Download Organization of the Honey Bee Mushroom Body

THE JOURNAL OF COMPARATIVE NEUROLOGY 450:4 –33 (2002)
Organization of the Honey Bee Mushroom
Body: Representation of the Calyx Within
the Vertical and Gamma Lobes
NICHOLAS J. STRAUSFELD*
Division of Neurobiology, Arizona Research Laboratories, The University of Arizona,
Tucson, Arizona 85721
ABSTRACT
Studies of the mushroom bodies of Drosophila melanogaster have suggested that their
gamma lobes specifically support short-term memory, whereas their vertical lobes are essential for long-term memory. Developmental studies have demonstrated that the Drosophila
gamma lobe, like its equivalent in the cockroach Periplaneta americana, is supplied by a
special class of intrinsic neuron—the clawed Kenyon cells—that are the first to differentiate
during early development. To date, however, no account identifies a corresponding lobe in the
honey bee, another taxon used extensively for learning and memory research. Received
opinion is that, in this taxon, each of the mushroom body lobes comprises three parallel
divisions representing one of three concentric zones of the calyces, called the lip, collar, and
basal ring. The present account shows that, although these zones are represented in the
lobes, they occupy only two thirds of the vertical lobe. Its lowermost third receives the axons
of the clawed class II Kenyon cells, which are the first to differentiate during early development and which represent the whole calyx. This component of the lobe is anatomically and
developmentally equivalent to the gamma lobe of Drosophila and has been here named the
gamma lobe of the honey bee. A new class of intrinsic neurons, originating from perikarya
distant from the mushroom body, provides a second system of parallel fibers from the calyx
to the gamma lobe. A region immediately beneath the calyces, called the neck, is invaded by
these neurons as well as by a third class of intrinsic cell that provides connections within the
neck of the pedunculus and the basal ring of the calyces. In the honey bee, the gamma lobe
is extensively supplied by afferents from the protocerebrum and gives rise to a distinctive
class of efferent neurons. The terminals of these efferents target protocerebral neuropils that
are distinct from those receiving efferents from divisions of the vertical lobe that represent
the lip, collar, and basal ring. The identification of a gamma lobe unites the mushroom bodies
of evolutionarily divergent taxa. The present findings suggest the need for critical reinterpretation of studies that have been predicated on early descriptions of the mushroom body’s
lobes. J. Comp. Neurol. 450:4 –33, 2002. © 2002 Wiley-Liss, Inc.
Indexing terms: learning and memory; social insects; brain organization
Although published in curtailed form, Dujardin’s (1850)
pioneering account of the honey bee mushroom bodies makes
explicit comparisons with the brains of other insect taxa,
which he had studied since 1846 by using a unique pigmentation method to reveal surface features of their neuropils.
Comparisons of the brains of various Hymenoptera with
those of Orthoptera, Odonata, and Diptera allowed his now
famous dictum that, because the mushroom bodies of honey
bees were larger and more convoluted than those of other
taxa, they must support the social behavior of these insects
and, thus by inference, should underlie learning, memory,
and “intelligence” (Dujardin, 1853).
Over 150 years after Dujardin’s discovery, three taxa
now separately offer unique insights into the neurological,
biochemical, and genetic basis of mushroom body function.
© 2002 WILEY-LISS, INC.
These are the cockroach Periplaneta americana, the honey
bee Apis mellifera, and the fruit fly Drosophila melano-
Grant sponsor: National Science Foundation; Grant number: IBN9726957; Grant sponsor: Human Frontiers Science Program; Grant number: RG0143/2001-B.
*Correspondence to: Nicholas J. Strausfeld, Arizona Research Laboratories Division of Neurobiology, University of Arizona, 611 Gould-Simpson
Science Building, Tucson, AZ 85721.
E-mail: [email protected]
Received 18 December 2001; Revised 11 March 2002; Accepted 13 April
2002
DOI 10.1002/cne.10285
Published online the week of July 1, 2002 in Wiley InterScience (www.
interscience.wiley.com).
VERTICAL AND GAMMA LOBES IN HONEY BEES
gaster. Current opinion ascribes sophisticated roles in
adaptive behaviors to mushroom bodies. These roles include, for Periplaneta, place memory and predictive motor
actions (Mizunami et al., 1998a,b) as well as contextdependent sensory integration (Li and Strausfeld, 1997);
for Apis, short- and long-term associative memory (Erber
et al., 1980; Mauelshagen, 1993); and, for Drosophila,
associative memory (Davis, 1993; de Belle and Heisenberg, 1994; Heisenberg, 1994, 1998) and “intelligent” decision making (Tang and Guo, 2001). A recent genetic and
behavioral analysis of the ala mutant of Drosophila has
proposed that long-term memory requires the integrity of
the mushroom bodies’ vertical lobes (Pascual and Préat,
2001), whereas short-term memory requires the gamma
division of the medial lobe (Zars et al., 2000).
Notwithstanding accumulated evidence that mushroom
bodies are likely to play cardinal roles in plastic and
acquired behaviors, there has been almost no attempt to
determine what structural features mushroom bodies
have in common in different taxa. One goal of the present
account is to rectify this omission by showing that the
organization of certain cell types into a discrete integrative neuropil called the gamma lobe occurs in the honey
bee as it does in Drosophila and Periplaneta. It is proposed
that this lobe is phylogenetically the oldest part of this
brain center.
In the honey bee, each mushroom body has a pair of
cup-like neuropils called the calyces (Fig. 1A–D), which
are elongated dorsoventrally over the rostral surface of
the protocerebrum (Fig. 1H). The calyx neuropil is concentrically organized, as was originally recognized by Dujardin (1850) and by Kenyon (1896a,b), one of the earliest to
use Golgi impregnations. Jawlowski (1959a,b, 1960) was
the first to identify four concentric regions in the calyces,
later termed the lip, collar, and the layered basal ring
(Mobbs, 1982).
Each of these regions is targeted by a characteristic set
of afferents from various peripheral sensory neuropils.
