cells (PG cells, sSA cells, and a portion of
the ET cells) are actually interneurons and do not innervate brain
regions outside the olfactory bulb.
The PG cell is the most abundant type of neuron in the
GL (Parrish-Aungst et al., 2007). These cells have the smallest
cell body (5–10 μm in diameter) among the three morpholog-
ical types. PG cells typically project their dendrites to a single
glomerulus, and only occasionally to multiple g lomeruli. Their
dendrites ramify in a smaller portion of the glomerulus than the
dendrites of ET cells. PG cells are generally thought to bear an
axon (Pinching and Powell, 1971a), but axonless subtypes may
also exist (Kosaka and Kosaka, 2011). The length of the axon
is variable, and can extend as far as 5–6 glomeruli ( 600 μm;
Pinching and Powell, 1971a). Axons of PG cells terminate in the
interglomerular
space.
The sSA cell was first reported by Pinching and Powell
(Pinching and Powell, 1971a). The percentage of sSA cells among
JG cells is thought to be small, although no estimate has been
provided. The somata of sSA cells are slightly larger than those
of PG cells, at 8–12 μm. These cells have dendrites that course
exclusively in the interglomerular space, with an axon that extends
as far as 1–2 glomeruli (Pinching and Powell, 1971a,c). No den-
drodendritic connections have been found among this population
(Pinching and Powell, 1971b,c). However, some recently described
sSA cells have a somewhat distinct morphology from previously
described sSA cells (Aungst et al., 2003; Kiyokage et al., 2010).
This recently described population will be discussed in detail
below.
The ET cell has the largest cell soma among the three types of
JG cells, at 10–15 μm(Pinching and Powell, 1971a). The primary
dendrites of ET cells are generally mono-glomerular, with a small
subpopulation being di-glomerular (Ennis and Hayar, 2008). In
contrast to PG cells, ET cell dendrites occupy a large volume of
the glomerulus. To date, at least two mor phologically distinctive
subgroups of ET cells have been reported (Macrides and Schnei-
der, 1982; Schoenfeld et al., 1985). One group has no secondary
dendrites; the cell body is only found in the GL; and the axon is
apparently restricted within the olfactory bulb. The other group of
ET cells has secondary dendrites that extend to the EPL, with cell
bodies generally found in the deeper one-third of the GL, or in the
EPL near the boundary with the GL. The axons of the latter group
project either to the internal plexiform layer (IPL) of the other
side (medial-lateral) of the same bulb, or to the anterior olfactory
nucleus (AON) pars externa (pE). The AONpE connects the cir-
cuits associated with homotypic glomeruli receiving input from
olfactory sensory neurons that express the same odorant recep-
tor between right and left olfactory bulb (Liu and Shipley, 1994;
Lodovichi et al., 2003; Yan et al., 2008). As discussed later, these
two groups may also be distinct in other attributes.
These JG neurons, including the subtypes discussed in the fol-
lowing subsections, are summarized in Figure 2A and Ta b l e 1.
More detailed description about basic neuronal circuits in the GL
can be found in an excellent review by Wachowiak and Shipley
(2006).
DIVERSITY OF PERIGLOMERULAR CELLS REGARDING SYNAPTIC
ORGANIZATION
PG cells can be divided into two subtyp es based on its synap-
tic organization. A glomerulus in the olfactory bulb is composed
of two distinct kinds of anatomical compartments (Kosaka et al.,
1996, Kasowski et al., 1999). One compartment includes the pro-
cesses of olfactory sensory neurons (ON zone), and the other
compartment lacks these processes and is instead occupied by the
processes of bulbar neurons (non-ON zone). Some PG cells extend
their dendrites to both zones (referred to as type-I PG cells), but
other PG cells extend their dendrites only to the non-ON zone
(referred to as type-II PG cells; Kosaka et al., 1998). This obser-
vation suggests that type-II PG cells do not receive direct inputs
from olfactory sensory neurons.
In parallel with the above-described anatomical heterogeneity,
experiments using slice preparations suggested two types of synap-
tic organization in PG cells, based on their physiological properties
(Shao et al., 2009; Kiyokage et al., 2010).Thefirsttypeofneu-
ron receives single spontaneous excitatory post-synaptic currents
(EPSCs) and exhibits consistent and shorter delays in response to
the electric stimulation of olfactory nerve bundles in the ONL. The
second type of neuron receives a burst of spontaneous EPSCs and
exhibits longer and varying delays to peri-threshold nerve stimu-
lation. The former neuron is likely driven by direct monosynaptic
inputs from olfactory sensory neurons (ON-driven cells), whereas
the latter is likely driven by polysynaptic inputs through olfactory
bulb neurons, such as ET cells (ET-driven cells; Shao et al., 2009).
It is tempting to relate type-I and type-II PG cells to ON-driven
cells and ET-driven cells, respectively. However, the relationship
may not be that straightfor ward. Type-II PG cells could be exclu-
sively ET-driven, given that they have no connection to olfactory
sensor y neurons. On the other hand, t ype-I PG cells, which may
connect to both olfactory sensory neurons and other cells, could
in principle be driven by either pathway. Therefore, the dominant
pathway is probably determined by as yet unidentified, additional
factors (Kiyokage et al., 2010).
DIVERSITY OF PERIGLOMERULAR CELLS REGARDING NEURONAL
TRANSMITTER
The neurotransmitters of PG cells are described largely on the
basis of expression of related molecular markers. Among the
many candidate mar kers related to neurotransmitters (Kosaka
et al., 1998), glutamic acid decarboxylase (GAD), and tyrosine
hydroxylase (TH) have attracted the most attention. Two isoforms
of GAD (GAD65 and GAD67) are of particular interest. First, the
expression of these proteins in a neuron implies its physiological
function as a GABAergic neuron. Second, one or both of these
isoforms is expressed in more than half of the entire JG cell pop-
ulation (Parrish-Aungst et al., 2007; Whitman and Greer, 2007).
Even though the relative proportions of each isoform in a par-
ticular cell and/or their co-expression are of broad interest, no
conclusive figures are yet available. One technical difficulty may
be a potential mismatch between the population positive for GAD
immunolabeling, genetic markers [e.g., green fluorescent protein
(GFP)], and the true population of GABAergic neurons. Another
problem is that a fraction of the PG cell population is not labeled
by the general neuronal marker, NeuN (Panzanelli et al., 2007;
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FIGURE 2
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Subtypes of neurons in the olfactory bulb. (A) Schematic
illustration of the subtypes of periglomerular cells (PG cells; purple),
superficial short-axon cells (sSA cells; red), and external tufted cells (ET cells;
blue). Two subtypes of PG cells are based on their synaptic connections.
Type-I PG cells receive synaptic inputs on their dendrites from both olfactory
sensory neurons and neurons in the olfactory bulb. Type-II PG cells only
receive inputs from neurons in the olfactory bulb. PG cells can be further
divided into neurochemical subtypes (not shown in the figure). Classically,
sSA cells had an axon and dendrites.The dendrites did not enter a glomerulus.
