Structure and Function of the Moth Mushroom Body

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Abstract
Sjöholm, M. 2006. Structure and Function of the Moth Mushroom Body. Doctor’s
dissertation.
ISSN 1652-6880, ISBN 91-576-7087-0
The mushroom bodies are paired, high-order neuropils in the insect brain involved
in complex functions such as learning and memory, sensory integration, context
recognition and olfactory processing. This thesis explores the structure of the
mushroom bodies in the noctuid moth Spodoptera littoralis using neuroanatomical
staining methods, immunocytochemistry and electron microscopy, and investigates
how the intrinsic neurons of the mushroom body, the Kenyon cells, respond to
olfactory stimulation of the antennae using whole-cell patch clamp technique.
The mushroom body in S. littoralis contains about 4,000 Kenyon cells, and
consists of a calyx, pedunculus and two lobes, one medial and one vertical. The
calyx houses dendritic branches of Kenyon cells and the pedunculus and lobes
contain the axons and terminals of these neurons respectively. The calyx is
doubled and concentrically divided into a broad peripheral zone, which receives
input from antennal lobe projection neurons, and a narrow inner zone, which
receives yet unidentified input. The lobes are parsed into three longitudinal
divisions, which contain a separate subset of Kenyon cells each. The Kenyon cells
are divided into three morphological classes, I-III. Class I Kenyon cells have
widely branching spiny dendritic arborisations in both zones of the calyx and
occupy the two most posterior subdivisions of the lobes called / and ´/´.
Class II Kenyon cells have narrow clawed dendritic trees in the calyx and invade
the most anterior division in the lobes, called . Class III Kenyon cells have
clawed, diffusely branching dendrites in the calyx and provide a separate system of
axons and terminal branches, partly detached from the rest of the mushroom body,
called the Y tract and lobelets. Kenyon cells within the classes display differential
labeling with antisera against neuroactive substances. Kenyon cells make synaptic
contact with one another and with other neuron types in the mushroom body.
Extrinsic inhibitory and putative modulatory neurons were identified.
Whole-cell patch clamp recordings revealed that Kenyon cells exhibit broadly
tuned subthreshold activation by odor stimulation and a few cells responded with
action potentials to specific biologically relevant odor combinations.
Keywords: amino acids, Lepidoptera, neuroanatomy, neuropeptides, pheromone,
plant odor, synapse.
Author’s address: Marcus Sjöholm, Department of Crop Science, SLU, SE-23053
ALNARP, Sweden. [email protected]
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Appendix
Papers I-V
This thesis is based on the following papers, which will be referred to in the text
by their Roman numerals.
I. Sjöholm, M., Sinakevitch, I., Ignell, R., Strausfeld, N.J. & Hansson, B.S. 2005.
Organization of Kenyon cells in subdivisions of the mushroom bodies of a
lepidopteran insect. Journal of Comparative Neurology 491, 290-304.
II. Sjöholm, M., Sinakevitch, I., Strausfeld, N.J., Ignell, R. & Hansson, B.S. 2006.
Functional division of intrinsic neurons in the mushroom bodies of male
Spodoptera littoralis revealed by antibodies against aspartate, taurine,
FMRFamide, Mas-allatotropin and DC0. Accepted for publication in Arthropod
Structure & Development.
III. Sinakevitch, I., Sjöholm, M., Strausfeld, N.J. & Hansson, B.S. 2006.
Glutamate-, GABA-, serotonin-, allatostatin- and TRP-like immunoreactivity in
the mushroom bodies of the moth Spodoptera littoralis. Submitted manuscript.
IV. Sjöholm, M. & Hansson, B.S. Synaptic organization in the mushroom bodies
of the moth Spodoptera littoralis, a TEM study. Submitted manuscript.
V. Sjöholm, M., Ignell, R. & Hansson, B.S. Odor-evoked sub- and suprathreshold
activation in Kenyon cells in the moth mushroom body. Manuscript.
Paper I is reprinted with permission from Wiley-Liss, Inc. a subsidiary of John
Wiley & Sons, Inc.
Objective
The objective of this thesis was to describe the structural and chemical anatomy
and explore the function of the mushroom bodies in the brain of the Egyptian
cotton leaf worm moth, Spodoptera littoralis. This was achieved by using standard
neuroanatomical staining techniques, immunocytochemistry targeted toward
putative neuroactive substances viewed with laser scanning confocal microscopy
and transmission electron microscopy. Lastly, whole-cell patch clamping was used
in order to investigate the role of the mushroom body intrinsic neurons in olfactory
processing.
Introduction
The insect brain is, for its size, an amazing piece of computational tissue. An adult
honey bee, Apis mellifera, has just under one million neurons and a fly, Musca
domestica some 340,000 neurons in their forebrain (Witthöft, 1967; Strausfeld
1976) which consists of the fused proto-, deuto- and tritocerebra. In addition,
insects possess several neural ganglia located along the ventral nerve chord, which
stretches throughout the length of the body. These ganglia handle sensory-motor
integration and coordination locally. The ganglia of the forebrain, on the other
hand, are responsible for integrating and perceiving information detected by the
principal peripheral sensory organs, i.e. the eyes, antennae, palpae and mouth parts
and information about the internal state of the insect. The brain allows the insect to
filter and translate a plethora of often meaningless events in its immediate
environment into meaningful information such as vision, odor and taste. This
information is used by the insect to deal with choice situations and to make
decisions that meet the current needs of the animal. Additionally, the brain is
responsible for storing information and comparing new situations with already
experienced ones. Together, all these functions are used to initiate and guide
adequate and essential behaviors, which help the insect to survive in an often
unpredictable environment. A strong candidate within the insect brain for
mediating several of these functions is a specific structure called the mushroom
body (de Belle and Heisenberg, 1994; Mizunami et al., 1998b, c; Strausfeld, et al.,
1998; Tang and Guo, 2001), a paired, lobed, high-order neuropil found in all insect
groups but one.
