Adaptation of Microglomerular Complexes in the
Honeybee Mushroom Body Lip to Manipulations of
Behavioral Maturation and Sensory Experience
Sabine Krofczik, Uldus Khojasteh, Natalie Hempel de Ibarra, Randolf Menzel
Institut für Biologie - Neurobiologie, Freie Universität Berlin, Königin-Luise Str. 28-30,
14195 Berlin, Germany
Received 8 October 2007; accepted 2 January 2008
ABSTRACT: Worker honeybees proceed through
a sequence of tasks, passing from hive and guard duties
to foraging activities. The underlying neuronal changes
accompanying and possibly mediating these behavioral
transitions are not well understood. We studied changes
in the microglomerular organization of the mushroom
bodies, a brain region involved in sensory integration,
learning, and memory, during adult maturation. We
visualized the MB lips’ microglomerular organization
by applying double labeling of presynaptic projection
neuron boutons and postsynaptic Kenyon cell spines,
which form microglomerular complexes. Their number
and density, as well as the bouton volume, were measured using 3D-based techniques. Our results show that
the number of microglomerular complexes and the bou-
INTRODUCTION
Age- and experience-dependent neuronal changes are
known to accompany naturally occurring behavioral
plasticity in vertebrates and invertebrates (Kolb and
Whishaw, 1998; Farris et al., 2001). Structural plasticity during experience-dependent maturation of the
nervous system is common to many sensory systems
Correspondence to: R. Menzel ([email protected]).
Contract grant sponsor: Deutsche Forschungsgemeinschaft; contract grant number: Me 365/27-1.
' 2008 Wiley Periodicals, Inc.
Published online 29 April 2008 in Wiley InterScience (www.
interscience.wiley.com).
DOI 10.1002/dneu.20640
ton volumes increased during maturation, independent
of environmental conditions. In contrast, manipulations
of behavior and sensory experience caused a decrease in
the number of microglomerular complexes, but an
increase in bouton volume. This may indicate an
outgrowth of synaptic connections within the MB lips
during honeybee maturation. Moreover, manipulations
of behavioral and sensory experience led to adaptive
changes, which indicate that the microglomerular organization of the MB lips is not static and determined by
maturation, but rather that their organization is plastic,
enabling the brain to retain its synaptic efficacy. ' 2008
Wiley Periodicals, Inc. Develop Neurobiol 68: 1007–1017, 2008
Keywords: Apis mellifera; plasticity; microglomerular
complexes; projection neurons; kenyon cells
(Wilson et al., 2006). Regarding olfactory systems, in
rodents it has been shown that olfactory learning
influences olfactory sensory neuron architecture
(Kerr and Belluscio, 2006). Striking results were
obtained for eusocial insects, which provide the opportunity for a socio-neuroethological analysis of
neuronal and behavioral maturation. The honeybee
Apis mellifera has a discrete system of age-related
division of labor (Roesch, 1925). Worker honeybees
perform brood rearing (nursing) for the first week of
their adult life, engage in other hive maintenance
duties when they are 2- to 3-weeks-old, and subsequently carry out foraging tasks (Winston, 1987).
This schedule is rather flexible, to meet the colony’s
varying needs, which are influenced by demographic
and environmental conditions (Seeley, 2002). Such
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Krofczik et al.
flexibility makes it possible to experimentally manipulate age- and experience-dependent changes in the
bee brain, particularly in the mushroom bodies (MBs)
(Withers et al., 1993; Durst et al., 1994; Fahrbach
et al., 1998; Ismail et al., 2005).
The MBs are multisensory integration centers that
play a dominant role in odor learning (Heisenberg,
1998; Menzel and Giurfa, 2001). Their calyces
receive second-order sensory input with different
modalities and thus are believed to integrate sensory
information, possibly leading to context-dependent
forms of learning (Menzel and Giurfa, 2001). Olfactory input is conveyed to the MB lip region by excitatory projection neurons connecting the primary olfactory neurophil, the antennal lobe (AL) with the MB
and transferring sensory information to the dendrites
of the mushroom body-intrinsic neurons, the Kenyon
cells (Mobbs, 1982; Sun et al., 1997; Abel et al.,
2001). In the lip region of the MB calyces projection
neuron boutons and Kenyon cell spines form microglomerular complexes. Each microglomerular complex contains a presynaptic projection neuron bouton
as its central core, surrounded by a shell of Kenyon
cell dendritic tips (Gronenberg, 2001; Roessler et al.,
2002; Yusuyama et al., 2002; Frambach et al., 2004;
Groh et al., 2004, 2006). The MBs integrate information from different neural circuits and previous studies revealed that they undergo processes reflecting
age- and experience-related plasticity (Withers et al.,
1993; Durst et al., 1994; Fahrbach et al., 1998; Ismail
et al., 2005). Investigations of the MB neuronal architecture showed that foraging experience correlates
with the complexity of the Kenyon cell dendritic
branching pattern (Farris et al. 2001). However, the
question whether the microglomerular organization
of the MB lips is influenced by the bee’s age or its
behavior remains elusive. This study addresses this
question by applying a combination of behavioral
manipulations, pre- and postsynaptic labeling and
3D-based quantification and volume measurement
techniques to investigate the MB lips’ microglomerular organization during adult maturation.