More recently reported types of sSA cells are positive for tyrosine
hydroxylase (TH) and connected to a few to tens of glomeruli. Subtypes of ET
cells are determined by morphology: those without and those with secondary
dendrites. The ET cells with secondary dendrites are more frequently found
around the border between the GL and the superficial external plexiform layer
(s-EPL). (B) Schematic illustration of subtypes of tufted cells (green) and
mitral cells (blue), as well as interneurons (red) in the external plexiform layer
(EPL) and the mitral cell layer (MCL). Tufted cells are classified as external,
middle, and internal tufted cells based on their soma location. ET cells are
discussed in the GL section of the main manuscript and thus are not shown
here. The EPL itself can be divided into multiple sub-layers, including the
s-EPL, the intermediate EPL (i-EPL), and the deep EPL (d-EPL). The two
subtypes of mitral cells are based on the depth of their secondary (basal)
dendrites in the EPL sub-layers and are referred to as type-I and type-II mitral
cells, respectively. On average, middle tufted cells have smaller cell somata
than internal tufted cells, and internal tufted cells have smaller cell somata
than mitral cells. Among interneurons, only large short-axon cells have axons.
Some of the neurons that had originally been categorized as short-axon cells
have an axon-like process (see main text), but they are not shown in this
figure to avoid any confusion. Van Gehuchten cells are axonless cells, while
inner horizontal and multipolar-type cells are identified by their morphological
features and locations. The majority of interneurons have been studied by
parvalbumin (PV) labeling. In addition, somatost atin-immunoreactive (SRIF-ir)
cells are located in the i-EPL/d-EPL and extend their dendrites specifically to
the d-EPL. (C) Schematic illustration of six subtypes of granule cells (GC;
black) and three subtypes of deep short-axon cells (dSA cells; red) in the
granule cell layer (GCL). The subtypes of granule cells and dSA cells are
determined based on the location (depth) of their cell somata in the GCL and
the layer or sublayer to which their dendrites extend. These subtypes of GC
are referred to as type-I, type-II, type-III, type-IV, type-V, and type-S cells. The
subtypes of dSA cells are referred to as GL-dSA, EPL-dSA, and GCL-dSA cells,
reflecting the distribution of their axons. Abbreviations: GL, glomerular layer,
ONL, olfactory nerve layer; IPL, internal plexiform layer.
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Table 1
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Glomerular layer.
Cell type Periglomerular cell
(PG cell)
Superficial short-axon cell
(sSA cell)
External tufted cell
(ET cell)
Subtype Type-I Type-II Classic TH+/GAD67+ No secondary
dendrite
With secondary
dendrite
Soma size 5–10 μm 8–12 μm 10–15 μm
Soma location Interglomerular space Interglomerular space Interglomerular space
GL-EPL border
Dendrite extension Single glomerulus
(infrequently two glomeruli)
Interglomerular
space
Multiple glomeruli Single glomerulus
(infrequently two glomeruli)
Superficial EPL
Input ET cell, PG cell, sSA cell, tufted cell,
mitral cell, GL-dSA cell, centrifugal fiber
PG cell, sSA cell,
centrifugal fiber
ET cell (same
glomerulus)
OSN, ET cell
(VGLUT3+, same glomerulus)
OSN GC
Output Tufted cell, mitral cell, PG cell, OSN PG cell, sSA cell
(classic), tufted
cell, mitral cell
ET cell (other
glomeruli)
PG cell, sSA cell (TH+/GAD67+), ET
cell (same glomerulus), mitral cell,
tufted cell
neuron in AONpE
Transmitter GABA, dopamine (in the TH+
subtype)
Unknown GABA, dopamine Glutamate, GABA (in the VGLUT3+
subtype)
CCK, vasopressin
Known neurochemical
subtypes
TH+ CB+,CR+ Unknown Unknown VGLUT2+,
VGLUT3+
Other known
molecules expressed
(in subpopulation)
GAD65, GAD67, PV, neurocalcin,
GABA
A
-R α5 subunit
Unknown GAD67, TH CB, GAD67, GABA
A
-R α1 and α3
subunits
CCK, vasopressin
Proportion to the total
JG cell population
Majority Unknown
(minority)
Less than 10%* Unknown
Function
Inhibition within the glomerulus Unknown Inhibition across
glomeruli
Excitation within the glomerulus
Connecting circuits
associated with the
glomeruli of the
same ORN
Additional notes
May have been
included in TH-
positive PG cell in
some studies
Also referred to as
“superficial tufted
cell”
References Pinching and Powell (1971a), Pinching
and Powell (1971c), Kosaka et al.
(1998), Kosaka and Kosaka (2005),
Wachowiak and Shipley (2006),
Kosaka and Kosaka (2007a),
Parrish-Aungst et al. (2007), Panzanelli
et al. (2007), Shao et al. (2009)
Pinching and
Powell (1971a),
Pinching and
Powell (1971c)
Aungst et al.
(2003), Kiyokage
et al. (2010)
Macrides and Schneider (1982),
Panzanelli et al. (2005), Tatti et al.
(2014)
Liu and Shipley
(1994), Tobin et al.
(2010)
*TH+ neurons (TH+ PG cell and TH+ sSA cell) represent 10% of entire JG cell population (Parrish-Aungst et al., 2007).
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Par rish-Aungst et al., 2007; Whitman and Greer, 2007). These lim-
itations make it difficult to reliably estimate the number of all PG
cells and their expression of GAD.
TH is an essential enzyme for the synthesis of dopamine. TH-
positive cells account for approximately 10% of all JG cells, and
most of these, if not all, co-express GAD. Indeed, the majority of
the cells, and perhaps the entire population, is positive for GAD67,
while a minor percentage is positive for both GAD65 and GAD67,
or possibly for GAD65 alone. TH-positive neurons were consid-
ered as belonging to the PG cell population (Kosaka et al., 1998;
Kosaka and Kosaka, 2005). However, recent studies revealed that
some of TH-positive neurons are in fact morphologically similar
to sSA cells (see below).
MOLECULAR DIVERSITY OF PERIGLOMERULAR CELLS
A number of studies address the molecular diversity of JG cells
(Panzanelli et al., 2007; Parrish-Aungst et al., 2007). Although
many of these studies did not originally intend to focus on spe-
cific morphological cell types, ET cells are negative for most of
the molecular markers employed in these studies (but see below).
Other studies limited their analyses to only the GABAergic neu-
rons. As a consequence, while the vast majority of these studies
intended to address the molecular diversity of PG cells, they
might have inadvertently included GABAergic sSA cells and/or
GABAergic ET cells.
The calcium-binding proteins, and particularly calretinin (CR)
and calbindin (CB), have been extensively studied for their expres-
sion in PG cells. The expression of CR, CB, and TH is mutually
exclusive, suggesting that each of these markers corresponds to a
specific subtype of PG cell. Nearly all TH-positive and CB-positive
PG cells, as well as the majority of CR-positive PG cells, are posi-
tive for GAD (Kosaka and Kosaka, 2007a; Sawada et al., 2011). TH,
CB, and CR are co-expressed in PG cells with both GAD isoforms,
and therefore are not exclusively associated with any one isoform.