The antennal lobes are represented in the lip and basal
ring by subsets of olfactory projection neurons whose axons ascend to the protocerebrum by means of the inner
antennocerebral tract and then extend laterally to discrete regions of the ipsilateral protocerebrum (Schröter
and Malun, 2000; Abel et al., 2001). These axons pass in
front of the calyces, into which they send collaterals. In
the honey bee, but not in Drosophila, the visual system is
represented in the calyces by the endings of projection
neurons that separately originate from the medulla and
lobula (Gronenberg 1986, 2001). The calyx collar is supplied by visual afferents from the ipsilateral lobula and
from both the ipsi- and contralateral medullae. These
terminals confer a layered arrangement of presynaptic
processes in the collar zone (Ehmer and Gronenberg, personal communication). An outer region of the collar, and
the basal ring, additionally receive terminals from mechanosensory interneurons, and possibly gustatory elements
(Strausfeld, unpublished observations).
Golgi and immunocytological studies show that each of
the concentric regions of the calyx is further divided into
narrower domains called zones. Antisera against gastrincholecystokinin (GCCK) and FMRFamide (Strausfeld et
al., 2000) demonstrate that the lip is divided into at least
three zones. Immunostaining (Strausfeld et al., 2000) and
ethyl gallate staining show the collar to be divided into at
least three zones: a narrow outer zone adjacent to the lip,
5
here called the outer collar zone; a broad zone called the
intermediate collar zone; and a third narrow zone (the
inner collar zone) adjacent to the basal ring (Fig. 1D). The
basal ring comprises a bistratified central zone flanked by
two narrower outer ring zones; a broad zone extending
across the calyx wall flanked by a shallower wedge-shaped
zone (Fig. 1D).
The calyx is represented in the lobes by the parallel
arrangements of axon-like processes of neurons that are
intrinsic to the mushroom bodies and whose cell bodies fill
and surround the bowls of the calyces (Fig. 1B,D–G).
These neurons, called Kenyon cells (Kenyon, 1896b), provide bifurcating axons to both lobes or send a single axon
to only one lobe (Fig. 1H). Early observations of the ant
Formica (Leydig, 1864) described the vertical lobe as being organized into three broad parallel divisions. However, although uncommented by observers, reduced silver
studies of the honey bee showed the medial lobes to be
approximately two thirds the width of the vertical lobes
(Mobbs, 1982, 1985 loc. cit. Fig. 9), a difference also figured in Rybak and Menzel (1993). A longitudinal division
of the lobes into many parallel strata has been revealed by
antisera against FMRFamide and GCCK (Schürmann and
Erber, 1990; Strausfeld et al., 2000). The lobes provide
multimodal efferent neurons that extend to the protocerebrum (Homberg, 1984; Schildberger 1984; Mauelshagen,
1993; Li and Strausfeld, 1997, 1999) or recurrently back to
the calyces (Gronenberg, 1987; Grünewald, 1999). Golgi
impregnations of various taxa, including the honey bee,
have shown that the lobes are also richly supplied by
afferent terminals from the protocerebrum (Li and Strausfeld, 1997; Ito et al., 1998; Strausfeld et al., 2000).
From studies of reduced silver sections, Mobbs proposed
that Kenyon cell axons from the lip, collar, and basal ring
divide the vertical and medial lobes into three equally
wide subdivisions (Mobbs, 1982). Kenyon cell axons in the
lower third of the vertical lobe (Mobbs’s layers 4 – 6) were
supposed to derive from the lip, axons in the middle of the
vertical lobe (Mobbs’s layer 3) were thought to originate in
the collar, and axons in the upper layers (Mobbs’s layers 1,
2) were ascribed to Kenyon cells in the basal ring. The
right side of Fig. 1A summarizes the conclusions of Mobbs.
The study of Mobbs has provided the basis for many
subsequent physiological and anatomical investigations
(Gronenberg, 1987; Schäfer et al., 1988; Bicker, 1991;
Rybak and Menzel, 1993, 1998; Menzel et al., 1994;
Grünewald, 1999; Strausfeld et al., 2000). However, an
immunocytological study demonstrated that there was an
apparent mismatch between immunoreactive zones in the
calyces and immunoreactive layers in the lobes (Strausfeld et al., 2000). Specifically, whereas two zones in the
calyx lip are strongly immunoreactive to antibodies
against GCCK and FMRFamide, there are no corresponding strongly immunoreactive zones in layers 4 – 6 of the
vertical lobe as designated by Mobbs (Strausfeld et al.,
2000, loc. cit. Fig. 6A–C). According to Mobbs (1982, loc.
cit. Fig. 15), these layers are supposed to receive Kenyon
cell projections from the lip. An explanation offered for
this discrepancy was that Kenyon cell axons might permutate their relationships in the lobes such that some
GCCK- and FMRFamide-immunopositive neurons from
the lip zone would be displaced upward into Mobbs’s layer
3 (1982, loc. cit. Figs. 10, 49) and mix with axons from the
collar (Strausfeld et al., 2000).
6
N.J. STRAUSFELD
Such positional shifts by axons representing a specific
zone of the calyx would be unprecedented. In the cockroach Periplaneta americana concentric divisions of the
calyces, defined by their afferent inputs and the dendritic
domains of class I Kenyon cells, are each represented in
the lobes by immunoreactive layers that subdivide the
lobes into three parallel divisions (Strausfeld and Li,
1999a,b). These divisions lie behind an anterior component that is supplied by a unique species of intrinsic
neuron, the clawed Kenyon cell, whose population represents the whole calyx rather than a restricted zone of it. In
Periplaneta, the clawed Kenyon cells are the first to differentiate and are already resolvable in the embryonic
mushroom body (Farris and Strausfeld, 2001). Having
developed first, the cell bodies of class II Kenyon cells are
displaced upward and outward by the subsequently generated Kenyon cells beneath them. Thus, the perikarya of
clawed Kenyon cells come to lie either above the calyx rim
or outside the neuropil’s perimeter (Farris and Strausfeld,
2001). As will be discussed below, in the honey bee, these
developmental events likewise result in the cell bodies of
clawed Kenyon cells lying outside the calyx cup (Farris et
al., 1999). These neurons are here compared with Kenyon
cells of Drosophila that differentiate first (Lee et al.,
1999), which in the adult possess clawed dendritic specializations and whose axons constitute the gamma lobe.