The mushroom bodies are in most insects intimately associated with the primary
olfactory centers of the brain, the antennal lobes. In fact, a substantial part of the
neural input to the mushroom bodies is olfactory. Olfaction is one of the most
important senses to insects (Hansson 1995). Insects often rely on odors to locate
and evaluate food and oviposition sites or to find a suitable mate (e.g. Visser,
1986; Baker 1989; Renwick 1989; Anderson et al. 1993). Particularly in nocturnal
insects like moths, odors provide the major contribution of the sensory world. A
good example of the impact the sense of smell has on insects is the great
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Fig. 1. Schematic overview of the insect brain, exemplified by the moth S. littoralis.
The right hemisphere shows the mushroom body and its different components,
Kenyon cell bodies (KC), calyx (cx), pedunculus (Ped), vertical lobe (V), medial lobe
(M), Y tract (Y). In the mushroom bodies, two types of Kenyon cells are
reconstructed. The left brain half gives examples of areas that are associated with the
mushroom body: antennal lobe (AL), projection neuron somata (PN), local
interneuron somata (LN), antennal lobe glomerulus (gl), inner antennocerebral tract
(iACT), outer antennocerebral tract (oACT), lateral protocerebrum (l Pr). Other
denoted structures: medulla (Me), lobula, (Lo), lobula plate (Lo pl), central complex
(cc). Scale bar = 1 mm.
Extrinsic innervation
It is often stated that the calyx is the main sensory input region but this seems to
hold true only when the insect is equipped with a functional olfactory system and
antennal lobes (Strausfeld et al., 1998). In anosmic species or in insects with
rudimentary antennae, like the Odonata and some aquatic insects, the calyces are
absent or greatly reduced although the pedunculus and lobes may be well
developed (Farris 2005b; Strausfeld et al., 1998). In these groups, as well as in
calyx-equipped insects, the MB lobes and pedunculus receive multimodal afferent
input originating from different parts of the brain (Ito et al., 1998; Strausfeld et al.,
1998; Strausfeld and Li, 1999a; Li and Strausfeld, 1997). Amongst odor-sensitive
insects, even within the same order or species, the elaboration of the calyx is
highly variable and seems to be partly determined by afferent innervation (Fig. 3).
Some of the most derived examples of MB calyces are found in social
hymenopterans like the honey bee. The calyx of the honey bee is concentrically
divided into three distinct zones: lip, collar and basal ring, each further parsed into
smaller areas defined both by the input and by KC make-up (Abel et al., 2001;
Ehmer and Gronenberg, 2002; Gronenberg, 2001; Mobbs, 1982; Strausfeld 2002)
(also see below). Each zone is supplied by modality-specific input such that the lip
receives olfactory input from the antennal lobe projection neurons, the collar is
supplied by visual interneurons from the optic neuropils and the basal ring receives
11
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as well as in the basal insect firebrat, Thermobia domestica (Farris, 2005a), only a
single primary calyx develops for each MB. Acheta has, however, also a large
accessory calyx, called the posterior calyx, constituted by putative class III KCs. In
yet other groups, like Dermaptera and Neuroptera, three MBNB clusters give rise
to as many calyces (for review see Farris, 2005b). In D. melanogaster, only one
single calyx is observed in the imago but studies using genetic markers have
revealed that this single calyx derives from four original MBNBs, each
hypothetically giving rise to an embryonic hemicalyx that are secondarily fused
(Ito et al., 1997; Kurusu et al., 2002; Tettamanti et al., 1997; Zhu et al., 2003).
Holometabolous and hemimetabolous insects display some clear differences in
MB structure. As mentioned above, KCs are deposited sequentially during
development, which produces a columnar or layered appearance of the lobes. In
Holometabola, with D. melanogaster as example, KCs are produced during the
larval stages in consecutive bouts (Lee et al., 1999). First the class II KCs are born
and then two types of class I, each innervating a separate division in the lobes.
This results in a tripartite structure, with more morphological and biochemical
subdivisions overlaid on this basic pattern (Strausfeld et al., 2003). In the honey
bee, the basic structure also consists of a few large sequentially produced
divisions, the lobe containing class II KCs and representing the whole calyx, and
three class I divisions, each representing one of the calyx zones (Mobbs, 1982;
Strausfeld 2002; Strausfeld et al., 2000). Each division is, however, further parsed
into multiple strata with biochemically and morphologically differentiated KCs
(Strausfeld, 2002) and the basic sequential pattern is further modulated by axon
rearrangement during development (Farris et al., 2004). In hemimetabolous
insects, new (class I) KCs are produced during each instar throughout the
preimaginal development, and sometimes into adulthood, resulting in a
continuously laminar appearance of the lobes. However, in the first instar, before a
large laminar array of class I KCs has developed, the structure resembles that in
holometabolous insects and tracing of KCs that occupy functional regions in the
calyx to the lobes reveals that the cockroach MB is also parsed into a few large
presumably functional divisions (Strausfeld et al., 1998; Strausfeld and Li, 1999b).