TH-positive PG cells are type-I PG cells, while CB-positive and CR-
positive PG cells are type-II PG cells (Kosaka and Kosaka, 2005,
2007a). Multiple research groups have made attempts to deter-
mine the proportions of subtypes expressing these neurochemicals
in the mouse (Panzanelli et al., 2007; Parrish-Aung st et al., 2007;
Whitman and Greer, 2007; Sawada et al., 2011). Although the
results are not ideal for direct comparisons between studies, the
percentages of each marker fall within certain ranges: 30.0–44.0%
for CR, 12.6–20.0% for TH, and 9.8–15.0% for CB. The diffi-
culty of direct comparison mainly stems from varying definitions
of overall cell populations. As described previously, enumeration
of PG cells is technically challenging. Each laboratory has taken
different paths to estimate the overall population of PG cells in
the olfactory bulb, including counting all neurons positive for
any neuronal markers tested (Parrish-Aungst et al., 2007); count-
ing all cells labeled with the fluorescent dye DRAQ-5, except for
ET cells (Whit man and Greer, 2007); counting cells immuno-
histochemically positive for a mixture of anti-GAD65/67 and
anti-GABA (Kosaka and Kosaka, 2007a); or using transgenic mice,
such as GAD67-GFP knock-in mice (Panzanelli et al., 2007)or
vesicular GABA transporter (VGAT)-venus mice (Sawada et al.,
2011), in which GABAergic neurons are labeled w ith fluorescent
proteins.
The expression of additional neurochemical markers, such as
neurocalcin, parvalbumin (PV), and GABA
A
receptor α5 sub-
unit, have also been explored in several studies (Panzanelli et al.,
2007; Parrish-Aungst et al., 2007; Whitman and Greer, 2007).
Although these markers have not been investigated as exten-
sively as the molecular markers, CR, CB, and TH, no evidence
for co-expression with CR, CB, TH, or any other co-tested
markers has been found. Accordingly, PG cells positive for
some of these mar kers may also represent distinct cell subtypes.
There are also reports of other markers that may only partially
co-localize in PG cells (Kosaka and Kosaka, 2012). Such stud-
ies imply that the number of cellular molecular subtypes will
continue to grow in the future based on the combination of
multiple molecular markers. However, expression of many molec-
ular markers (e.g., calcium-binding proteins) does not suggest
the function of the cell subtype in and of themselves. Func-
tional characterization of each cell subtype will therefore be
essential.
DIVERSITY OF SUPERFICIAL SHORT-AXON CELLS
There is little literature explicitly describing the diversity of sSA
cells. However, two types of sSA cells have apparently been docu-
mented in the literature. The first type is the sSA cells described
by Pinching and Powell (Pinching and Powell, 1971a; see above).
Here we mainly discuss the second type that was reported more
recently.
Retrograde tracing showed that a subpopulation of JG cells
possess long neuronal processes (several 100 μm to 1 mm).
This population of neurons is positive for both TH and GAD67
(TH+/GAD67+ neurons; Kiyokage et al., 2010; Kosaka and
Kosaka, 2011). Kiyokage et al. (2010) proposed that interglomeru-
lar process-bearing TH+/GAD67+ neurons might be classified as
sSA cells. These cells were once reported as glutamatergic sSA cells
by the same group (Aungst et al., 2003).
However, the morphology of this proposed TH+/GAD67+ sSA
cell is clearly distinct from that of the “classic sSA cell reported
by Pinching and Powell (Kosaka and Kosaka, 2011; Pinching
and Powell, 1971a,c). A TH+/GAD67+sSA cell has an axon that
extends for 1 mm, and its dendrites make contacts with up to
50 glomeruli. On the other hand, a classic sSA cell has an axon
that extends for just one to two glomeruli, and its dendrites avoid
glomeruli. This discrepancy suggests that TH+/GAD67+sSA cells
are a different breed from the classic sSA cell, even though their
morphology is typical of the short-axon cell. It seems possible
that there are two (or m ore) types of short-axon cell-like neu-
rons in the GL, considering the heterogeneity of short-axon cells
in the EPL and the GCL, as discussed in more detail later in the
review.
Finally, TH-positive JG cells show further heterogeneity in
soma size, where the size forms a bimodal distribution (Pignatelli
et al., 2005; Kosaka and Kosaka, 2008b). The “small-soma group
is typical of PG cells, whereas the “large-soma” group exhibits
a slig htly larger soma than the PG cells. It is worthwhile to
point out that TH+/GAD67+sSA cells are included in the large-
soma group (Kosaka and Kosaka, 2008b). Therefore, we suggest
that TH+/GAD67+ cells account for only a subpopulation of
TH-positive JG cells.
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MORPHOLOGICAL DIVERSITY OF EXTERNAL TUFTED CELLS
As discussed previously, ET cells are historically divided into
two morphologically distinct subtypes: those without and
those with secondary (basal) dendrites (alternatively, the cells
can be divided into three subtypes by further dividing the
dendrite-bearing cells according to morphological differences
in the secondary dendrites; Macrides and Schneider, 1982).
More recently, these morphological differences were shown
to correlate with physiological distinctions (Antal et al., 2006).
Therefore, the non-basal-dendrite-bearing and basal-dendrite-
bearing ET cells may form two (or potentially more) separate
populations.
In several studies, the basal-dendrite-bearing cells are referred
to as superficial tufted cells (Ezeh et al., 1993; Kiyokage et al.,
2010). Note that tufted cells are most typically classified into
three types: external, middle, and internal tufted cells (see below).
On the other hand, even though some ET cells with secondary
dendrites are located in the superficial part of the EPL, they
can still be distinguished from the middle tufted cells, par-
ticularly because few axons from these cells travel beyond the
AON. It is therefore of great interest to determine whether the
synaptic organization within the glomerulus is the same or dif-
ferent among each subtype of ET cells and the middle tufted
cells.
DIVERSITY OF EXTERNAL TUFTED CELLS REGARDING NEURONAL
TRANSMITTER
ET cells have long been considered exclusively glutamatergic.
However, the latest study has revealed a novel and interesting sub-
type of ET cells, which is identified by its expression of vesicular
glutamate transporter (VGLUT)3. A portion of this subtype is
found to be not only glutamatergic but also GABAergic (Tatti
et al., 2014). The synaptic targets of ET cells include other JG
cells and projection neurons (both tufted and mitral cells) in the
same glomerulus that connect through dendrodendritic synapses.
For projection neurons, ET cell mediated di-synaptic inputs are
a major source of excitatory inputs, in addition to direct inputs
from olfactory sensory neurons (Wachowiak and Shipley, 2006;
Najac et al., 2011; Gire et al., 2012). VGLUT3-positive subtype
has been exhibited unique connectivity, that is, it excites tufted
cells but not mitral cells. VGLUT3-positive subtype also inhibits
the other subtype of ET cells i n the same glomerulus (Tatti et al.,
2014).