Our results show that the microglomerular organization of the MB lips changes according to the behavioral
and sensory experience of the bee, which might imply
alterations of the synaptic connectivity between presynaptic PN boutons and postsynaptic KC spines.
MATERIALS AND METHODS
Honeybees
Honeybees (Apis mellifera carnica) were obtained from
colonies kept in our lab according to standard apicultural
Developmental Neurobiology
techniques. Experiments were performed during the
summers of 2004 and 2005. Developing adult honeybees
were collected by removing brood combs containing latestage pupae from a selected colony (headed by a naturallymated queen) and transferring them—while protecting
them from light—into an incubator (348C, 60–80% relative
humidity). The comb was inspected daily for eclosion of
bees, which were marked with different-colored numbered
tags (Opalithplättchen, Imkershop, Wolkenstein, Germany).
Immediately after eclosion the bees were either reintroduced into their original colonies or into a host colony kept
within a net cage inside a glasshouse or used for immunocytochemistry (hereafter referred to as 1-day-old bees, 1d).
Daily observations were made at the hive entrance for several sessions of 1–2 h each to identify the onset of orientation flights and later foraging behavior. Bees returning to
the hive were designated foragers according to standard
criteria (Lindauer, 1952; Winston, 1987). Pollen foragers
showed conspicuous pollen loads or were dusted with
pollen, whereas nectar foragers were identified by their
swollen abdomens and checked for a nectar load by gently
pressing the abdomen (nail probe). Observations of nursing
behavior were made either by opening the hive or via an
observation hive. Nurse bees were identified when they
inserted their heads into brood cells (Robinson, 1987).
Experimental Part I: Natural
Colonies Under Different
Environmental Conditions
The colonies used for this experiment were housed either in
a hive outside—referred to as the \rich" environment—or
in a hive contained within a flight net situated in a glasshouse (the \poor" environment). In the rich environment
the colony contained a naturally mated queen, adult nonmarked bees and about 700 marked bees collected immediately after eclosion. Foragers were marked with an additional paint mark to ensure a minimum of 2 days of foraging experience. The poor environment colony contained a
single-inseminated active queen in a small hive with about
1000 adult bees and 300 marked bees collected immediately after eclosion. Bees foraged at artificial gray odorless
sugar feeders or collected pollen from a dark chamber. Behavioral observations were carried out as described above
and bees of dedicated ages (4, 14, 18, and 37 days for the
rich environment bees and 14, 18, and 37 days for the poor
environment) were collected for immunocytochemistry (see
Fig. 1).
Experimental Part II: Manipulations of
the Bees’ Social Behaviors and
Sensory Experience
The single-cohort colony of about 3000 bees used for this
experiment was housed in an observation hive containing a
single-inseminated queen. Observations of flight and foraging behavior were made as described above. Additional
observations of nursing and overall activity were made via
Plasticity of Microglomerular Complexes
1009
Figure 1 Experimental design to investigate changes of microglomerular complexes during adult
maturation. Bees were either reared in a natural colony (top row) under rich or poor environmental
conditions or in a single-cohort colony (bottom row). Bees from the natural colony were collected
during the first (4-day-old nurses) and after the second week (14-, 18-, and 37-day-old foragers).
Bees from the single-cohort colony were collected during the first (5-day-old hive-bound bees,
nurses, and precocious foragers) and after the second week (14-day-old hive-bound bees, nurses,
and foragers as well as 18-day-old hive-bound bees and foragers. All samples were used for immunocytochemistry to visualize microglomerular complexes (confocal images) and to apply 3D quantification and volume measurement. All measurements were undertaken on optical slices whose
plane was defined by the central body (cb) [Scale bars: 100 lm; inset, 50 lm].
the glass window of the observation hive. Extended nursing
activities performed by the marked bees were observed
until Day 14 presumably because new eclosed bees took on
further nursing activities. About 1000 bees were marked
with numbered bee tags of different colors. Following the
procedure described by Withers et al. (1995), plastic discs
were glued to the thoraces of an additional 200 bees (hereafter referred to as hive-bound bees). The thickness of the
plastic disk prevented hive-bound bees from entering the
comb to perform nursing behavior. The hive entrance was
restricted such that hive-bound bees could not leave. Foragers were additionally marked with paint. The experiment
was undertaken until Day 18, since after this period the survival rate of hive-bound bees decreased progressively. This
observation might be explained by the fact that hive-bound
bees were detained to perform defecation/orientation flights
which might have affected their metabolic activity and
positive phototaxic behavior essential for the bees’ natural
life span.