Size and shape
One obvious discrepancy between MBs in different species is the size. It ranges
from extremely well developed MBs containing hundreds of thousands of KCs like
in the honey bee and cockroach, to small ones consisting only of a few thousand
cells, like in the fruit fly. These differences in size do not seem to be directly
linked to phylogenetic relationships but rather to the behavioral ecology of the
insect (Farris, 2005b). The MB size may also differ between sexes and castes or
between individuals of different age and even experience within the same species,
as is observed in honey bees and ants (Ehmer and Gronenberg, 2004; Gronenberg,
1999; Gronenberg and Hölldobler, 1999; Farris et al., 2001; Seid et al., 2005;
Withers et al., 1993). However, age-related growth of the MB in adult
holometabolous insects does not generally involve neurogenesis but is the result of
growth and elaboration of neural processes. The deep convolution of the calyx
seen in many behaviorally complex insects has been interpreted as a result of the
14
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for seconds, KCs respond with a short fast burst of only a few spikes (Laurent and
Naraghi, 1994; Perez-Orive et al., 2002; Stopfer et al., 2003; Szyszka et al., 2005).
KCs also seem to sparsen the signal, meaning that while PNs may be widely tuned,
KCs are highly specific and respond only to one or a few stimuli (Perez-Orive et
al., 2002; Stopfer et al., 2003; Szyszka et al., 2005; Wang et al., 2004). Perhaps
contradictory to these observations is the finding that there is a massive divergence
as well as convergence of the signal from PNs to KCs. PNs are contacted by many
KCs and KCs receive input from numerous PNs (Perez-Orive et al., 2002; Szyszka
et al., 2005).
Clearly, some highly efficient mechanisms are at work to accomplish the
observed temporal and qualitative specificity in the response of KCs. One of these
mechanisms may be provided by GABAergic feed-forward inhibition to the calyx
via interneurons originating in the lateral protocerebrum (Perez-Orive et al., 2002).
Such neurons have been found in several species and since PNs also terminate in
the lateral protocerebrum these neurons are thought to mediate a delayed inhibitory
input to the KCs, partly explaining the short response. Another observation is that
KCs seem to have active membrane conductance properties in their dendrites
(Perez-Orive et al., 2004), possibly mediated by voltage-sensitive Ca2+-channels
(Grünewald, 2003; Schäfer et al., 1994). This could favor detection of coinciding
input events by transiently amplifying local excitatory postsynaptic potentials
(EPSP) into dendritic spikelets, which elicit a response in the neuron only if they
occur simultaneously, as opposed to passive graded potentials, which summate the
input over a longer time. This could contribute to the sparse activation, since even
if a KC receives input from many PNs, it would only respond if a given number of
them fire simultaneously. Interestingly, similar active membranes properties have
been suggested in parasol cells, which are the intrinsic neurons of hemi-ellipsoid
bodies in crustaceans (Mellon, 2003) (see above). But why would PNs fire
synchronously? From studies in locusts and honey bees, it has been suggested that
part of the function of the LNs in the AL, additional to spatiotemporal shaping of
the output, would be to actively drive oscillating synchronous activity in the PNs
(Stopfer et al., 1997; Laurent and Naraghi, 1994; Macleod and Laurent, 1996).
This is reflected in oscillations of the local field potential (LFP) in the AL, which
only occurs during olfactory stimulation. By pharmacologically blocking GABAAlike receptors in the AL, it was shown that synchrony and oscillations disappeared
and fine discrimination between similar odors was impaired (Stopfer et al., 1997).
In locusts, coherent LFP oscillations are also seen in the calyx and recordings from
PNs have shown that spiking activity is phase-locked to the LFP oscillations
(Laurent and Davidowitz, 1994). If these observations are universal, it would mean
that information-carrying PN spikes that arrive at the KCs are phase-locked and
synchronous and occur in oscillating bouts, separated by delayed, phase-shifted
cyclic inhibition from the GABAergic feed-forward neurons in the lateral
protocerebrum (Perez-Orive et al., 2002). However, most of the data supporting
these ideas has been obtained in locusts, which have an altogether different
anatomical structure of the AL compared to e.g. honey bees, flies and moths
(Anton and Homberg, 1999; Ignell et al., 2001). Moreover, similar patterns of
phase-locking between PN activity and LFP in the AL or the MB have not been
found in the brain of the moth Manduca sexta (Christensen et al., 2003).
20
Much remains to be understood in this system and considering that not even the
ultimate function and nature of the output from the antennal lobe is completely
known (e.g. see Ng et al., 2002 and Wilson et al., 2004), it is difficult to draw firm
conclusions about the MB’s role in the coding of odors. Possibly there are general
principles that apply to all insect groups but there may also be genus- or species
specific differences in these mechanisms, as suggested by the vast anatomical
differences in MB and AL structure. The present thesis will investigate some of
these mechanisms in S. littoralis and compare the findings with those obtained in
other species.
The model
Spodoptera littoralis (Boisd.)(Noctuidae: Lepidoptera), or the Egyptian cotton leaf
worm moth, is a nocturnal insect distributed over northern Africa, the Middle East
and throughout the Mediterranean countries. It is a pest on many crops and cause
great damage to cotton fields and alfalfa (Avidov and Harpaz, 1969). It is a true
generalist with respect to host plants and feeding larvae have been found on a wide
range of plant species from over 40 different families (Brown and Dewhurst 1975).