Furthermore, to date, two peptide hormones are known to
be released from subpopulations of ET cells. Liu and Ship-
ley (1994) reported a subpopulation of ET cells (referred to as
“superficially situated tufted cells”) that are involved in intr a-
bulbar (medial-lateral) connections by using the peptide hor-
mone, cholecystokinin (CCK), as a neurotransmitter. However,
it is not clear whether CCK is used only by this type of ET
cell, or by other ET cells as well. Tobin e t al. (2010) reported
a subpopulation of ET cells that releases a peptide hormone
vasopressin. These vasopressinergic ET cells are involved in
processing olfactory signal related to social recognition. These
CCKergic and vasopressinergic ET cells are most often located
around the boundary of GL and EPL, and have secondary
dendrites.
MOLECULAR DIVERSITY OF EXTERNAL TUFTED CELLS
The molecular diversity of ET cells began to be revealed recently.
For example, ET cells can be divided into specific subpopula-
tions based on immunoreactivit y for different GABA
A
receptor
subunits, such as the α1 and α3 subunits (Panzanelli et al.,
2005). Another study has classified ET cells into VGLUT2-
positive and VGLUT3-positive subpopulations. VGLUT2-positive
and VGLUT3-positive subpopulations are also distinctive about
the synaptic organization in the glomerulus (Tatti et al., 2014).
In the latter study, a portion of VGLUT3-positive ET cells coex-
pressed CB, but few of them coexpressed CR or TH. Although
these molecules may not be strictly specific as molecular markers,
they would be enormously helpful for the future studies on ET
cells.
ADULT-BORN NEURONS IN THE GLOMERULAR LAYER
Subpopulations of olfactory bulb neurons are generated during
adulthood and continue to be replaced throughout the life of the
organism. JG cells are included in these subpopulations, although
the proportion of adult-born neurons in the GL is much lower than
that in the GCL. These neurons are generated in subventicular zone
(SVZ) or rostral migratory stream, and then migrate to the olfac-
tory bulb through the rostral migratory stream. Adult-born JG
cells are probably primarily composed of PG cells, based on their
morphology. Whitman and Greer (2007) described two morpho-
logically distinct adult-born JG cells, one with a dendritic arbor
limited to one or two glomeruli, and the other with more extensive
multi-glomerular dendrites. The latter cells also had slightly larger
cell bodies. Nonetheless, TH-positive neurons with interglomeru-
lar processes (i.e., the TH+/GAD67+ sSA cells discussed earlier)
may not be generated in adulthood (Kosaka and Kosaka, 2009).
As in the overall PG cell population, neurochemical subtypes
have been revealed in adult-born JG cells. The vast majority of
these subtyp es are likely GABAergic (Whitman and Greer, 2007;
Sawada et al., 2011). However, a recent study reported the pres-
ence of adult-born, g lutamatergic JG cells (Brill et al., 2009). Even
though their numbers are small, further characterization of these
neurons would be of great interest.
Regarding other molecular markers, adult-born PG cells
express all three typical molecular markers discussed above: TH,
CR, and CB. Whitman and Greer (2007) determined the percent-
age of each PG cell subtype in adult-born JG cells of a specific
age by using the thymidine analog, 5-bromo-2
-deoxyuridine
(BrdU), to label cells generated at the time of injection. At 46 days
post-BrdU injection, a date at which adult-born PG cells are mor-
phologically mature, BrdU-positive cells ( 46 days old) contain
higher p ercentages of TH-positive, CR-positive, and GAD67-
positive neurons compared with BrdU-negative cells (cells of all
other ages, including embryonic cells). This finding raises the pos-
sibility that the relative percentage of PG cell subtypes may not be
static, but instead keeps changing throughout life.
EXTERNAL PLEXIFORM LAYER AND MITRAL CELL LAYER
PROJECTION NEURONS IN THE EXTERNAL PLEXIFORM LAYER AND
MITRAL CELL LAYER
Odor signals, which are processed within the glomerulus, prop-
agate in the EPL along the primary dendrite of two types of
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projection neurons, mitral and tufted cells (Figure 1). The signal
arrives at the cell body of tufted cells in the EPL and mitral cells
in the mitral cell layer (MCL), then horizontally back-propagates
through the secondary dendrites in the EPL. The back-propagation
signal is considered not to be attenuated, but rather to be con-
ducted throughout the dendrites until it is blocked by local
inhibition from granule cells (Xiong and Chen, 2002). The hor-
izontal signal propagation is believed to inhibit the activity of
other mitral/tufted cells via the dendrodendritic interaction with
granule cells and other interneurons. This process is called lateral
inhibition. The EPL is mostly occupied by dendritic fibers that are
secondary dendrites of mitral/tufted cells and apical dendrites of
granule cells.
Mitral and tufted cells share many morphological properties.
For example, they both extend a single primary dendrite to one
of the several thousand glomeruli in the olfactory bulb. This
observation signifies that each projection neuron receives odor
information originating from only one type of odorant recep-
tor. Therefore, even at the level of projection neurons, each
neuron follows the “single cell single odorant receptor” rule.
Furthermore, neurons associated with the same glomerulus (sis-
ter cells) receive homogeneous input from the same olfactory
sensory neurons and thus are thought to have similar odorant
response properties. However, their odor-tuning specificity is vari-
able, depending on odor concentration and spatial location (Tan
et al., 2010; Kikuta et al., 2013). In addition, sister cells reportedly
have various temporal activ ity patterns (Dhawale et al., 2010).
Mitral and tufted cells also share certain biophysical properties.
For instance, the dendrodendritic recipro cal synapses between
tufted and granule cells are both AMPA and NMDA receptor-
mediated synapses but NMDA receptor is essential for the den-
drodendritic inhibition (Christie et al., 2001), as are the synapses
between mitral and granule cells (Isaacson and Strowbridge, 1998;
Chen et al., 2000).Therefore,tuftedcellstendtoberegardedas
smaller mitral cells, and they are often, especially in physiologi-
cal studies, categorized into a single group termed “mitral/tufted
cells.” In other organisms frequently used in olfactory research,
such as the fly and the fish, mitral, and tufted cells are not mor-
phologically well segregated and are accordingly categorized into
a single group of projection neurons (Satou, 1990; Bargmann,
2006).
However, in the mammal, the morphologies of mitral and
tufted cells are discriminable, especially by the location of the
cell body and the extension pattern of secondary dendrites in the
EPL (Mor i et al., 1983; Orona et al., 1983). The secondary den-
drites of the majority of tufted cells extend to the superficial/outer
EPL, while those of mitral cells mostly extend to the deep/inner
EPL. Hence, the EPL could be subdivided into at least two sub-
layers, the superficial/outer and the deep/inner layers, althoug h a
clear marker for the sublayers is not known. Interestingly, an early
report identified a third intermediate sublayer, which is labeled by
cytochrome oxidase (Mouradian and Scott, 1988).
Cell bodies of tufted cells are sparsely distributed in the EPL,
and it is rare for them to find adjacent tufted cells. To the contrar y,
the cell bodies of mitral cells are surrounded by adjacent mitral
cells in the MCL. Given the idea that more closely situated neurons
tend to interact more strongly via reciprocal synapses with granule
cells, such interactions are expected to be stronger among mitral
cells than among tufted cells. The difference may, at least partially,
be responsible for functional discrepancies between mitral and
tufted cells (e.g., differential odor selectivity; Kikuta et al., 2013).