At each adult developmental age (5, 10, 14, 18 days)
hive-bound, nurse, and forager bees were collected and dissected for immunocytochemistry (see Fig. 1).
Intracellular Markings of
Projection Neurons
Electrodes (Hilgenberg) were pulled with a Brown-Flaming
horizontal puller (P-2000 Sutter Instruments, Novarto, CA),
and their tips were filled with 4% tetramethylrhodamin-biotin dextran (Micro-Ruby; Molecular Probes, Eugene, OR)
in 0.2 M potassium acetate. The electrodes were inserted
Figure 2 Identification, and 3D quantification of microglomerular complexes within the MB lip. (A) Segmentation
of the lip neuropil based on anti-synapsin IR. Anti-synpasin
IR gray-value image and segmented lip neuropil (hatched
purple) superimposed. (B) Segmentation (hatched green)
of anti-synapsin IR positive regions. (C) Identification of
microglomeruli complexes by alignment with a phalloidin
gray-value image. (D) High-resolution image of a single
PN bouton and anti-synapsin IR superimposed (yellow). (E)
Surface model of the segmented anti-synapsin IR positive
region, the PN bouton. (F) Illustration of the six-voxel
neighborhood algorithm applied by the connected components module of the software Amira. A single voxel i is
compared with its neighboring voxel (iU, iW, iN, iS, iE,
iD) based on thresholding. Voxels of the same gray value
are assigned to a cluster representing a single microglomerulus. The 3D area of each cluster represents the anti-synapsin IR interpreted as bouton volume and the number of clusters the number of microglomerular complexes. Scale bars:
A: 20 lm, B and C: 5 lm, D and E: 0.1 lm.
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into the AL for recordings from projection neurons at about
50–180 lm depths. Electrode resistances in the tissue
ranged from 140 to 200 MO. A silver wire placed into the
eye served as the indifferent electrode. To ensure that the
recording was performed from a projection neuron MicroRuby was injected by using depolarizing pulses of 1–2 Hz,
0.2-s duration and 2–4 nA. Complete filling of the PN
required dye injection for 30–45 min. After intracellular
filling, the dye was allowed to diffuse for 3 h.
Immunocytochemistry
Collected bees were immobilized by cooling. Brains were
dissected in bee physiological saline solution (in mmol1:
136 NaCl, 3 KCl, 10 Na2HPO4, 2 KH2PO4, 105 sucrose,
pH 6.7) and fixed in 4% paraformaldehyde (PFA) in 0.1 M
phosphate-buffered saline (PBS) (overnight at 48C).
Brains were washed three times in PBS, embedded in 7%
low-melting agarose (Sigma) and sectioned in a frontal
plane (100 lm) with a vibrating microtome (Leica, VT
1000 S). Agarose sections were transferred into marked
titrimetric plates and preincubated in PBS with 0.2%
Triton-X100 (PBST) and 2% normal goat serum (NGS;
Sigma, St. Louis, MO) for 1 h at room temperature. For
visualization of MCs within the calycal lip we used a double labeling technique adapted from Groh et al., 2004.
This technique allows simultaneous pre- and postsynaptic
labeling in the lip region of the MB calyx by combining
anti-synapsin with f-actin labeling. Preparations were
incubated simultaneously with a monoclonal antibody
against the Drosophila synaptic-vesicle-associated protein
synapsin I, (Klagges et al., 1996) (1:50; SynOrf1; kindly
provided by E. Buchner, University of Wuerzburg, Wuerzburg, Germany) and with 0.2 units of Alexa Fluor 488
phalloidin (Molecular Probes, A-12379) in PBS for 2 days
at 48C. To visualize synapsin, preparations were rinsed
five times in PBS and incubated with a Cy5-conjugated
goat anti-mouse secondary antibody (Jackson Immunoresearch, West Grove, PA; dilution 1:250 in PBS) overnight
at 48C. For triple labeling of the intracellular-stained PN,
neuronal f-actin, and synapsin, the preparations containing
marked PNs were fixed and processed as described above.
To intensify the intracellular staining of the PN, 1:500
diluted streptavidin-Cy3 (Invitrogen, Kalsruhe, Germany)
was added to the primary antiserum.