The present study demonstrates that the lip of the calyx
is not represented in the lower third of the vertical lobe
but in the lower half of a layer in the lobe that Mobbs
(1982) originally ascribed to projections from the calyx’s
collar. The present results provide evidence that the lower
third of the vertical lobe receives axons of clawed Kenyon
cells that together represent the entire calyx. By comparison to the cockroach and fruit fly, the division of the
honey bee vertical lobe receiving axons from clawed
Kenyon cells is called the gamma lobe. The identification
of a gamma lobe in the honey bee advocates a reinterpretation of previous studies that refer to the internal organization of this center.
MATERIALS AND METHODS
Honey bees were collected while visiting flowers or were
culled from combs provided by the USDA Carl Hayden
Bee Research Laboratories in Tucson. Although categorized as workers, the precise age of each individual was
not known.
All descriptions of neurons are from Golgi preparations.
The architecture of the mushroom bodies and surrounding
neuropils are described from Bodian (1936) reduced silver
preparations and from ethyl gallate–stained material.
Laminae in the mushroom bodies were identified by using
antisera against FMRFamide and gastrin-cholecystokinin
(GCCK). The methods are described in detail elsewhere
(Strausfeld and Li, 1999b; Strausfeld et al., 2000).
Two variants of the combined Golgi method were used,
both of which use 1:5 parts 25% glutaraldehyde and 2.5%
potassium dichromate as the initial fixative and chromation fluid. Usually, the second chromation, which is used
4 –5 days after the first, uses 1 part 1% osmium in 99 parts
potassium dichromate. A useful modification increased
the osmium concentration to 1 part 4% osmium in 99 parts
potassium dichromate. This modification stained large
numbers of clawed Kenyon cells. Clawed, rosette, and
chandelier-type Kenyon cells in the basal ring were selec-
tively stained by using a modification of the Golgi rapid
method. After 12 hours fixation in 2.5% glutaraldehyde in
0.1 M Millonig’s phosphate buffer (at pH 7.2), brains were
washed twice 30 minutes in buffer and then twice 30
minutes in 2.5% potassium dichromate. Brains were incubated for 5 days in a mixture of 95 ml of 2.5% potassium
dichromate and 5 ml of 2% osmium tetroxide before incubation in 0.75% silver nitrate for 3 days.
Neuron reconstruction and image
processing
Line drawings of neurons were reconstructed at ⫻1000
by using a camera lucida apparatus. Images were scanned
at 1,200 dpi and stored as TIFF files. These were overlaid
on outlines of the mushroom bodies and surrounding protocerebrum obtained from digitized tracings of Bodianstained preparations, sectioned at the same orientation as
Golgi preparations (Fig. 1C). Outlines were made by using
Smartsketch (Futureware Software, Inc., San Diego, CA).
Files were then converted to TIFF files.
Dendritic trees and terminals of Golgi-impregnated
Kenyon cells, and certain neurons in the vertical lobes,
were reconstructed from stacked optical sections captured
with a Sony DKC 5000 CCD digital camera linked to an
Apple G4 computer equipped with graphics software. Images were captured at an initial magnification of ⫻600, by
using a planapochromat oil immersion objective. For each
25-␮m-thick section, 25 optical images were captured and
layered in register. These were made transparent by using
the Photoshop darkening function. Shadows were removed, and the images were then flattened. For larger
areas, between four and six such reconstructions were
made separately and then seamlessly montaged. These
procedures result in images of Golgi-impregnated neurons
(Figs. 2–5) that show each process exactly in focus
throughout a depth of 25 ␮m (in some cases 50 ␮m, after
overlying two consecutive histologic sections).
Matches between dendrites or terminals and immunostained laminae in the lobes were achieved by false coloring flattened Golgi-impregnated elements within a crosssection of the vertical lobe having the same orientation as
an antiserum-stained cross-section at the same level. The
two images were overlaid, and the diameters of the lobes
were isometrically adjusted, if necessary, to equal each
other without altering layer relationships. After using the
darkening function, the two images were then flattened.
Matches between Kenyon cell projections and immunostained layers were done by comparing the position of the
cross-section of a Kenyon cell axon, or bundle of axons, at
a level of the lobe that corresponded to the immunostained
sections.
Terminology
The generic names “vertical lobe” and “medial lobe” are
used here so as to avoid applying the same term to nonhomologous parts of the mushroom bodies. For example,
the terms ␣ lobe and ␤ lobe, coined by Vowles (1955) for
the honey bee, are not used here to avoid confusion with
the ␣ and ␤ lobes of Drosophila, which are genetically
discrete subdivisions contributing to at least three parallel components of the vertical and medial lobes. The terms
vertical and medial lobes have also been used for the
cockroach mushroom bodies (Li and Strausfeld, 1997). In
honey bees, the medial lobe extends obliquely ventrad
from the pedunculus toward the midline of the brain. The
VERTICAL AND GAMMA LOBES IN HONEY BEES
lobe branching from the pedunculus and extending frontally (Fig. 1H) is the vertical lobe, which in Drosophila and
Periplaneta is bent upward when the brain is viewed from
the front, whereas in the honey bee the lobe points directly
forward (Fig. 1A,H). With respect to the neuraxis of the
honey bee, the vertical lobe extends ventrally (Fig. 1H) so
that its lower third, as seen in frontal sections of the brain,
is sensu stricto its most posterior third.
The term “lobe” is used to describe one of the branches
of the pedunculus. The term “division” is used to describe
the main parallel partitions in the lobes, each of which
represents one of the major regions of the calyx (Fig. 1A,
left) and provides its own systems of efferent neurons (Fig.