Targets of axons also differ between tufted cells and mitral cells
(Haberly and Price, 1977; Skeen and Hall, 1977; Scott et al., 1980).
The axons of tufted cells project to the anterior olfactory cortex,
including the olfactory peduncle, olfactory tubercle, and ventro-
rostral subdivision of the piriform cortex. By contrast, those of
mitral cells project widely throughout the entire olfactory cortex
(Nagayama et al., 2010; Igarashi et al., 2012). Thus, there may be
two types of axon bundles in the lateral olfactory tract, where one
bundle is composed of thicker axons, and the other is composed
of thinner axons [Price and Spri ch, 1975; see also Bartolomei and
Greer (1998) for PCD mice]. It is speculated that the thicker axons
represent projections from mitral cells, while the thinner axons
represent projections from tufted cells.
Functionally, tufted cells have lower thresholds to induce spike
discharges by electrical stimulation of olfactory sensor y neurons
(Ezeh et al., 1993). The same result was also obtained with odorant
stimulation of sensory neurons (Igarashi et al., 2012; Kikuta et al.,
2013). In addition, tufted cells show a higher firing frequency than
mitral cells (Nagayama et al., 2004), and tufted cells respond to a
broader range of odorants than mitral cells (Nagayama et al., 2004;
Kikuta et al., 2013). Recent reports also indicated that tufted cells
respond during an earlier phase of the respiratory cycle, while
mitral cells are activated during a later phase of the respiratory
cycle (Fukunaga et al., 2012; Igarashi et al., 2012).
Mitral and tufted cells are gener ated during different periods
of development. Whereas most mitral cells are born between
embryonic days 10 and 13, tufted cells are born during a later
period (embryonic days 13–16; Hinds, 1968; Imamura et al.,2011).
The distinction in the timing of the genesis of tufted cells and
mitral cells may affect to the differential locations of their somata,
extension patterns of secondary dendrites, axon projections, and
terminal locations (Inaki et al., 2004;
Imamura et al., 2011).
Below, we discuss the structural and functional features of
subgroups of tufted cells, mitral cells, and interneurons. The
morphological features of these var ious kinds of neurons are
summarized in Figure 2B and Ta b l e 2 .
SUBGROUPS OF TUFTED CELLS
Currently, tufted cells tend to be categorized into three sub-
groups: ET cells, middle tufted cells, and internal tufted cells.
ET cells have relatively small cell bodies (10–15 μm) and are
located around the boundar y between the GL and the EPL (Pinch-
ing and Powell, 1971a). As discussed previously, ET cells can be
separated into two distinct populations, one with no secondary
dendrites, and the other with extended secondary dendrites in the
superficial EPL.
Middle tufted cells are located in the intermediate and super-
ficial EPL, which lies underneath the boundary between the GL
and EPL. As might be expected, the cell body of these cells is of a
medium size (15–20 μm; Shepherd et al., 2004). The majority of
the middle tufted cells extend relatively short secondary dendrites
in the superficial and intermediate EPL. They also extend axon
collaterals in the IPL (Orona et al., 1984), which probably make
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Table 2
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External plexiform layer and mitral cell layer.
Projection neurons
Cell type Tufted cell Mitral cell
Middle Internal Type-I Type-II
Soma size 15–20 μm >20 μm >20 μm
Soma location Superficial-intermediate EPL Deep EPL MCL
Dendrite extension Superficial-intermediate EPL Deep EPL Intermediate EPL
Output within OB Dendrites of PG, granule (probably type-I and -III) and
other interneurons in EPL
Dendrites of PG, granule (Probably type-I and -II) and
other interneurons in EPL
Output out of OB Anterior part of olfactory cortex Entire olfactory cortex
Transmitter Glutamate
Molecular markers Tbx21, Pcdh21
Population 50 tufted cells/glomerulus 20 mitral cells/glomerulus
Physiological properties Higher sensitivity to the odor stimuli Lower sensitivity to the odor stimuli
Odor evoked spike activity during early phase of
respiratory cycle
Odor evoked spike activity during late phase of
respiratory cycle
Additional notes Sometimes, it is called
intermediate tufted cells
Sometimes, it is called
displaced mitral cells
References Mori et al. (1983), Orona et al. (1984), Ezeh et al. (1993), Royet et al. (1998), Nakajima et al. (2001), Nagai et al. (2005),
Shepherd et al. (2004), Igarashi et al. (2012), Fukunaga et al. (2012)
Interneurons
Cell type Van Gehuchten cell types Multipolar types
Van Gehuchten cell Somatostatin-
immunoreactive cell
Inner horizontal cell Other types Large short axon cell
Soma size 12 μm 10 μm 9–12 μm 9–15 μm 14 μm
Soma location
Throughout the EPL Intermediate-deep EPL Deep EPL and MCL Throughout the EPL Superficial EPL
Dendrite extension Throughout the EPL Deep EPL MCL and just above MCL Throughout the EPL EPL and GL
Output Unknown Probably mitral cell probably mitral cell Mitral/tufted cell Unknown
Transmitter
Almost all of EPL interneurons are GABAergic
Molecular markers CR Somatostatin, CR, VIP CR
Axon No Ye s
Additional notes Fusiform shape soma Relatively rare to be found
References
Schneider and Macrides (1978), Kosaka et al. (1994), Brinon et al. (1998), Kosaka and Kosaka (2008a), Lepousez et al. (2010)
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contact with the other side (lateral-medial) of olfactory bulb like
ET cells (Belluscio et al., 2002).
Internal tufted cells have relatively large cell bodies (>20 μm)
and are located in the deep por tion of the EPL (Orona et al., 1984).
Internal tufted cells are sometimes called displaced mitral cells,
because they reportedly have st ructural and functional properties
similar to mitral cells. They also extend secondary dendrites in the
intermediate and superficial EPL,as found with middle tufted cells.
No tufted cells have been observed to extend secondary dendrites
in the deep EPL.
SUBGROUPS OF MITRAL CELLS
Mitral cells have large cell bodies (>20 μm) and are found in
the MCL. The majority of these cells extend long secondary den-
drites predominantly in the deep EPL and are termed type-I mitral
cells (Mori et al., 1983; Orona et al., 1984). However, some mitral
cells extend secondary dendrites predominantly in the interme-
diate EPL and are termed ty pe-II mitral cells (Orona et al., 1984;
Mouradian and Scott, 1988). In addition, subsets of mitral cells
extend secondary dendrites in the superficial EPL (Imamura and
Greer, unpublished data) and may not represent either type-I or
type-II mitral cells. This new type of mitral cell may thus make a
unique odor-processing contribution and receive dendritic inhi-
bition in the superficial EPL (like tufted cells), as well as strong
somatic inhibition in the MCL from type-S granule cells (also see
GCL section). Recently, several reports have suggested the pres-
ence of different types of mitral cells based on their structur al and
functional properties (Padmanabhan and Urban, 2010; Angelo
and Margrie, 2011; Angelo et al., 2012; Kikuta et al., 2013). By
using in vivo two-photon imaging microscopy, mitral cells were
recently grouped into three subtypes according to cell body shape:
triangular, round, and fusiform type (Kikuta et al., 2013). Due to
the lack of detailed evidence about the secondary dendrite exten-
sion pattern for each of these three subtypes, it is still unclear
whether these cells are related to type-I or ty pe-II mitral cells.