Confocal Microscopy
Vibratome sections were imaged with a confocal laser scanning microscope (Leica TCS SP2) using a Leica HCX PL
APO CS 40 3 1.25 oil UV lens objective and a voxel size
of 0.1 3 0.1 3 0.5 lm3. For clear visualization of microglomerular complexes a defined zoom factor between 2 and
3 was used for each preparation. The preparation containing
the intracellularly recorded and marked projection neuron
was imaged with a HC PL APO 20 3 0.70 IMM/CORR
UV lens objective with a voxel size of 0.5 3 0.5 3 1 lm3
and a zoom factor of 1.4. Synapsin was visualized by a secDevelopmental Neurobiology
ondary antibody conjugated to Cy5, which was excited with
the 633-nm line of a HeNe laser. F-actin stained with phalloidin was visualized by a conjugated Alexa-488, which
was excited using the 488-nm line of an ArKr laser. The
projection neuron staining was intensified by using streptavidin-Cy3, which was excited with the 543-nm line of a
He-Ne laser.
3D Quantification and Volume
Measurement
Quantitative neuroanatomical techniques were applied to
optical sections at a defined plane in the region of the central body (Fig. 1 right panel) and further analyzed with the
Amira 3.1.1 software (Mercury Computer Systems). The
3D quantification and volume measurements were accomplished on images normalized to a voxel size of 0.15 3
0.15 3 1 lm3 and a resolution of 796 3 512 3 11. The
confocal image stacks were cropped according to the area
of the MB lip region, which was identified using anti-synpasin immunoreactivity (IR) (see Fig. 1 inset). Fully automated threshold-based identification and quantification
failed due to the complexity of the structures, and therefore
semiautomatic techniques were applied. Because of the
high number of preparations and the time-consuming semiautomatic process the analysis was performed on a total
thickness of 11 lm. In each preparation lip and bouton volume as well as number and density of microglomerular
complexes were measured.
Figure 2 illustrates the stepwise process of the 3D-based
volume measurement and quantification technique. To visualize the image stacks in 3D we converted the 8-bit gray
scale stacks into another format, the so-called Labelfield
file, which is closely related to a uniform scalar field. This
conversion was done by applying a technique referred to as
‘segmentation’. Thereby each voxel was assigned to a material representing either the MB lip or a microglomerular
complex [Fig. 2(B,C)]. Each microglomerular complex was
identified by segmenting the anti-synapsin IR positive
regions in the x, y, and z direction and aligning them with
the phalloidin staining [Fig. 2(B,C)]. Segmentation was performed blindly by two independent investigators without
knowledge about the animals’ group affiliation. The Labelfield containing the structures of interest was then converted
into a 3D scalar field and visualized as a surface model
[Fig. 2(E)]. The 3D volumetric measurements and quantification were accomplished by using the ‘connected components’ module of the Amira software. Based on gray-value
thresholding this module searches for connected voxel in a
3D image volume meaning that voxel of the same gray
value are interpreted as connected and thus belonging to the
same region (here to the class of microglomeruli). The segmented anti-synapsin IR positive regions represent the
microglomeruli throughout the entire defined MB lip region
[Fig. 2(D,E) show a single PN bouton (red) counterstained
with anti-synapsin (green), superimposed yellow]. The connected components module operates on a 6-voxel neighborhood screening algorithm, which compares a central voxel
Plasticity of Microglomerular Complexes
(i) with its six neighboring voxels (iU, iW, iN, iS, iE, iD)
[Fig. 2(F)] by means of their gray-values (for further details
see Holden et al., 1995). Figure 2(E,F) illustrate this algorithm by representing each voxel as a transparent cube in a
3D space. The algorithm operates throughout the 3D image
stack and classifies each voxel as either lip or microglomerulus. Voxel of a microglomerulus [Fig. 2(E) surface
model], are assigned to one cluster in the 3D space. The 3D
area of each cluster represents the segmented synapsin-IR
positive region (here interpreted as the bouton volume) and
the number of clusters the number of microglomerular
complexes.
Data Analysis
Statistical analysis was performed using SPSS 13. The density of microglomerular complexes was calculated on the
basis of the 3D lip volume and the number of microglomerular complexes within this volume (figures show density/
10 lm3). The mean and standard deviation of number and
density of microglomerular complexes and bouton volumes
were calculated for same-aged animals. For comparison of
animals within one group Kruskal-Wallis non-parametric
statistics were performed. Statistical analysis of differently
aged animals was performed by applying Mann-Whitney U
comparisons. Relationships between the measured parameters we analyzed by applying Pearson correlation. Illustrations were created with Amira 3.1.1, MatLab, Adobe Illustrator CS2, Adobe Photoshop 7.0 and Corel Draw 11.
RESULTS
Projection Neuron Boutons Represent
the Microglomerular Complex Core
The present study focuses on the analysis of the
microglomerular organization within the MB lips as
related to the animals’ age, behavior and experience.