Both the male and the female are heavily dependent on olfaction in order to find
resources, which for the female are suitable, healthy plants to oviposit on and for
the male a receptive female to mate with. Both sexes nectar-feed when possible. S.
littoralis is a short-lived insect and the adult dies within two weeks after eclosion.
Male Spodoptera littoralis.
21
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predation (Miller and Surlykke, 2001; Roeder, 1962). However, in the presence of
female sex pheromone male moths are willing to take a much higher risk of being
eaten than without the pheromone. An intricate trade-off behavior dictated by the
relative salience of the pheromone signal and bat sound has been demonstrated
(Skals et al., 2005).
In females, odor-guided behavior has been studied in oviposition experiments,
showing that gravid females can distinguish between and prefer a healthy hostplant over one that is under herbivore attack (Anderson and Alborn, 1999;
Anderson et al., 1993; Jönsson and Anderson, 1999). Plant-odor compounds are
similarly detected by the antenna and in the brain of both sexes, suggesting that the
ability to locate host-plants is not exclusive to females. (Anderson et al., 1995;
Anton and Hansson, 1994, 1995). Finally, associative learning to odors is effective
in both females and males (Fan et al., 1997; Fan and Hansson 2001) with one
significant difference. Both sexes readily learn to associate plant odors and
individual pheromone components with a sugar reward but only females, although
less robustly, will learn the complete female pheromone blend, indicating partly
different processing strategies of olfactory signals between the two sexes (Hartlieb
et al., 1999).
Questions
•
How is the structure of the MB in S. littoralis related to that found in
other insects? What similarities and dissimilarities can be seen?
•
How does the MB structure and size relate to the behavioral ecology of
this moth? S. littoralis is nocturnal and primarily guided by odors. Is this
reflected in the afferent input to the MBs? The moth is short-lived and
leads, compared to many other insects, a rather simple life with a
hypothetical low demand on elaborate learning and memory or social
competence. Is this reflected in a small or simple MB or is the structural
complexity seen in many other species a general feature necessary for
normal basic functions?
•
How do moth KCs respond to odor stimulation of the antennae? Are they
highly selective like those KCs previously described in e.g. locusts and
honey bees? Do moth KCs sparsen the signal coming from AL PNs?
23
Summary of Results
Mushroom Body Structure in Spodoptera littoralis
Gross morphology of the moth mushroom body (Papers I-IV)
Reduced silver staining (Bodian, 1937) clearly showed the position and size of the
MBs relative to other identifiable structures in the protocerebrum. It also revealed
the different parts of the MB, namely: the calyx, pedunculus, Y tract, vertical and
medial lobes and the Y tract bulbs or lobelets (Fig. 1). The calyx is composed of
two partly fused cups, or calyces proper, one medial and one lateral. The double
calyx, about 200 :m across, is located in the dorsoposterior part of the
protocerebrum and multiple KC somata cover its dorsoposterior surface and also
invade the cavities formed in the center of each calyx (Fig. 3). The calyces consist
of fine, glomerular neuropil and from each calyx, a thick bundle of KC neurites,
the pedunculus necks, emerges and projects anteriorly and ventrally. Immediately
after the exit from their respective calyx, the necks merge to form the pedunculus,
which travels down and forward in the direction of the dorsal margin of the AL.
The pedunculus reaches a length of about 300 :m before it swells into a small
bulbous structure, the spur, and bifurcates into two perpendicularly oriented
neuropils, the vertical and medial lobe (Fig. 2). The vertical lobe curves backwards
and up through the surrounding brain tissue until it reaches the dorsofrontal
surface of the brain. The medial lobe reaches toward the brain midline, bending
slightly down and backwards until it touches its contralateral partner in the
opposite hemisphere. Additional to the pedunculus, a thinner second tract, the Y
tract, emerges from the calyx’ dorsolateral margin and passes diagonally in front
of the pedunculus on its way to the base of the medial lobe, where it terminates in
a pair of club-like bulbs, here called lobelets. The vertical and medial lobes are
clearly heterogeneous and silver-staining preparations allowed us to describe the
clear separation and organization of three longitudinal divisions of each lobe in
detail for the first time in a moth (Fig. 4). These divisions were named after
similarly oriented divisions in the MB of previously described species.
Accordingly, the most anterior division of both lobes is called (originally used in
Sphinx ligustri, Pearson, 1971). The most posterior division is in the vertical and
medial lobes called and respectively, and the intermediary division ´ and ´
respectively (these terms were adopted from Drosophila melanogaster,
Heisenberg, 1980; Crittenden, 1998; Strausfeld et al., 2003). The divisions of the
lobes lie adjacent to one another as they diverge from the pedunculus, but as the
distance from the pedunculus base increases the division separates from the two
anterior divisions and bends backwards. The two remaining divisions of the
vertical lobe stay close together and toward the surface of the brain, the division
envelopes the tip of the ´ (Paper II). In the medial lobe the divisions stay attached
throughout their lengths. Further, silver staining resolved the spatial relationship
between the medial lobe and the Y tract bulbs, revealing that the ventral lobelet
penetrates through the medial lobe neuropil and emerges on its ventral surface (the
term Y tract was adopted from Pearson’s (1971) study of the moth Sphinx ligustri).
24
Fig. 3. Organization of the calyx. The top row shows Bodian silver stained
preparations, horizontal section to the left and posterior view on the right. The calyx
neuropil has a fine glomerular structure. Note KC somata in the calyx cavities. The
middle row shows on the left a phalloidin stained calyx and on the right a threedimensional reconstruction made after a phalloidin stained brain. Note the separation
into an outer zone and an inner rim (bracket). Bottom row shows brains with dyefilled antennal lobe projection neurons, horizontal on the left and posterior on the
right. Note the lack of antennal innervation in the inner rim.