Mitral cells vary in molecular expression profiles. Subsets
of the cells express the α3 subunit of the GABA
A
receptor
(Panzanelli et al., 2005), and variably express the voltage-gated
potassium channel (e.g., Kv1.2) and the hyperpolarization-
activated cyclic nucleotide gated channel (e.g., HCN2; Padman-
abhan and Urban, 2010; Angelo and Margrie, 2011). Because
HCN2 channel expression levels may be strongly associated with
the parental glomerulus, olfactory sensory neuronal activity likely
influences channel expression in mitral cells (Angelo et al., 2012).
These data suggest the possibility that mitral cells can be subdi-
vided based on the expression levels of specific molecules. Recent
reports revealed that intrinsic biophysical properties also vary
among mitral cells, such as firing frequency (Padmanabhan and
Urban, 2010) and the I
h
sag current (Angelo et al., 2012). The I
h
sag current is probably associated with HCN2 expression levels.
These studies highlight the possibility that the activity of mitral
cells is controlled not only by inhibitory neurons in the olfactory
bulb circuit, but also by intrinsic physiological properties.
As noted above, several reports indicate variations in mitral cell
morphology, molecular expression profiles, and biophysical prop-
erties. However, it is uncertain whether these properties are related
to one another. Connecting information and drawing a detailed
profile of each mitral cell subtype will undoubtedly promote an
understanding of odor coding.
INTERNEURONS IN THE EXTERNAL PLEXIFORM LAYER AND MITRAL
CELL LAYER
Several types of local neurons exist in the EPL (Schneider and
Macrides, 1978; Shepherd et al., 2004). The majority are GABAer-
gic neurons and make reciprocal synaptic contacts with mitral cells
(Kosaka et al., 1994; Toida et al., 1994). These neurons reportedly
express several calcium-binding proteins, such as PV, CB, CR, and
neurocalcin (Brinon et al., 1999). Almost all EPL interneurons
express CR. One-third are PV-positive neurons, which are well-
studied, especially regarding their structural features (Lepousez
et al., 2010; Huang et al., 2013). Nonetheless, PV is expressed in
multiple morphological subty pes and thus, cannot be used as a
definitive marker for a specific neuronal subtype. We first dis-
cuss PV-positive neurons below, and then briefly introduce several
morphologically identified subtypes of EPL interneurons.
Parvalbumin-positive neurons
PV-positive cells in the EPL have quite varied structural prop-
erties. The y can be categorized into five g roups: sSA cells, Van
Gehuchten cells, multipolar-type cells, inner short-axon cells, and
inner horizontal cells (Kosaka et al., 1994). The latter two are
regarded as particular subtypes or variations of the multipolar-
type cell. Although some of the neurons are termed short-axon
cells, it is not clear whether these PV-positive cells have axons or
not (also see the following section,“short-axon cells in the external
plexiform layer”; Kosaka et al., 1994; Kosaka and Kosaka, 2008a).
PV-positive cells show reciprocal interactions with mitral cells,
receiving excitatory inputs from mitral/tufted cells and returning
inhibition to mitral/tufted cells, as seen with granule cells (Toida
et al., 1994; Huang et al., 2013; Kato et al., 2013; Miyamichi et al.,
2013). An interesting finding is that PV-positive cells have quite
wide odorant selectivit y, and odors can activate them in a broad
area. Therefore, PV-positive neurons are expected to contribute to
the non-specific gain control signals in the olfactory bulb circuit
(Kato et al., 2013; Miyamichi et al., 2013).
A recent study reported that 80% of corticotropin-releasing
hormone (CRH)-positive neurons express PV, and that the num-
ber of CRH-expressing cells is almost the same as the number of
PV-positive cells in the EPL ( Huang et al., 2013). This implies that
the majority of CRH- and PV-expressing cells represent the same
population. CRH-positive cells have a relatively low input resis-
tance, a high capacitance, and a high firing rate response to current
injection. Therefore, CRH-positive interneurons are considered
to comprise a population of medium-sized and fast-spiking
interneurons in the EPL (Huang et al., 2013).
Short-axon cells in the external plexiform layer
Historically, short-axon cells in the EPL were visualized by using
Golgi techniques (Schneider and Macrides, 1978), and their struc-
tural details were studied by using PV immunostaining (Kosaka
et al., 1994; Brinon et al., 1998). In some PV-positive cells, the
axon initial segments of axon-like processes lack βIV-spectr in, an
essential protein for spike propagation in axon. This observation
suggests that the processes might not function as typical axons.
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Therefore, only large, multipolar cells with distinctive axons are
defined as short-axon cells among PV-positive cells (Kosaka and
Kosaka,2008a). Interestingly, trans-synaptic tracing methods indi-
cated that some of the short-axon cells in the EPL are presynaptic
to adult-born neurons (Arenkiel et al., 2011). This implies that
short-axon cell-like interneurons in this region may act to recruit
or navigate newly generated granule cells into proper connections
with mitral/tufted cells.
Van Gehuchten cells
This neuron type is considered an axonless cell. Van Gehuchten
cells have medium-sized somata (12 μm) and few dendrites with
polarized extension patterns. The thicker and longer dendrites
(<150 μm) extend toward one side of the EPL, and the thinner and
relatively shorter dendrites (<100 μm) extend toward the opposite
pole (Schneider and Macrides, 1978). Half of these cells are PV-
positive (Kosaka et al., 1994; Toida et al., 1996). Neurocalcin- or
CB-positive cells are rarely found (Brinon et al., 1998).
Somatostatin-immunoreactive neurons
These neurons are recently identified and share some of the same
morphological features as Van Gehuchten cells. They are also con-
sidered axonless cells. The soma size is approximately 10 μm.
The majority of somatostatin-immunoreactive (SRIF-ir) neurons
(95%) are located in the intermediate and deep EPL and extend
dendrites specifically into the deep EPL (Lepousez et al., 2010).
Because most mitral cells (type-I) extend secondary dendrites
in the deep EPL, the SRIF-ir neurons may connect specifically
with mitral cell secondary dendrites. SRIF-ir neurons are also
GABAergic (99.4%), CR-positive (99.9%), and vasoactive intesti-
nal polypeptide (VIP)-positive (96.7%) cells. Half of these cells
are also PV-positive.