Previous studies using PN mass labeling revealed that
PN boutons synapse onto f-actin rich KC dendritic
spines (Frambach et al., 2004; Groh et al., 2004). In
this present study we combined intracellular staining
of single PNs with immunocytochemistry to visualize
the location of single PN boutons and their postsynaptic KC spines. Figure 2(D,E) shows a single
marked PN (red) counterstained with the synapsin
antibody SynOrf1 (green) (superimposed yellow) and
the f-actin marker Alexa-488 phalloidin (blue,
appears diffuse due to high resolution of the image,
see also Fig. 1 inset). The triple labeling technique of
a single PN, anti-synapsin and the f-actin marker
phalloidin shows that the antibody SynOrf1 marks
presynaptic terminals of olfactory PNs, which are surrounded by KC dendritic spines. More importantly,
this technique identifies microglomerular complexes
1011
as seen in electron microscopic sections (Ganeshina
and Menzel, 2001; Ganeshina et al., 2006).
Microglomerular Organization of the MB
Lips During Adult Maturation
Previous studies have shown that MB calycal neuropils undergo volumetric expansions during adult
honeybee maturation (Withers et al., 1993; Durst
et al., 1994; Fahrbach et al., 1998, 2003; Farris
et al., 2001; Ismail et al., 2005). The goal of our
study was to investigate whether the MB lip’s
microglomerular organization undergoes structural
changes accordant to the sensory-guided behaviors
during maturation. Therefore we analyzed the
number and density of microglomerular complexes
as well as the bouton volume in bees at different
adult stages. The bees used (see Materials and Methods, Experimental part I) showed an onset of orientation flights between day 5 and 6. Pollen foraging
was already observed in a few animals between
Days 9 and 11, with an increase around Day 15.
Since we did not observe significant changes
between nectar and pollen foragers (data not shown),
the number and density of microglomerular complexes and bouton volume were pooled into one
forager (Fo) group.
Animals performing age-related tasks in a natural
environment [rich environment, Fig. 3(A,B)] showed
an increase in the number of microglomerular complexes accompanied by an increase in bouton volume.
The number of microglomerular complexes and the
bouton volume stayed almost constant until the 18th
day [Fig. 3(A), white bars, Fig. 3(B)], and then
increased significantly (number of microglomerular
complexes: Kruskal-Wallis, v2 ¼ 9.966, df ¼ 4, p ¼
0.041, Mann-Whitney U p < 0.05; bouton volume:
Kruskal-Wallis v2 ¼ 13.217, df ¼ 4, p ¼ 0.01, MannWhitney U p < 0.05) until the age of Day 37 [Fig.
3(A,B)]. Similar age-dependent effects were observed
in the group of animals reared under poor environmental conditions [Fig. 3(C,D)]. The number of
microglomerular complexes showed a not significant
but slight increase whereas the bouton volume
increased significantly most pronounced on Day 37
[Fig. 3(C), white bars; Fig. 4(D); bouton volume:
Kruskal-Wallis v2 ¼ 8.799, df ¼ 3, p ¼ 0.032]. In
comparison to the results observed in bees reared
under rich environmental conditions, the number of
microglomerular complexes increased to a higher
degree, whereas the density of microglomerular complexes stayed constant [Fig. 3(A,D), white and gray
bars on day 37].
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Krofczik et al.
Figure 3 Changes of the MB lips’ microglomerular organization in bees reared in either a rich
(Fo) or poor (poor) environment. (A), (C) Bar diagram shows changes in the number (left x-axis,
white bars) and density (right axis, gray bars) of microglomerular complexes during adult maturation
(1-day-old bees (d1), 4-day-old nurse (Nu) bees, and 14-, 18-, and 37-day-old forager (Fo) bees
reared in rich or poor environmental conditions. (B), (D) Bar diagram shows the bouton volume of
bees at different stages of adult maturation (as above). Asterisks and different letters indicate significant differences (Mann-Whitney U, p < 0.05). Sample size is depicted on each bar.
We did not observe statistical differences for the
measured parameters comparing same-aged forager
bees reared under either rich or poor environmental
conditions. Thus either the increase of the number of
microglomeruli and bouton volume is independent of
the rich versus poor environmental difference or other
parameters (e.g. flight activity, internal conditions of
the colony) regulate the number of microglomeruli
bouton volume.
Microglomerular Organization During
Manipulated Behavioral Maturation and
Sensory Experience
Manipulations of the bees’ olfactory-guided behavior
were applied to determine whether maturation of the
microglomerular organization of the MB lips is influenced by the bee’s behavior and its olfactory experience. For this purpose bees were reared in a singlecohort hive in which same-aged bees were prevented
Developmental Neurobiology
from nursing and foraging (hive-bound, hb), or
allowed to engage in extended nursing (Nu) and precocious foraging (pFO) (see Materials and Methods,
Experimental part II). In contrast to bees reared in a
natural colony, the changes in the microglomerular
organization of the MB lips depend on the behavioral
maturation and olfactory experience [Fig. 4(A–F)].