Fluorescent staining with phalloidin, which selectively binds to F-actin (Rössler
et al., 2002), confirmed the observations of the silver stained preparations but also
revealed that each calyx is further parsed into two concentric neuropils, a broader
peripheral ring containing a central thinner one, the latter referred to as the rim
(Fig. 3) (Paper I). The double concentric arrangement in the calyx and the tripartite
(omitting the Y tract) organization in the lobes present a clear mismatch. How do
the calycal subdivisions relate to the ones in the lobes? And how are the two
calyces, lateral and medial, represented in the lobes?
These questions were partly answered by applying different antisera that
differentially labeled specific parts of the MB (Papers II and III). Antibodies
25
Fig. 4. Organization of mushroom body lobes and their subdivisions. A-E: Selected
images from a series of reduced silver-stained frontal sections, from anterior to
posterior in the brain showing the different divisions of the MB lobes and their threedimensional organization. /-, ´/´-, -lobes, dorsal and ventral lobelets (d lobl, v
lobl) and Y-tract (Y) are indicated in each section. F: Posterior view of a 3Dreconstruction of a phalloidin-stained mushroom body to illustrate the relative
locations of mushroom body subdivisions. Pedunculus (Ped; green), Y-tract (Y;
blue). Scale bars = 100 m. Reprinted with permission from Wiley-Liss, Inc.
26
against the peptide Mas-allatotropin only labeled the two posterior divisions
selectively, thus facilitating tracing the KC bundles supplying these lobes through
the pedunculus to the calyx. This labeling revealed that the two bundles supplying
the / and ´/´ divisions merge about half way to the calyx and fibers from each
bundle mix diffusely. Closer to the calyx two new bundles, the pedunculus necks,
emerge and enter the calyx neuropil. This indicates that there is an equivalent
contribution from the lateral and medial calyces to each of the lobe divisions and
this was further substantiated in a Golgi-impregnated preparation. The
correspondence between the concentric arrangement in the pedunculus necks and
the lobes was further established by immunolabeling with an antiserum against the
amino acid taurine. This antiserum labeled the division and the posterior margin
of the / divisions, in other words the most anterior and the most posterior
portions of the lobes respectively. In the pedunculus necks labeling was observed
in the very periphery (corresponding to the division) and in the core of each neck
(corresponding to /). Additionally, the Y tract and lobelets were strongly labeled
by taurine antiserum. In the calyx these three labeling patterns overlapped and only
the stronger labeling belonging to Y tract neurons could be discerned from the
background (see summary in fig. 8).
In the central core of each neck and at the very edge of the /, a minute nontaurinergic region was observed. In previous studies, KCs in this position have
been shown to be immunopositive to antibodies against the amino acid glutamate
(Sinakevitch et al., 2001; Strausfeld et al., 2003). These KCs are probably the
youngest neurons since developing KCs generally grow out through the core of the
pedunculus (Farris and Sinakevitch, 2003). To see whether this pattern also applies
to moth we used a glutamate antibody, which indeed labeled central core fibers
leaving the calyx, confirming observations in other species (Paper III). An
antiserum against the neuropeptide FMRFamide revealed differential labeling
within the / and ´/´ divisions such that the anterior margins of both divisions
were labeled. The entire division was also immunoreactive to this antiserum
(Fig. 8). Lastly, two antibodies labeled the entire MB more or less indifferently.
These were anti-DC0 and anti-aspartate, both previously found to ubiquitously
label MBs in several insects (Farris, 2005b; Farris and Strausfeld, 2003;
Sinakevitch et al., 2001; Skoulakis et al., 1993; Strausfeld et al., 2003).
The gross morphology of the MB in S. littoralis superficially resembles
examples from other Lepidoptera (Pearson, 1971; Strausfeld et al., 1998) but the
present description is more detailed than previous accounts. Outside the
Lepidoptera, the most reminiscent MB organization is probably found in flies
(Strausfeld, 1976; Strausfeld et al., 2003). The arrangement in the lobes with
clearly separated parallel neuropils is very similar, although large differences exist.
For example, the conspicuous Y tract and resulting lobelets are absent in flies, as
in most other insects for that matter. In addition, flies have a simplified calyx that
lacks the double cup arrangement seen in the moth. Another closely resembling
group might be the Neuroptera, which also possess a Y tract but this group has not
been thoroughly studied (Farris, 2005b). Our results confirm that the relationship
between centrally located and peripheral KCs in the calyx is maintained in the
lobes and represented by posterior and anterior KCs respectively. As in other
27
insects, it is likely that this arrangement in the moth reflects a sequential
generation of KCs during development.
28
Intrinsic neuron types in the moth mushroom body (Papers I-IV)
The total number of KCs in each hemisphere was estimated to 4,060, with 3,400
sending their neurites through the pedunculus and 660 through the Y tract (paper
IV). This is low compared to a similar-sized insect, ~170,000 in honey bee
(Witthöft, 1967) but similar to that in D. melanogaster, ~2,500 (Technau and
Heisenberg, 1982). The KCs were categorized into four groups according to their
dendritic morphology and branching pattern in the calyx and lobe divisions. These
qualities were examined by using Golgi impregnation and dye-injection in single
KCs. Within these groups, subsets of neurons occupying specific zones within the
divisions were identified according to the differential affinity of several antibodies
to these zones as described above (Paper II-III).