1C). Divisions of the lobes can be almost separate from
each other, as are components of the medial lobe in Drosophila. Or they can be contiguous, as in the honey bee
and cockroach. In the honey bee, divisions of the lobe are
further divided into parallel strata.
Kenyon cells are classified according to the convention
adopted for the cockroach. Kenyon cells whose perikarya
are contained within the calyx (Fig. 1D–F) and whose
dendritic branches penetrate the inner wall of the calyx
are classified as class I Kenyon cells (Fig. 1B,H). These
have spiny, blebbed, or varicose dendritic trees. Class II
Kenyon cells possess clawed dendrites, and their
perikarya lie outside the calyx flanking the collar and
basal ring (Fig. 1B,G,H).
RESULTS
Two classes of Kenyon cells
supply the calyces
As their name suggests, the calyces are cup-shaped
neuropils approximately four times longer than they are
wide, extending around and over the rostral hemisphere of
each protocerebral lobe (Kenyon, 1896a,b). The two calyces of each mushroom body are joined by short necks to a
common pedunculus, which then divides into a medial and
vertical lobe, the former tapering to the midline of the
brain where it ends just in front of the lower margin of the
central complex’s ellipsoid body. The vertical lobe extends
forward to the front surface of the brain where its truncated end lies approximately 150 ␮m above the antennal
lobe (Fig. 1A,H).
The walls of the calyces are composed of synaptic neuropil ranging in depth from 80 ␮m to 120 ␮m. Their inside
surfaces are lined with layers of axons from Kenyon cells
(Figs. 2– 4). The morphometric study of Witthöft (1967)
estimated that in the worker honey bee approximately
170,000 Kenyon cell bodies reside in the bowl of each calyx
and above its rim.
Kenyon cell perikarya within the calyx are grouped into
three assemblages (Fig. 1B,D–G). The smallest perikarya
are arranged as a central stack above the basal ring. This
stack is surrounded by an assemblage of larger and more
basophilic perikarya, which are divided into three clusters. A small proximal cluster supplies dendrites to the
outer basal ring zone. A middle cluster supplies dendrites
to the outer, intermediate, and inner collar zone. An outer
cluster supplying dendrites to the lip extends above and
over the rim of the calyx. All these neurons are classified
as class I Kenyon cells. Their perikarya provide neurites
that extend to the inner wall of the calyx where they
branch. One branch penetrates the calyx neuropil to pro-
7
vide a dendritic tree, the other branch projects along the
inner surface of the calyx wall to finally pass through the
calyx neck and into the pedunculus (Figs. 2A, 3A–D).
These axon-like processes each send a tributary into the
vertical lobe and another into the medial lobe.
Counts from ethyl gallate sections estimate that an
additional 14,000 Kenyon cell bodies lie outside each calyx
(Fig. 1B,D,G). These neurons are classified as class II
Kenyon cells. Their small perikarya form a layer between
2 and 20 cell bodies deep around the outer calyx wall.
Their neurites penetrate the outer wall to project directly
through the calyx neuropil normal to its surface (Fig.
3F,G). Each of these extensions provides several short
dendrite-like branches ending in distinctive claw-like specializations (Figs. 2A, 3B,F,G) and then proceeds axon-like
along the inner calyx wall.
The mixed Golgi Colonnier-rapid method most commonly impregnates class I Kenyon cells. As in the mushroom bodies of Periplaneta, various types of dendritic specializations have been identified in the honey bee: spines,
thorns, and varicosities decorated with tiny spicules. The
most common of these structures are spines and thorns,
which belong to Kenyon cells that appear to make the
major contribution to the calyx’s lip and collar neuropils
(Figs. 2, 3). Although the Golgi method cannot provide
quantifiable data because it is stochastic, it is not unusual
to impregnate several spiny cells in such close proximity
in these regions that their dendritic fields occupy the same
domain (Fig. 2G), a feature also noted by Mobbs (1982).
This finding suggests that spiny class I neurons are the
most numerous Kenyon cells. Class I neurons that are
decorated with small spines or various types of varicosities and spicules supply the basal ring zones (Fig. 5). Each
of these cell types is described below, with reference to its
parent zone. Clawed (class II) Kenyon cells are found in all
calycal zones where they form palisades of dendrites,
those of neighboring cells overlapping only slightly (Fig.
3F,G).
Kenyon cells of the lip and their projections
to the lobes
The dendritic domains of spiny class I Kenyon cells
reflect the calycal zones in which they lie. This was also
alluded to by Mobbs (1982), although his study distinguished only three zones. In the calyx lip, however, there
are at least three, possibly four, distinct cell morphologies
amongst class I Kenyon cells whose dendritic trees occupy
domains that appear to reflect the organization of axon
collateral endings from different classes of antennal lobe
projection neurons. Thus, certain Kenyon cell dendrites
extend across the whole of the lip’s width (Fig. 2A,B).
Others are constrained to a part of the lip, lining its outer
half (Fig. 2C) or its outer and lower edges (Fig. 2D–F). One
species of these marginal Kenyon cells also sends
branches into the outer collar zone, suggesting that a
separate supply of afferents terminates in a discrete zone
of neuropil adjoining the lip (Fig. 2E). Class I Kenyon cells
send their bifurcating axons into the medial and vertical
lobes. In the vertical lobe, axons of class I cells from the lip
occupy the lower half (Fig. 2G–I) of the layer designated
by Mobbs (1982, his layer 3 shown here in Fig. 1A, right)
as receiving axons from the collar. Mobbs’s 1982 account
proposed that the calyx lip supplied his layers 4 – 6 (Mobbs
1982; loc. cit. Fig. 10).
8
Fig. 1. Components and orientation of the honey bee mushroom
bodies. A: Comparison of present results (left) and those of Mobbs
(1982), right. The medial and lateral calyces (M Ca, L Ca) are each
divided into three major regions, lip, collar, and basal ring (Li, Co,
BR). Left: These are represented by three divisions in the vertical lobe
(V) that lie above the adjoining gamma lobe (␥). The medial lobe (M;
old nomenclature, beta lobe) extends toward the brain’s midline.