Hive-bound bees show a significant steady decrease
in the number [Fig. 4(A), white bars, Kruskal-Wallis
v2 ¼ 10.275, df ¼ 4, p ¼ 0.036] and density [Fig.
4(A), gray bars, Kruskal-Wallis v2 ¼ 11.347, df ¼ 4,
p ¼ 0.023] of microglomerular complexes until Day
14 (Mann-Whitney U, p ¼ 0.05) with a slight
increase on Day 18 (Mann-Whitney U, p ¼ 0.034).
The bouton volume showed a slight continuous
increase until Day 18 [Fig. 4(B)] Bees engaged in
extended nursing showed a minor decrease in the
number [Fig. 4(C), white bars] and density [Fig. 4(C),
gray bars] of microglomerular complexes and a significant increase in bouton volume most pronounced
on Day 14 [Fig. 4(D), Kruskal-Wallis, v2 ¼ 10.413,
Plasticity of Microglomerular Complexes
1013
Figure 4 Changes of the MB lips’ microglomerular organization in bees with manipulated social
behaviors and sensory experience. (A) Microglomerular organization in bees prevented from nursing
and foraging activities (hive-bound, hb bees). Bar diagram shows changes in the number (left x-axis,
white bars) and density (right axis, gray bars) of microglomerular complexes of 1-day-old bees and
differently-aged hive-bound bees (5-, 10-, 14-, and 18-day-old). (B) Bar diagram shows the bouton
volume of hive-bound bees at different ages (as above). (C) Microglomerular organization in bees
performing nursing (Nu) and extended nursing (eNu) behavior. Bar diagram shows changes in the
number and density of microglomerular complexes of 1-day-old bees, 5-day-old nurse bees, and
extended nurses (Day 10 and 14). (D) Bar diagram shows the bouton volume of nurses and extended
nurses (as above). (E) Microglomerular organization in bees performing precocious foraging (pFo)
behavior. Bar diagram shows changes in the number and density of microglomerular complexes of 1day-old bees and differently-aged forager bees (5-, 10-, 14-, and 18-day-old). (F) Bar diagram shows
the bouton volume of differently aged precocious foragers (as above). Asterisks and different letters
indicate significant differences (Mann-Whitney U, p < 0.05). Sample size is depicted on each bar.
df ¼ 3, p ¼ 0.015, Mann-Whitney U, p ¼ 0.034].
Bees engaged in precocious foraging showed a significant decrease in the number [Fig. 4(E) white bars,
Kruskal-Wallis, v2 ¼ 10.880, df ¼ 4, p ¼ 0.028] of
microglomerular complexes until Day 14 (MannWhitney U, p < 0.05). The bouton volume showed
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Krofczik et al.
Figure 5 The MB lips microglomerular organization in bees of the same age but reared either in
a natural colony under rich environmental conditions (Fo) or poor environmental conditions (poor)
or reared in a single-cohort colony (hb, hive-bound, eNu, extended nurses, pFo precocious foragers). (A) Bar diagram shows the number (left axis, white bars) and density (right axis, gray bars) of
microglomerular complexes of 14-day-old bees reared in a natural colony (Fo and poor) and in a
single-cohort colony (hb, eNu, pFo). (B) Bar diagram shows the bouton volume of bees reared in a
natural and a single-cohort colony (as above). Different letters indicate significant differences.
Sample size is depicted on each bar. 300 3 120 mm2 (600 3 600 DPI).
a significant increase [Fig. 4(F), Kruskal-Wallis,
v2 ¼ 12.903, df ¼ 4, p ¼ 0.012] until Day 14 followed
by a slight decrease (Mann-Whitney U, p < 0.05).
Comparing the changes in the number of microglomerular complexes and bouton volume observed in the
three groups our results showed that the number of
microglomerular complexes is negatively correlated
with the bouton volume in hive-bound (Pearson correlation coefficient ¼ 0.607, p ¼ 0.024) and extended
nurse bees (Pearson correlation coefficient ¼ 0.910,
p ¼ < 0.001) but not in precocious forager bees.
The most striking difference between the three
groups of bees (hive-bound, extended-nursing and
precocious foraging bees) relates to the different
stages of maturation in which changes in the microglomerular organization occurred. In hive-bound bees
and precocious forager bees the number and density
of microglomerular complexes continuously decreased [Fig. 4(A,E)], whereas in extending nursing
bees the significant decrease occurred from Day 10 to
14 [Fig. 4(C)]. Statistically the number and density
of microglomerular complexes differs significantly
comparing hive-bound and precocious foragers
with extended nurses aged 10 days (Mann-Whitney
U, p < 0.04).