Alpha/beta Kenyon cells
KCs supplying the / have their cell bodies located centrally over each calyx and
the primary neurites line the inner surface of the rim neuropil of the calyx before
they enter through the bottom of the calyx cups and join the pedunculus necks.
From the primary neurite, several dendritic branches reach into the calycal
neuropil, penetrating through both the inner rim and into the outer wall (Fig. 5).
These branches are decorated with numerous stubby spines and radiate in one
main direction from the calyx center. In the pedunculus, the / KCs travel
through the core bundle and may give rise to a single short collateral about half
way toward the lobes. At the base of the pedunculus, the neurites send out a few
short collaterals in the spur region, and then bifurcate giving rise to one collateral
invading the vertical division and one invading the medial . The lobe tributaries
are equipped with terminal varicose branches along their lengths (Paper I). These
KCs are, by their morphology and location, defined as class I KCs (Farris and
Sinakevitch 2003). As described previously, these neurons are ubiquitously
immunoreactive to anti-aspartate, anti-DC0 and anti-Mas-allatotropin. Subsets
within this group show immunoreactivity toward anti-taurine, anti-glutamate and
anti-FMRFamide (Paper II-III).
Alpha´/beta´ Kenyon cells
KCs occupying the ´/´ division of the lobes share many characteristics with the
/ KCs and also fall under the class I definition. Cell bodies of these neurons are
found over the calyces and their primary neurites enter through the calyx cavity.
The shape of the dendritic tree differs somewhat from the / and forms a large
umbrella-shaped canopy of spiny branches radiating from the primary neurite in all
directions (Fig. 5). The dendritic ramifications of a single ´/´ KC cover a
Fig. 5. Kenyon cells in S. littoralis. Roman numerals I-III denotes their assumed
class. Target divisions in the lobes are shaded grey for clarity. Top left a class II KC
branching in the divisions of the lobes. Note the narrow dendritic tree with clawed
specializations. Top right a class I KC branching in the / divisions. Bottom left a
class I KC in the ´/´ divisions. Note the differential dendritic branching patterns in
the two class I KCs. Bottom right a camera lucida reconstruction of KCs projecting
through the Y tract and branching in the lobelets. Their dendritic arbors could not be
resolved in this preparation.
29
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bee (Locatelli et al., 2005). The function of allatostatins in the insect CNS remains
unclear.
As previously discussed, additional modulatory substances, e.g. octopamine and
dopamine have been found to exert important effects on MBs in other species.
These substances are likely to be found also in S. littoralis, but this has not yet
been specifically investigated.
Synaptic organization in the moth mushroom body (Paper IV)
Transmission electron microscopy of different parts of the MB reveals several
interesting features of the intrinsic organization of the moth MB. The glomerular
structure in the calyx neuropil represents microglomeruli composed of a central
large presynaptic swelling, here called bouton (Schürmann 1974), surrounded by
numerous smaller profiles of various sizes (Fig. 6). The large central bouton is
likely to belong to afferent sensory interneurons and the size and shape of these
boutons resembles that of the AL PN varicosities (see above). This has previously
been confirmed with immuno-EM against acetylcholine related molecules in
Drosophila (Yasuyama et al., 2002). The smaller of the surrounding postsynaptic
profiles are putative KC dendrites and these are devoid of synaptic vesicles. The
degree of divergence from a single bouton onto KC profiles is very high and we
estimate that each bouton contacts up to 40 dendrites belonging to individual KCs.
Other medium-sized profiles, presynaptic to both KCs and boutons, as well as
postsynaptic to boutons, are inferred to comprise inhibitory and/or modulatory
neurons that innervate the calyx (Ganeshina and Menzel, 2001; Leitch and
Laurent, 1996; Yasuyama et al., 2002). The high amounts of dense core vesicles in
some of them further suggests this, since dense core vesicles are thought to reveal
the presence of peptides or biogenic amines (Ganeshina and Menzel, 2001).
In the pedunculus, KCs are organized in tightly packed fascia, separated by thin
layers of glial substance. Within these fascia, KCs often engage in en-passant
synaptic contacts with other KCs or with large extrinsic profiles. KC-KC synapses
are often complex and involve several KC profiles connected through serial,
converging and diverging synapses. KCs exhibit varying contents of dense core
vesicles, possibly representing the differential distribution of peptides and other
substances in groups of KCs as revealed with immunolabeling (Paper II-III). This
is also seen in the lobes, where the content of both dense core- and clear vesicles is
high in putative KCs. However, the identification of specific neuron types is
significantly more difficult in the lobes than in the calyx and pedunculus. In the
lobes, complicated chains of serial and converging/diverging synapses are
observed and they probably involve several different neuron types that could not
be separated on morphological grounds.
The Y tract contains axon-like profiles similar to the KCs in the pedunculus but
they are of slightly larger diameter and individually separated by glia in the upper
part of the tract. In the lobelets, the same kind of intricate synaptic arrangements
are observed as in the lobes. One clear difference between the Y tract KCs and
KCs invading the medial lobe is that the former contain more and larger
37
mitochondria. This could easily be seen as a darker shade in the lobelet compared
to the medial lobe in a low-power micrograph. The reason for this difference
remains elusive but previous studies have suggested a relationship between a high
continuous neuronal activity and a high content of taurine, which may have an
excitotoxicity-protective function (Discussed in Sinakevitch et al., 2001). A high
content of large mitochondria could suggest an elevated neuronal activity in these
KCs and as we have shown already, these neurons are intensely labeled with
taurine antiserum.