Mobbs’s (1982) account describes the lip, collar, and basal ring represented by three bands in the alpha lobe (old terminology for vertical
and gamma lobe) comprising, respectively, layers 1 and 2, layer 3, and
layers 4 through 6. A Lo, antennal lobe; S L Pr, I L Pr, and I M Pr, the
superior lateral, inferior lateral, and inferior medial protocerebrum,
respectively; U, upper; L, lower. B: Organization of class II (clawed)
and class I Kenyon cell bodies. Groups of class I cell bodies are
indicated by the calycal region they supply: Li (lip), Co (collar), o BR,
i BR (outer and inner basal ring). C: Efferent neurons from the
mushroom bodies, such as these vertical lobe efferents, are shown
N.J. STRAUSFELD
with their relevant protocerebral regions. L Ho, lateral horn; S M PR,
superior medial protocerebrum. D: Ethyl gallate–stained medial calyx
showing the dense packing of Kenyon cell bodies. Selected areas
(E–G) are enlarged in E–G below. w o BR, wedge component of outer
basal ring; i, inner; o, outer; Ne, neck zone. E: Cell bodies supplying
the lip and collar. F: The extremely small cell bodies supplying basal
ring neuropil. G: Small perikarya outside the calyces belonging to
class II Kenyon cells. H: Side views of the brain and mushroom body.
The axons of class I Kenyon cells (two shown) supply the vertical (V)
and medial lobes (M). Class II Kenyon cell axons supply the gamma
lobe (␥). The gamma lobe (␥) lies under the vertical lobe, when viewed
sagittally or frontally (as in A). The neuraxis, shown in red, shows the
brain tilted upward. Its calyces are rostral (Ros), and the vertical and
gamma lobe extend ventrally (Ven). Dor, dorsal; Cau, caudal; U,
upper; L, lower; SOG, suboesophageal ganglion. Scale bars ⫽100 ␮m
in A (applies to A–C,H), 100 ␮m in D.
Fig. 2. Kenyon cells of the calyx lip. A,B: Spiny class I Kenyon
cells with arbors throughout the lip. A clawed Kenyon cell is shown in
A, and two partially shown in C (arrows). C,D,F: Spiny Kenyon cells
with arbors lining the upper and distal margins of the lip. Dashed
lines mark lip zone border. E: Spiny Kenyon cells with dendrites
restricted to the inner lip (Li) and adjacent outer collar zone (o Co). F:
Two columnar Kenyon cells invading the outer collar. The lip zone is
shown with parts of a blebbed terminal from an antennal lobe projection neuron and a Kenyon cell dendrite restricted to its distal margin.
G: A cluster of lip Kenyon cells with axons projecting into the pedunculus neck. BR, basal ring. H,I: Cross-sections showing the axon
bundle of G terminating in the lip division (Li bracket) of the vertical
lobe. Other smaller groups of axons, also from the lip of the medial
calyx, are in the middle of the lobe. Double arrowheads point to axons
from the basal ring. The gamma lobe is shown bracketed (␥). Note that
from one level to the next (H to I) the lateral relationships amongst
axons within and between bundles remain the same. Scale bars ⫽ 10
␮m in A (applies in A–F), 50 ␮m in G, 50 ␮m in H (applies in H,I).
Fig. 3. Kenyon cells of the calyx collar. Dendritic trees shown
through calyx neuropil. Inner margin of neuropil is up, outer margin,
down. A–E: Spiny class I Kenyon cells have wedge-shaped (A) or
rectangular bistratified narrow (B) or broad (C,E) dendritic trees in
the intermediate collar zone. The single clawed cell shown in B (arrowed) illustrates the difference between class I and class II Kenyon
cells. D: An unistratified spiny Kenyon cell of the collar zone. Equivalent inner levels of the calyx are shown bracketed in B–E. Unistratified Kenyon cells (D) omit this inner level, which receives inputs from
lobula interneurons. F,G: Palisades of clawed Kenyon cells in the
intermediate (F) and outer (G) collar zones. Cell bodies of these
neurons (some indicated by arrows in G) lie outside the calyx.
H: Wedge-shaped spiny dendritic trees of two Kenyon cells serving the
outer collar zone, which lies between the intermediate collar zone (Co)
and lip (Li). I: The collar and basal ring (BR) are separated by an
inner collar zone consisting of narrow columnar dendritic trees of
spiny class I Kenyon cells. J: Bistratified diffuse Kenyon cells in this
zone have branches within the inner layer of the calyx that extend
laterally into the basal ring and through the inner collar zone to the
intermediate collar zone. K: Encrusted type I Kenyon cells have
pyramidal-shaped dendritic trees that lie at the junction between the
outer collar zone (margins, dashed lines) and the lip. These cells also
provide lateral branches in the inner layer of the calyx that extend to
the intermediate collar zone and lip. Scale bars ⫽ 10 ␮m in A (applies
in A–F,H–K), 10 ␮m in G.
VERTICAL AND GAMMA LOBES IN HONEY BEES
Clawed class II Kenyon cells extend through the lip
zones, each having a narrow columnar dendritic domain
(Fig. 2A,C). As will be described below, it is these that
supply the gamma division occupying the lower third of
the vertical lobe.
Kenyon cells of the collar
The most commonly observed Kenyon cells in the collar
have spiny dendritic trees, each with a wedge-shaped (Fig.
3A,D) or rectangular (Fig. 3B,C,E) domain with respect to
its proximal-distal orientation within the calyx. However,
within a narrow zone adjacent to the lip (the outer collar
zone), spiny Kenyon cells have rectangular dendritic trees
that are elongated perpendicular to the wall of the calyx
(Fig. 3H). An inverted cone-like arrangement of
dendrites—narrower near the inner surface of the calyx
and broader at its outer surface (Fig. 3I)— hallmarks
Kenyon cells within a narrow zone adjacent to the basal
ring (the inner collar zone). However, there are also
bistratified Kenyon cells in both the inner and outer collar
zones that have broadly spread inner branches and much
narrower apical processes (Fig. 3J,K).