GRANULE CELL LAYER
The GCL of the olfactory bulb is occupied mostly by granule cells,
which are inhibitory interneurons with a small cell body (6–8 μm
in diameter; Price and Powell, 1970b). Their somata are localized
mostly in the GCL, but some granule cells are also found in the IPL
and the MCL. An apical dendrite typically extends radially toward
the surface of the olfactory bulb and rarely branches in the GCL,
until it ramifies in the EPL. In the EPL, granule cells have dendritic
spines that form reciprocal synapses with the secondary dendrites
of mitral and/or tufted cells. Due to their axonless morphology, the
output of granule cells relies solely on dendrodendritic synapses.
The other interneurons in the GCL are deep short-axon (dSA)
cells. Short-axon cells comprise heterogeneous populations, and
they were previously classified into multiple subpopulations based
on their soma location and morphology (Schneider and Macrides,
1978; Shepherd et al., 2004). Among these short-axon cells, the
Blanes cells, Golg i cells, Cajal cells, and horizontal cells have their
cell bodies in the deeper regions of the olfactory bulb, including the
MCL, IPL, and GCL. These cells are now collectively re-classified as
dSA cells (Ey re et al., 2008). Compared with granule cells, dSA cells
have larger cell bodies (10–20 μm in diameter), and their dendrites
do not usually extend beyond the MCL. On the other hand, they
have axons that project to different layers of the olfactory bulb (see
below).
Granule cells and dSA cells are divided into subgroups, mostly
based on morphological differences between the groups. These
cells are discussed in detail below, and their properties are
summarized in Figure 2C and Ta b l e 3 .
MORPHOLOGICAL DIVERSITY OF GRANULE CELLS
Intracellular horseradish peroxidase (HRP) injection into the rab-
bit olfactory bulb cells revealed at least three morphologically
distinct subpopulations of granule cells based on their dendritic
extension patterns in the EPL (Mori et al., 1983; Mori, 1987). The
type-I granule cell (GI) ramifies spiny dendrites at any depth of
the EPL. Dendrites of the type-II granule cell (GII) extend only
in the deep EPL, while the ty pe-III granule cell (GIII) ramifies
spiny dendrites predominantly in the superficial EPL. Therefore,
type-II and ty pe-III granule cells are thought to preferentially reg-
ulate the activity of mitral cells and tufted cells, respectively. In
an early work, Orona et al. (1983) injected HRP into the EPL of
the rat olfactory bulb to label granule cells extending dendrites
to the injection site. Injection of HRP into the superficial EPL
preferentially labeled the granule cells localized in the superficial
portion of the GCL (superficial g ranule cells). In contrast, HRP
injection into the deep EPL labeled additional granule cells in the
deep GCL (deep granule cells). These results st rongly indicate a
correlation between somata location and dendrite-ramifying area.
Nonetheless, the location of somata is not a definitive determi-
nant of type-I, II, or III cells, because some superficial g ranule
cells ramified dendrites in the deep EPL, while some deep granule
cells sent dendrites up to the superficial EPL.
Several other types of granule cells do not fit into type-I, II, or
III categories. Naritsuka et al. (2009) discovered a novel type of
cell in the GCL of a transgenic mouse expressing GFP under the
control of the nestin promoter. The cell was named a type-S cell
based on its strong GFP expression. Although neuronal progen-
itor cells usually express nestin, the type-S cell was defined as a
mature neuron due to its expression of NeuN, a marker of mature
neurons, and the existence of dendritic spines. The cell was also
considered a granule cell based on its GABAergic phenotype and
axonless morphology. The especially unique feature of the type-S
granule cell was that the apical dendrites did not penetrate into the
EPL, but rather formed reciprocal synapses with the perisomatic
region of mitral cells. Therefore, the dendrites might regulate the
production of action potentials in mitral cells. This hypothesis
could be tested by experiments using electrophysiology and/or
optogenetics.
Recently, Merkle et al. (2014) reported four previously
unknown interneuron types that are generated in the adult mouse
ventricular zone-SVZ and migrate to the GCL. Based on soma
size/location, dendritic arbors bearing spines, and axonless mor-
phology, two of the cells were termed type-IV granule cells (GIV)
and type-V g ranule cells (GV). The type-IV granule cell was fre-
quent dendritic branching in the GCL, and often failed to reach
beyond the IPL. Somata of type-V granule cells lacked basal den-
drites and were restricted to the MCL. These cells extended spiny
shrub-like apical dendrites predominantly into the deep EPL. Due
to their char acteristic features, the authors also called type-IV
and -V granule cells deep-branching granule cells and shrub gran-
ule cells, respectively. However, neurotransmitter and existence of
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Table 3
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Granule cell layer.
Granule cell
Cell type Type-I Type-II Type-III Type-IV (deep-branching) Type-V (shrub) Type-S
Soma size 6–8 μm
Soma location
(primary)
Unspecified Deep GCL Superficial GCL to
MCL
Unspecified MCL Middle GCL
Dendrite extension
All through EPL Deep EPL Superficial EPL Frequently branch in GCL Deep EPL (no basal
dendrite)
MCL
Transmitter GABA GABA? GABA
Output Mitral/tufted cell Mitral cell? Tufted cell? Unknown Mitral cell? Mitral cell soma
References Mori et al. (1983), Orona et al. (1983) Merkle et al. (2014) Naritsuka et al. (2009)
Deep short-axon cell
Cell type GL-dSA EPL-dSA GCL-dSA
Soma size
14–20 μm 11–15 μm 10–20 μm
Soma location (primary) IPL GCL GCL
Dendrite extension Predominantly confined to the IPL MCL, IPL, GCL MCL, IPL, GCL
Axon extension GL (some in EPL) EPL (some in IPL and superficial GCL) GCL (some to the olfactory cortex)
Output PG cell (granule cell) Granule cell Granule cell
Transmitter GABA
Additional notes Horizontal cells, Golgi cells Blanes cells, Cajal cells Horizontal cells, Golgi cells
References Schneider and Macrides (1978), Eyre et al. (2008, 2009)
reciprocal synapses are still necessary to be specified, which will
increase our knowledge of granule cell diversity.
MOLECULAR DIVERSITY OF GRANULE CELLS
To date, only GABA has been identified as a neurotransmitter of
the granule cell. Although glycine evokes an inhibitory response in
both mitral/tufted cells and granule cells, and immunoreactivity
for glycine, the glycine receptor, and glycine transporters is found
in the olfactory bulb (van Den Pol and Gorcs, 1988; Tromb le y
and Shepherd, 1994), there is no direct evidence suggesting the
existence of glycinergic neurons in the GCL (Zeilhofer et al., 2005).
Therefore, glycinergic axons may originate outside the olfactory
bulb.
In contrast to PG cells, granule cells are molecularly less diverse.
Among known molecular markers expressed by subpopulations
of PG cells (i.e., TH, CR, CB, and PV), only CR is expressed
by a subset of g ranule cells that are localized in superficial GCL
(Batista-Brito et al., 2008). In addition, 5T4, a leucine-rich-repeat
transmembrane protein, also labels a subpopulation of superficial
granule cells (Imamura et al., 2006; Yoshihara et al., 2012). Because
dendrites of the 5T4-positive granule cells preferentially ramify in
the superficial EPL, these cells most likely represent type-III gran-
ule cells. On the other hand, 5T4-positive granule cells are mostly
found in the MCL and IPL, but r a rely in the GCL, suggesting that
they account for only a subset of type-III granule cells.