Comparing the results observed in bees reared in
a natural colony with those observed in bees from
the single-cohort colony, our results show different
dependencies between the number or density of
microglomerular complexes and the boutons’ volume
(see Fig. 5). The single-cohort colony may have been
in suboptimal conditions, since a considerable proportion of bees (the hive-bound bees) were conDevelopmental Neurobiology
strained to the hive and their survival rate was
reduced. These conditions could well have led to an
imbalance in the social life within this colony. Nevertheless, in the context of our question it was interesting to ask whether the parameters of the microglomerular complexes change in hive-bound bees in a
similar or a different manner, and no attempt was
made to trace the factors acting on bees in a singlecohort colony and, in particular, on hive-bound bees.
In bees reared in a natural colony the number and
density of microglomerular complexes and the bouton volume increased, whereas in bees reared in a single-cohort colony the number and density of microglomerular complexes decreased and the bouton
volume increased. This negative correlation was most
pronounced on Day 14 in hive-bound bees and
extended nurses, whereas the effect was weaker in
precocious foragers. Comparing manipulated with
non-manipulated bees on Day 14 our results revealed
that foragers from the natural colony—irrespective of
the rearing conditions (rich and poor)—exhibit a significantly higher number [Fig. 5(A) white bars, Kruskal-Wallis, v2 ¼ 9.820, df ¼ 4, p ¼ 0.04] and density
[Fig. 5(A) gray bars, Kruskal-Wallis, v2 ¼ 12.276, df
¼ 4, p ¼ 0.015] of microglomerular complexes and a
significantly lower bouton volume [Fig. 5(B), Kruskal-Wallis, v2 ¼ 16.040, df ¼ 4, p ¼ 0.003].
DISCUSSION
This study provides the first 3D-based analysis of the
MB lips’ microglomerular organization during adult
Plasticity of Microglomerular Complexes
behavioral maturation of the honeybee (see Fig. 1).
We visualized microglomerular complexes within the
mushroom body lips by simultaneously labeling preand postsynaptic components (Figs. 1 and 2) following a study by Groh et al. (2004), in which microglomerular complexes in adult worker and queen bees
were studied. A 3D-based quantification and volumetric measurement technique allowed us to quantify
changes in the microglomerular organization of the
MB lip, using light microscopical techniques. We
also aimed for a better distinction between effects of
environmental experience and those that result from
manipulations of behavioral maturation and sensory
experience, especially the transition from nursing to
foraging behavior.
Previous studies focused on age- and experiencerelated neuroanatomical changes associated with the
mushroom bodies’ volume, but not with their microglomerular organization. The fact that age and foraging experience is associated with an increase of
mushroom body neuropil (lip, collar, basal ring) volume has been observed in several studies (Withers
et al., 1993; Durst et al., 1994; Fahrbach et al., 1998;
Ismail et al., 2005). More recently Farris et al. (2001)
have shown that foraging experience is associated
with an increase in the complexity of the Kenyon cell
dendritic branching pattern. A comparison with the
microglomerular complexes analyzed in our study is
limited since these observations were made on the
visual part of the MB, the collar region, and not on
the lip region. With respect to the microglomerular
organization it was observed that this organization is
influenced by brood-temperature in worker bees and
queens, respectively (Groh et al., 2004, 2006). Adding to that our study now revealed that the quantitative microglomerular organization is also influenced
by age-dependent and behavioral maturation. Our
results showed that the number and density of microglomerular complexes and their bouton volumes as
measured by anti-synapsin IR increased during maturation (see Fig. 3) and depend on experience and
behavior particularly inside the hive. Hive-bound,
extended nurses and precocious foragers showed a
decrease in the number and density of microglomerular complexes associated with an increase in bouton
volume (see Fig. 4). The comparison between nonmanipulated and manipulated bees indicates that the
number and density of microglomerular complexes—
as well as the bouton volume—depend on the bees’
behavior, and not just their age. However such a comparison is complicated by the fact that the colonies in
the rich/poor environment experiments (experimental
part I) and the one in the experimental part II (age
cohorts of behaviorally manipulated animals in an
1015
observation hive) were exposed to a range of different
factors. Not only that the bees in these two different
conditions lived in colonies of different size and environments they were also exposed to different factors
which resulted from differences between a normal
hive and one of an observation hive with a glass window. These latter differences could have resulted in
differences in colony temperature and light exposure.
Therefore, the higher number and density of microglomerular complexes found in animals of the rich/
poor experiment cannot be traced to any single parameter. However, since we are dealing with a neural
structure, the mushroom body lip, which is dedicated
to the neural processing of olfactory in formation in
the bee brain we can at least conclude that the factors
relevant here have an impact on the organization of
this structure, most likely with the consequence of
altered synaptic transmission. Larger numbers of
microglomerular complexes and their higher density
combined with smaller volumes might indicate that
olfactory processing is more advanced and more
detailed, and such a neural substrate appears to be
more related to the normal colony life than to the
rich/poor conditions of the external environment.