The over-all impression of the synaptic organization in the S. littoralis MB is
that it is very similar to previously studied insects. The microglomerular
arrangement in the calyx resembles that in flies, bees and locusts, although
immuno-EM is needed to ascertain the profile identity (Ganeshina and Menzel
2001; Leitch and Laurent 1996; Yasuyama et al. 2002). The tight packing of KCs
into fascicles and abundance of en-passant synapses in the pedunculus confirms
observations in locust, honeybee, cricket and cockroach (Leitch and Laurent 1996;
Schürmann 1970; Shürmann 1974).
Our estimation that each PN bouton contacts roughly 40 dendrites each
belonging to a unique KC suggests a high degree of divergence, but how high?
Does a given PN synapse onto only 40 KCs? PNs normally branch widely in both
calyces, implying that there must be a divergence higher than 1:40. How high is
the convergence onto KC? Do these 40 hypothetical KCs only sample this one
PN? Each class I KC has about 550 dendritic spines. In M. sexta, the PNs have
about 100 boutons each (Homberg et al., 1988), meaning that even if a given KC
contacts every bouton on a single PN there is still 300-400 “empty” dendrites. The
widely branching dendritic tree of ´/´ KC suggests that it may sample PNs over
a large area in the calyx, further suggesting a higher convergence. There are in M.
sexta about 500-600 PNs reaching the calyx (Homberg et al., 1988), thus providing
some 60,000 boutons, each with 40 synaptic appositions, resulting in some
2,400,000 synaptic contacts. In S. littoralis, ~4,000 KCs provide 550 dendrites
each, which gives a total number of some 2,200,000 synapses. The distribution of
these synapses, with respect to how the signal diverges and converges, is difficult
to speculate about but it could be very high in both aspects.
The TEM data also showed that KCs may be both pre- and postsynaptic
throughout the pedunculus and in the lobes. This confirms the view that the MB is
by no means monopolar in the respect that it receives information at the calyx and
transmits it through the lobes, but must instead be regarded as a highly integrative
neuropil not only in the calyx, but throughout the length of the KCs.
Olfactory Response and Electrophysiology of Kenyon Cells
(Paper V)
To answer questions about how moth KCs respond to odor stimulation of the
antennae, we employed whole-cell patch clamp on KC somata in an in situ head
preparation of the moth (see Box 2). This allowed us to test the
38
electrophysiological membrane properties of the KCs, record odor-evoked activity
and to visualize the recorded neurons by including a tracer in the recording
electrode. An advantage of whole cell patch-clamp recording via the soma
compared to e.g. intracellular recording with sharp electrodes in the axon is the
possibility to monitor both sub- and supra-threshold activity and not only action
potentials.
Membrane properties of Kenyon cells
In order to evaluate the membrane properties we recorded from KCs both in
voltage-clamp and current-clamp mode. In voltage-clamp mode the net current
through the cell membrane was measured while stepwise depolarizing the
membrane. In current-clamp mode, increasing steps of depolarizing current were
injected into the cell and the resulting depolarization of the membrane potential
39
was monitored. Depolarizing the membrane elicited voltage-activated inward- and
outward currents of different types. Injection of depolarizing current evoked active
membrane potentials, both action potentials and smaller spikelets, which were
hypothesized to represent local voltage-activated currents (Perez-Orive et al.,
2002, 2004), possibly mediated through voltage-activated Ca2+ channels, which
have been identified in honey bee KCs (Grünewald 1999).
Odor-evoked activity of Kenyon cells
For testing the olfactory responses of KCs we used a wide range of odors, applied
both singly and in blends to the ispilateral antenna while recording the activity in
single KCs in current-clamp mode. The odors tested comprised different plant- and
flower odors as well as pheromone, known to elicit responses in the antenna and
AL. Odor stimulation elicited a short, phasic excitatory postsynaptic potential
(EPSP) with spikelets riding on top of it in 5 out of 28 recorded KCs (Fig. 10).
This was interpreted as sub-threshold activation caused by insufficient or
“incorrect” presynaptic signaling by PNs and the qualitative specificity of this
activation was very low. One single KC responded with action potentials to
stimulation with behaviorally relevant complex odor mixtures (Fig. 11). The
temporal characteristics of both sub-threshold activation and transmitted response
was very brief and with a fast onset. The activation typically lasted ~200 ms.
Odor-evoked responses in PNs have previously been shown to last ~500 ms under
similar experimental conditions, although large variation is seen in the temporal
characteristics of PNs in S. littoralis (Anton and Hansson, 1994, 1995; Sadek et
al., 2002). The temporal sharpening of the KC response could suggest feedforward inhibition to take place shortly after the response, as proposed by studies
in locust and honey bee (Perez-Orive et al., 2002; Szyska et al., 2005). In some of
the KCs that exhibited sub-threshold activity, an additional short depolarization
was observed about 800 ms after the response, suggesting that release of feedforward inhibition allowed lingering PN activity to once again depolarize the
membrane.