Curiously, few molecules specifically expressed by deep gran-
ule cells have been identified. As an exception, electrophysi-
ological analyses suggested that expression levels of Group I
metabotropic glutamate receptors (mGluRs) differed between
superficial and deep granule cells (Heinbockel et al., 2007). (RS)-
3,5-dihydroxyphenylglycine (DHPG), an agonist of Group I
mGluRs, directly depolarized both superficial and deep granule
cells in the wild-type mouse olfactory bulb slice. In mGluR5
/
and mGluR1
/
mice, only superficial and deep granule cells,
respectively, were depolarized with DHPG. Further confirmation
of differential expression of these molecules could be achieved
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Nagayama et al. Olfactory bulb neuronal types/subtypes
by immunohistochemical analyses with antibodies specific to
mGluR1 and mGluR5.
Recently, Saino-Saito et al. (2007) reported that CaM kinase IV
(CaMKIV) expression is restricted to deep granule cells. How-
ever, according to the immunohistochemical staining pattern,
CaMKIV-positive cells localized to the entire GCL, but rarely to the
MCL. The authors apparently defined granule cells in the whole
GCL as deep granule cells. Therefore, albeit an interesting obser-
vation, CaMKIV might not be specific to deep granule cells under
the definition in this review, in which the GCL is divided into
superficial and deep layers. One possible reason for the lack of
identification of any molecule specifically expressed by deep gran-
ule cells is simply that we have not yet examined the expression
of the appropriate molecules. However, an alternative possibility
is that granule cells innately have properties to differentiate into
deep granule cells, and are thus redirected toward superficial gran-
ule cells by the expression of alternative molecules, such as Pax6
(see below).
ADULT-BORN GRANULE CELLS
An estimated 10,000 new neurons are generated in the adult mouse
SVZ and enter into the olfactory bulb via the rostral migratory
stream every day. Approximately half of these new neurons are
integrated into the existing neuronal circuit as adult-born granule
cells (Petreanu and Alvarez-Buylla, 2002; Yamaguchi and Mori,
2005). On the other hand, many pre-existing granule cells are
eliminated from the circuit via apoptotic cell death every day.
Whether specific types of granule cells are replaced with adult-
born granule cells is an interesting question. Both morphologically
and molecularly, all types of granule cells mentioned above, except
for type-S cells, are generated in the adult olfactory bulb (Merkle
et al., 2007, 2014). To date, the replacement of type-S cells has
not been investigated. Although adult-born granule cells are dis-
tributed throughout the MCL, IPL, and GCL, several studies have
suggested that adult-born granule cells are located in the deep GCL
with higher density, while embryonically/perinatally born gr anule
cells are preferentially located in the MCL, IPL, and the superficial
GCL (Lemasson et al., 2005; Imayoshi et al., 2008). Intriguingly,
retroviral fate mapping in the rat revealed that more adult-born
granule cells ramified their dendrites in the deep EPL, even thoug h
their somata were located in the superficial GCL (Kelsch et al.,
2007). These studies raise the idea that the microcircuit formed in
the deep EPL is more plastic than that in the superficial EPL in the
adult olfactor y bulb.
DETERMINANTS OF GRANULE CELL DIVERSITY
What determines the final morphology and molecular phenotype
of granule cells? Deep granule cells are mainly generated during
postnatal stages, while superficial gr anule cells have a generation
peak around the perinatal period (Hinds, 1968). Thus, the tim-
ing of neurogenesis influences the fate of granule cells. However,
the factor that probably has the largest influence on granule cell
diversity is the SVZ region in which the granule cell is gener-
ated. Neural stem cells in different portions of the SVZ produce
different types of granule cells. Lineage tracing analyses of cells
generated in various SVZ regions revealed that superficial and deep
granule cells are preferentially produced in the dorsal and ventral
SVZ, respectively (Merkle et al., 2007). Kelsch et al. (2007) used
retroviral injection into the SVZ to show that the anterior and
posterior axis also influenced the production of superficial and
deep granule cells. In addition, Merkle et al. (2007) showed that
CR-positive granule cells were mostly produced from the rostral
migratory stream or the medial wall of the anterior SVZ. Further-
more, grafting experiments from these groups suggested that SVZ
stem cells are highly resistant to respecification by environmental
cues.
The molecular mechanisms regulating the production of spe-
cific g ranule cell types are not well known. Two transcription
factors, Pax6 and ER81, are reportedly expressed only in sub-
sets of superficial granule cells (Kohwi et al., 2005; Saino-Saito
et al., 2007; Haba et al., 2009). Moreover, Pax6-deficient stem cells
grafted into the wild-type SVZ produced many deep granule cells,
but failed to produce superficial granule cells or TH-positive PG
cells (Kohwi et al., 2005). These results may indicate that newly
generated granule cells have innate properties to differentiate into
deep granule cells, and are directed to become superficial gran-
ule cells via the expression of an additional set of transcription
factors.
DIVERSITY OF DEEP SHORT-AXON CELLS
Based on morphological diversity revealed by Golgi staining, dSA
cells in the rodent olfactory bulb were initially defined belong-
ing to one of four cell types: Blanes cells, Golgi cells, horizontal
cells, and Cajal cells. Schneider and Macrides (1978) described
the morphology and location of the various dSA cells in the ham-
ster olfactory bulb. Blanes cells are mostly found in the GCL or
the IPL and have the largest cell body (16–23 μm) of the four
dSA cells. They also have stellate dendrites covered with many
spines. Golgi cells likewise are found in the GCL. Their cell bodies
are slightly smaller than those of Blanes cells (12–22 μm), and
their dendrites rarely have spines. Both horizontal cells and Cajal
cells have the smallest cell bodies (15–18 μm), are restricted to
the IPL and MCL, and have smooth dendrites. To date, all dSA
cells are considered to be GABAergic, as are their post-synaptic
target cells. Recently, dSA cell morphologies were reconstructed
after electrophysiological recording in a rat olfactory bulb slice
(Eyre et al., 2008). Overall, the dendrites of all four dSA cell
types were restricted to the layers below the MCL. Despite the
Schneider and Macrides report of the extension of Cajal cell den-
drites to the EPL, this was not described by Eyre et al. Although
the input sources were not revealed in detail, both excitatory
and inhibitory inputs apparently modulated the activity of dSA
cells.
Eyre et al. (2008) next re-classified dSA cells into three sub-
populations according to their axonal distribution patterns in
the olfactory bulb. The first subpopulation corresponds to the
GL-dSA cell. The axons of these cells travel in the EPL up to
the GL, where they extensively ramify with a few axon collater-
als extending into the EPL and GCL. The major post-synaptic
targets of GL-dSA cells are PG cells in the GL, while symmetri-
cal synapses between axons of GL-dSA cells and the dendrites of
granule cells are found in the EPL and GCL. The somata of many
GL-dSA cells elongate in a direction parallel to the MCL, and are
mostly found in the MCL and IPL. The dendrites of these cells are
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