The dendrites of Kenyon cells reflect the stratified organization of afferents. For example, in the intermediate
collar zone, unistratified dendritic trees (Fig. 3D) interact
with layers of afferents from the medulla but omit deeper
bundles of terminals from the lobula, which lie nearer the
inner wall of the calyx (Fig. 4A,B). Other Kenyon cells
have stratified arrangements, with their deeper dendrites
providing a diffuse arbor and their outer dendrites providing a dense thicket of processes (Fig. 3B,C,E). All of these
arrangements appear to reflect the complexity of the afferent supply to this zone, which originates from several
sensory neuropils. In addition to afferents from the medulla and lobula, inputs include columnar terminals of
unknown origin (Fig. 4B) and whorled terminals deep
within the intermediate collar zone (Fig. 4E) that dye
tracings suggest are supplied from large glomerular-like
neuropils lying behind the antennal lobes (unpublished
data).
Diffuse dendritic trees in the intermediate collar zone
(Fig. 3A) branch through all the layers of afferents from
the medulla and lobula, as is also shown by the pale gray
profiles of Kenyon cells that straddle the afferent layers in
Figure 4A. In addition to spiny Kenyon cells, the outer
collar zone contains dendritic trees that are densely invested with small tuberous or spiny outgrowths (Fig. 3K).
These cells, which have been identified in the same preparations as spiny neurons, are here termed “encrusted”
class I Kenyon cells. Their inner branches extend laterally
into the intermediate collar zone and the inner margin of
the lip.
Class I Kenyon cells from the collar zones send bifurcating axons into the medial and vertical lobes where they
occupy the upper half of Mobbs’s layer 3 (compare Fig. 1A
left, right). Clawed class II Kenyon cells occur throughout
the outer, intermediate, and inner collar zones (e.g., Fig.
3B). Golgi rapid impregnation shows clawed Kenyon cells
arranged as palisades, here exemplified from the collar’s
intermediate (Fig. 3F) and outer zones (Fig. 3G).
Kenyon cells of the basal ring zones and
their projections
The basal ring is partitioned into an outer basal ring
zone and a bistratified inner basal ring zone (this includes
11
Jawlowski’s [1959a] layer IIIA), surrounding a central
area through which pass axons of Kenyon cells from the
collar, lip, and basal ring. Between the central area and
the inner basal ring are two narrower concentric areas
through which pass processes of feedback neurons. The
latter are here termed the inner neck and outer neck zones
(Fig. 4D) and are probably equivalent to the intermediate
tract shown in Mobbs (1982). As in the lip and collar, each
of the basal ring zones receives a characteristic complement of afferent processes (Fig. 4D).
Given so many discrete components, it is not surprising,
therefore, that the basal ring contains more morphological
types of neurons than elsewhere in the calyx. This was
also remarked on by Mobbs (1982; loc. cit. Fig. 29; see also
Mobbs, 1984), who identified at least three Kenyon cell
morphologies in it. The present study has identified six
types of class I Kenyon cells in this region (Fig. 5). The
two-layered inner zone of the basal ring contains four
types of Kenyon cells: bi- and unistratified spiny neurons
(Fig. 5A) and varicose bi- and unistratified chandelier cells
(Fig. 5B). Compared with the basal ring’s spiny neurons,
chandelier cells have extremely dense dendritic trees that
provide the largest contribution to basal ring neuropil.
Their axons are, however, the thinnest of all the Kenyon
cells, and their dendrites are more slender and delicate in
appearance than those of spiny neurons (compare insets,
Fig. 5B).
The outer basal ring zone consists of a columnar component surrounded by a smaller partition, wedge-shaped
in cross-section. The columnar zone contains densely
branching Kenyon cells, here called coppiced neurons due
to their trim appearance. These are equipped with dense
clusters of irregular spicules (Fig. 5C). The narrower
wedge-shaped zone contains palisades of small overlapping dendritic trees equipped with clusters of entwined
varicosities and spicules. These neurons, here termed rosette cells (Fig. 5J), are clearly distinguished from clawed
Kenyon cells that invade the same volume of neuropil
(Fig. 5G). Other clawed Kenyon cells extend through the
upper and lower layers of the bistratified inner basal ring
zone (Fig. 5E,F) and through the outer zone. Axons from
the basal ring terminate in what Mobbs termed layers 1
and 2 (Mobbs, 1982; shown here in Fig. 1A, right) of the
medial and vertical lobes (Fig. 5H,I). The present Golgi
study, therefore, confirms his conclusions, based mainly
on reduced silver studies, about the destination of basal
ring axons.
Representation of the calyx in the vertical
lobe by class I Kenyon cell axons
Single or clusters of Kenyon cell axons can occasionally
(in approximately 10% of preparations) be followed from
their origin in the calyces into the lobes (Figs. 2G–I, 5H,I).
Figure 6 summarizes tracings of groups of Kenyon cell
axons, in which cross-sections of the vertical lobes reveal
the location of their axons, allowing direct comparisons of
these locations, their origins in the calyces, and the stratification of the lobes revealed by immunostaining (Fig. 7).