Changes in anti-synapsin IR indicate changes in
the volume of the microglomerular complexes which
might imply altered synaptic functions at this input
sites to the olfactory part of the mushroom body. It is
thus tempting to interpret the correlation between
number/density and volume of microglomerular complexes as indicating a compensatory mechanism,
which increases effective synaptic transmission in the
microglomerli surviving a putative selection process.
A similar interpretation has been provided by Seid
et al. (2005) who found new synapses to be formed
in the lip region of the mushroom body of the ant
Pheidole dentate.
Compensatory functional changes triggered by
sensory deprivation have been observed in developing mammalian brains. Deprived olfactory bulbs
become more sensitive to odor stimulation: mitral
cells become more responsive and more glomeruli
are activated by odors (Leon, 1998). A recent study
by Tyler et al. (2007) revealed that deprivation
increased the strength of primary synapses by acting
at both presynaptic and postsynaptic sites. Their
results argue against a change in the number of olfactory sensory neuron synapses but rather suggest a
change in the efficiency of existing synapses. If similar conditions would apply to the system studied here
we have to postulate that the larger volume of boutons leads to more efficient synaptic transmission
probably because of the larger presynaptic (and possibly also postsynaptic) compartments. On the other
Developmental Neurobiology
1016
Krofczik et al.
side, if the relationship between pre- and postsynaptic
membrane area is kept constant, as it appears to be
the case in the first neuropil in flies (Meinertzhagen,
1993) the larger bouton volume would reflect a larger
number of presynaptic sites on the grown area of the
PN boutons. The observed changes in the number and
density of microglomerular complex might also
induce a remodeling process (Stepanyants et al.,
2002) at their postsynaptic sites, the formation or
elimination of KC dendritic spines (postsynaptic
sites) or changes in the arborization pattern of KCs
and inhibitory feedback neurons from the MB lobes,
both of which receive input and serve as presynaptic
sites to the PN bouton (Ganeshina and Menzel,
2001). This would imply changes of the microcircuit
structure and function around each bouton, not only
quantitatively with respect to synaptic weights, but
possibly also qualitatively by rewiring the connectivity within each microglomerular complex.
It is well documented that the age- and experience-dependent neural plasticity may be restricted to
sensitive periods. For example, sensory deprivation
or inadequate stimulation in zebra finches leads to an
extension of the sensory period, which is accompanied by a change in spine density (Bischof et al.,
2002). We found for the bee that reducing the sensory
input at a time when the animals are prepared to
change their behavioral tasks leads to the structural
changes explained above. In hive-bound bees these
structural changes evolved already on Day 10 [Fig.
4(A,B)] whereas in nurse bees at a later stage on Day
14 [Fig. 4C,D)]. The earlier onset of these structural
changes in hive-bound bees might be explained by
the fact that the brain is more readily prepared for
structural plasticity and thus more sensitive to sensory/behavioral impairment at a particular time of its
age dependent maturation program.
Age-related programs appear to prepare the animal
for particular sensory/motor conditions through hormonal controls and thus coordinate the formation of
the neural structures to the expected inputs and/or
outputs. Only under conditions of a full- balanced
colony life do animals develop normally, and the
working of coordinated developmental programs and
experience effects can only be uncovered by interfering with normal life conditions. Interfering with the
sensory/motor experience of any subgroup of animals
within the colony will also have an effect on the normal life history of the whole colony through reciprocal feedback loops up to the extreme case in which a
colony loses the balance of social life and deteriorates, as may have been the case in our colony 3. In
the domain of olfactory cues, which we studied here,
the general effect of such disturbance was the reducDevelopmental Neurobiology
tion in the number and density of microglomerular
complexes, accompanied by an increase of bouton
volume. It is tempting to conclude that the microglomerular complexes are the substrate for central nervous olfactory processing that provides the structural
modules for both developmental programs as well as
experience-dependent plasticity. Further physiological and structural studies need to be brought to the
single neuron and the network level, taking advantage
of the insect nervous system with its identified neurons and well-described neural nets (Abel et al.,
2001; Gronenberg, 2001; Kirschner et al., 2006). The
strength of synaptic transmission between projection
neurons and Kenyon cells can be monitored in Caimaging recordings (Szyszka et al., 2005), and electron microscopy studies can be combined with singlecell marking and immunolabeling (Ganeshina and
Menzel, personal communication). These and other
experiments, which are easily carried out in Apis
mellifera, will allow us to test the hypothesis that
microglomerular organization changes lead to a
rewiring of cell-to-cell connections, enabling the
brain to adapt its synaptic efficacy.
We thank L. Müßig and D. Drenske for motivated help
during experiments, P. Knoll for beekeeping, and B.
Brembs, G. Leboulle, N. Stollhoff and the Journal Club for
helpful comments regarding this manuscript.
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Developmental Neurobiology