If it is a general property of KC odor processing that excitatory input is
intercepted by periodic inhibition, this could be a way for the system to reset itself
and prepare for new information. Odors have, under natural conditions, a complex
structure and appear as small packages or filaments of odor-laden air intercepted
by clean air (Baker et al., 1988; Vickers et al., 2001). Being able to accurately
resolve the temporal structure of odor stimuli could be important in order to detect
Fig. 10. Odor-evoked subthreshold activity. KC # 32 displayed EPSP and spikelets
by stimulation with all odors. This cell was tested with all blends, the single odorants
in blends 1-3 and the main pheromone component before the contact was lost. The
blend of all plant odors evoked the largest EPSP (blends 1-5, grey bar). This KC also
exhibited a slight depolarization around 800 ms after stimulus onset in most of the
traces (vertical grey box). One spontaneous spikelet is seen preceding the stimulus in
the magnification of the pheromone trace (purple bar). This neuron was filled and
stained and branched in the ´/´ divisions. Current injection and stepwise
depolarization caused spikes and spikelets and fast inward currents, respectively.
40
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Conclusions
The results of the investigations of the moth mushroom body presented here
answer some of the questions posed earlier in this thesis. However, they also, as is
often the case, raise several new questions.
Functional Implications of Mushroom Body Structure
The small size of the MB in S. littoralis (~4000 neurons) can possibly be
correlated to the short and behaviorally simple life of this moth. Crucial sensory
cues are probably stable and reliable and the need for elaborate learning and for
making new associations between stimuli seems limited. Nevertheless, the
organization of the MB of S. littoralis is complex and seems to comprise all the
basic components found in MBs across insects and the blueprint at a cellular and
system level is conserved. Each calyx is anatomically and functionally divided into
two concentrically arranged regions. The marginal region receives input from
antennal lobe projection neurons and the central region receives yet unidentified
innervation. The double concentric organization in the calyx is translated into a
tripartite arrangement of parallel divisions in the vertical and medial lobes. Each of
these parallel divisions contain a morphologically discernable KC species and
within these KC categories, further differentiation into biochemically diverse
subtypes is seen. A fourth type of KC provides a separate system partly detached
from the rest of the MB.
Questions about how the structural organization might relate to function arise:
Do separate divisions of the spodopteran MB support different functions? Our
results show that input in to the calyx is separated but how this relates to the lobe
divisions is not clear. Extensive overlap between dendrites from different KC
groups is evident, but the specific combination of input to each division could be
unique. Each of the lobe divisions contains biochemically different KCs and the
differences in dendritic branching pattern between KC classes and subtypes
suggest that they integrate their input differently, not only with respect to modality
but also to activation threshold and temporal characteristics of firing (for reviews
see Gulledge et al., 2005; Kath, 2005). There is extensive crosstalk in form of
synapses between KCs in the pedunculus and lobes. Glial sheets separate bundles
of KCs but how the arrangement of these insulated fascia relates to divisions or
biochemical differentiation among KCs is unknown. Moreover, KCs are
postsynaptic to extrinsic neurons that probably modulate the activity in the KCs
further. It is likely that the divisions comprise parallel functional units that each
processes a unique combination of inputs through a number of computational
regimes by different KC subtypes, and lateral interactions within the MB and
extrinsic modulation influences the MB activity further.
44
Olfactory Responses in Moth Kenyon Cells
Moth KCs respond sparsely to relevant odor stimulation with a temporally narrow
burst of spikes. This is in line with findings in other insects and may represent a
general property of KCs across insect taxa. However, subthreshold activation of
KCs is odor-nonspecific, which provides a functional evidence of high
convergence of olfactory signaling onto KCs. Our experiments suggest that KCs
may possess active dendritic membrane properties, which could make them prone
to detecting synchronous input from AL PNs. This might be a way to sparsen the
converging signal but could also be involved in response plasticity of KCs.
One important issue not specifically addressed in this thesis is the output from
the KCs. Previous studies have shown that the dendritic arborization of efferent
neurons in the lobes may vary from limited, covering only one or few divisions to
very large, sampling the majority of or all KCs (Ito et al., 1998; Li and Strausfeld
1997, 1999; Rybak and Menzel, 1998; Schildberger, 1984; Strausfeld, 2002). How
do these efferent neurons “perceive” KC signaling? Indeed, what kind of
information do KCs transmit? The olfactory signal changes in the calyx from a
phasic-tonic, frequency-modulated signal in PNs to a brief all-or-nothing signal in
KCs. How is KC activity integrated by the downstream neurons? Is the activity
across assemblies of KCs temporally structured? Can ephaptic interactions take
place in the pedunculus and what information do KC-KC synapses transmit? MB
efferents, which sample a large part of the KCs, like the Pe1 neuron in honey bee,
can alter their response during olfactory conditioning (Mauelshagen, 1993). Is this
accomplished by summating the response from additional responding KCs or is the
change taking place downstream of KCs (Menzel and Manz, 2005)? How does
input and output relate to the multitude of KC subtypes, modal, morphological and
biochemical, and to the high degree of lateral interactions between KCs that
obviously is taking place?
Future Directions
Further investigation is needed to explain how information is transmitted through
the MB. For example, the relationship between input and activation must be
further elucidated. What criteria must be met in order for a KC to respond? The
lateral integration between KCs must also be explored. What kind of information
is transmitted through KC-KC synapses? How is the KC activity read by efferent
neurons? Some of these questions can possibly be answered by simultaneously
recording activity from multiple neurons at different synaptic levels of the odorsignaling pathway.
The insect MB provides a truly fascinating and most challenging neural structure,
the function of which we are only beginning to unveil. A deeper knowledge about
how MBs work might aid the understanding of higher brain functions in other
animals, including mammals and humans. The low cell number and relative
45
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Structure and Function of the Moth Mushroom Body

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