Neurons from the basal ring zones terminate in the
uppermost layer of what Vowles (1955) and Mobbs (1982)
termed the alpha lobe (Figs. 5H,I, 6A,B,H,I). However, the
present study departs from Mobbs’s 1982 account with
respect to the representation of the lip and collar. The
present study demonstrates that the collar zones send
Fig. 4. Calyx subdivisions are determined by afferent distributions. A,B: The intermediate collar zone (inner neuropil margin to the
right) contains layers of afferent endings from the medulla (arrows;
med aff) and lobula (lob aff). In A, the wedge-shaped dendritic tree
derived from a cell body (k) extends through medulla and lobula
afferents. In B, a unistratified dendritic tree (from cell body k) has
dendrites only within the layer of medulla afferents. The lip (Li) in A
contains parts of three clawed Kenyon cells (top arrow) originating
from cell bodies (cb) outside the calyx. The bracketed area next to the
lip is the outer collar zone, which in B is shown with ascending
afferent endings of unknown origin. The lip zone is supplied by nu-
merous efferents from antennal lobe projection neurons. C: A spiny
Kenyon cell of the outer collar zone. The brackets indicate its zone in
A,B. This cell has a ball-like group of branches in the inner layer of the
calyx (wide bracket), which corresponds to a glomerulus of terminals
(arrow in E), with one of the ascending processes (arrowhead in E).
D: The lateral half of the basal ring, shown with the inner collar zone
(i Co) and the outer and inner basal ring zones (o BR, i BR), each
demarcated by their characteristic afferents. The outer and inner
neck zones (o Ne, i Ne) occur within the bundles of outgoing Kenyon
cell axons. Scale bar ⫽ 10 ␮m in A (applies in A–D).
Fig. 5. Kenyon cells of the calyx basal ring. A: Bistratified and
unistratified varicose spiny neurons. B: Blebbed chandelier cells provide two layers of dendritic processes (1, 2) and their specializations
(lower inset) are distinct from those of varicose spiny cells (upper
inset). C,D: Diffuse and dense spiny cells (C to the left, D) contribute
processes to the lower layer of the inner basal ring. C: Coppiced
neurons occupy the columnar outer ring zone (o BR), which is flanked
by a smaller wedge-shaped component (w). Dendritic specializations
of coppiced cells are distinguished from those of clawed Kenyon cells
(E,F), as shown in the inset at F (clawed, left inset; coppiced right
inset). J: The wedge component is supplied by overlapping rosette
cells, the dendrites of which (inset in J) are clearly distinguished from
the clawed Kenyon cells in the same zone (G). G: Clawed Kenyon cells
of the wedge component of the outer BR with cell bodies by the outer
calyx wall (white arrows) and axons (black double-arrow) leaving the
calyx through the outer neck zone. H: Ensembles of chandelier cells
sending axons into the pedunculus. Note their origin from cell bodies
within the central column of perikarya. I: Axons of chandelier cells
from H invade the uppermost strata of the vertical lobe. Scale bars ⫽
10 ␮m in A (applies in A–G), 2.5 ␮m in insets in B, 5 ␮m in insets in
F,J, 50 ␮m in H,I, 10 ␮m in J.
Fig. 6. Trajectories of Kenyon cell axons from the calyces to the
lobes. Each panel shows the mushroom body seen from the front with
its medial lobe extending to the right. The root of the vertical lobe is
seen in cross-section and is outlined in red. The cross-section of the
vertical lobe, with its divisions, is again shown beneath the relevant
mushroom body. Axon from groups of Kenyon cell dendrites, the
positions of which are indicated in the calyces, are shown in the
medial lobe and their branches into the vertical lobe are shown as blue
dots in the cross-section beneath. A,B: Kenyon cell dendrites in the
basal ring provide axons that terminate in the uppermost part of the
vertical lobe (also BR in H,I). C: Intermediate collar Kenyon cells
terminate in the middle of the lobe (also Co in D). Li, lip. E: Cell bodies
of clawed Kenyon cells (IIK) lying outside the calyx provide axons only
to the gamma lobe. F: Two groups of Kenyon cells in the collar. The
one closest to the lip ends deeper in the collar division than does the
other. G: Kenyon cells from the lip (Li, dark blue) of the lateral (L Ca)
and medial (M Ca) calyx, project to the lip division of the vertical lobe.
Axons from adjoining outer collar zones (o Co) terminate immediately
above them. H: Outer basal ring neurons (o BR, the coppiced and
rosette types) send their axons into the lower stratum of the basal ring
division. I: Projections from the two calyces remain discrete in the
lobes, as shown by axons from the lips of the lateral and medial
calyces (see also G).
Fig. 7. Proposed correspondence between immunoreactive strata
and distributions of Kenyon cell axons in the vertical and gamma
lobes. A: Distribution of Kenyon cell axons from the basal ring zone
(BR), collar zone (Co), lip zone (Li) and gamma lobe (␥, gray strata).
Golgi-impregnated axons from the basal ring segregate to at least
three levels (dark and light gray), as do those from the collar and lip
zones. For example, Golgi impregnations show chandelier cell axons
arranged above those of varicose spiny cells. B: Each level is represented by a set of thinner strata, defined by their immunoreactivity to
antisera against FMRFamide and gastrin cholecystokinin (GCCK).
Strata 1–5 in the basal ring division are proposed to correspond to the
five types of Golgi-impregnated class I Kenyon cells of the basal ring.
C: The calycal zones providing these cell types are numbered in the
cross-section of the hemicalyx. Golgi-impregnated axons traced from
the collar appear to segregate to three levels, which correspond to six
immunoreactive strata (B). Axons from the inner collar zone invade
stratum 6 and part of 7. Axons from the intermediate collar zone
occupy strata 7 to 9. Axons from the outer collar zone occupy strata 9,
10. The stratum labeled incerta sedis may receive axons from a
species of Kenyon cell so far unidentified in the calyx. Golgiimpregnated class I Kenyon cell axons from the calyx lip appear to
segregate to three levels in the vertical lobe’s lip division, which
contains two strata immunoreactive to both antisera and one stratum
immunonegative to both. The gamma lobe is occupied by axons of
clawed Kenyon cells and the endings of basal ring intrinsic neurons.
The latter intermingle with class II axons in the lowest stratum,
which is weakly immunoreactive to anti-FMRFamide. Clawed
Kenyon cell axons in the three strata of the gamma lobe possibly
represent the lip, collar, and basal ring.
16
axons only to the upper half of Mobbs’s layer 3 (Fig.
6C,D,F). Neurons from the lip region project through, and
are constrained within, the lower half of this layer (see
Figs. 2G–I, 6D,G–I), which was originally thought to receive axons from the collar not the lip (Mobbs, 1982, 1984).
Class I Kenyon cells from the lip, collar, and basal ring
omit the lower third to one quarter of the vertical lobe.
This includes Mobbs’s layers 4 – 6 (loc. cit. 1982 ref. Fig.
10), which he supposed received Kenyon cell axons from
the lip (compare Fig. 1A left, right). Instead, as will be
detailed below, these layers receive class II Kenyon cell
axons from the entire calyx.
Thus, the vertical lobe can be partitioned into two elements: a lower third, here termed the gamma lobe and an
upper two thirds, the vertical lobe sensu stricto. This lobe
is longitudinally partitioned into three major components:
the lip (Li), collar (Co), and basal ring (BR) divisions (Fig.
7A).
Within these divisions, axons further segregate into
much narrower strata. This further segregation of Kenyon
cell axons had been suggested by Mobbs (1982), who proposed that different types of Kenyon cells from the basal
ring segregate out into what silver-stained material suggested were two narrower layers. In the present account,
tracings of chandelier cell axons, which are the thinnest
Kenyon cell axons, show their termination in a stratum of
the BR adjacent to the upper margin of the vertical lobe
(Figs. 6A, 7A). Axons from spiny stratified neurons of the
basal ring appear to end more deeply within the BR division (Figs. 6B, 7A). Similarly, axons from the lip zone
appear to segregate to approximately three strata in the
Li division (Fig. 6G,I), possibly reflecting the three types of
spiny Kenyon cell dendritic trees identified in the lip (see
Fig. 2). Axons from the narrow outer collar zone, which is
adjacent to the lip, terminate just above the upper margin
of the Li division (Fig. 6G), whereas axons from the intermediate collar zone and inner collar zone extend through
more superficial levels of the Co division (Fig. 6D,F).
This study supports two of Mobbs’s contentions. The
first was that axon projections from class I Kenyon cells
are orderly, meaning that, when neighboring bundles of
axons are followed from the calyx into the vertical lobe,
they maintain their relative positions to each other (Fig.
2H,I). As Mobbs (1982) originally proposed, bundles of
axons from the calyx transform the concentric arrangements of calycal zones into stratified arrangements of
Kenyon cell axons in the lobes. The second was that,
within each layer, Kenyon cells further segregate into
thinner strata according to their morphological type in the
calyx, a feature here confirmed for the BR and Co divisions.
The crucial difference between this study and that of
Mobbs (1982) is that the ␣ lobe (vertical lobe sensu lato) is
composed of two distinct entities: the vertical lobe proper,
formed by the axons of class I Kenyon cells, and the
anatomically distinct gamma lobe, which is seamlessly
fused to the vertical lobe above it and contains the axons
of the clawed class II Kenyon cells. The medial lobe, which
tapers toward the midline and is only two thirds the width
of the vertical lobe, comprises three divisions that receive
axon tributaries of class I Kenyon cells from the lip, collar,
and basal ring.
N.J. STRAUSFELD
Relationships of Kenyon cell axons in the
vertical and gamma lobes to
immunoreactive strata
Antisera raised against FMRFamide and GCCK, together reveal 17 strata (Strausfeld et al., 2000). The crosssectioned appearance of these strata is schematized in
Figure 7B, which compares FMRFamide and GCCK immunoreactivity with the distribution of Golgiimpregnated Kenyon cell axons (Fig. 7A) that were followed from the calyces into the lobes. Kenyon cell axons
from the basal ring define at least three levels of the BR
division. Three levels in the Co division are defined by
axons traced from the collar, of which the narrowest lower
layer represents the outer collar zone. Three levels of
Kenyon cell axons in the Li division are defined by axons
followed from the lip. Two lower strata, which constitute
the gamma lobe, receive clawed Kenyon cell axons from
the entire calyx.
The BR, Co, and Li divisions, as defined by the terminals of class I Kenyon cells, actually comprise more strata
than suggested by the observed segregation of Golgiimpregnated axons. That Golgi-impregnated axons cannot
always be confidently ascribed to an immunoreactive stratum is due to the observational difficulty of relating single
axons, amongst other axons, to the corresponding dendritic trees, which are often stained with several other
overlapping trees. Also, clusters of Kenyon cells are often
impregnated athwart adjacent zones so that it is impossible to determine the zonal affinity of their axons.
Nevertheless, it is possible to suggest which zones in the
calyx, and thus which species of Kenyon cell, match which
immunostained strata in the lobes. Ensembles of class I
Kenyon cell dendrites in different zones of the calyx have
characteristic affinities to anti-FMRFamide and antiGCCK (Strausfeld et al., 2000). Together with the characteristic profiles of dendritic trees, these features define the
finer nuances of calycal zonation and suggest a partitioning into at least 13 zones (Fig. 7C). As described by Strausfeld et al. (2000), these two antisera distinguish many
strata in the lobes, and it is suggested here that each of
these correspond to a particular type of Kenyon cell within
a particular zone of the calyx.
For example, the BR division consists of a broad antiGCCK–immunopositive stratum above an anti-GCCKimmunonegative one. The BR division is also characterized by a superficial stratum that is weakly antiFMRFamide immunopositive, above a narrower and more
strongly stained FMRFamide layer that is succeeded by
an immunonegative stratum. Immediately beneath this
are two thin strata having different levels of antiFMRFamide immunoreactivity. Do these five strata correspond to the five types of class I Kenyon cells identified
in the basal ring? Golgi studies speak for this: chandelier
cell axons from the inner basal ring zone have been traced
to stratum 1. Axons from spiny Kenyon cells have been
traced to strata 2 and 3. Coppiced and rosette cells appear
to send axons to strata 4 and 5.
The collar division of the vertical lobe has five immunoreactive strata, of which the uppermost (stratum 6 in Fig.
7B) is intensely immunoreactive to both anti-FMRFamide
and anti-GCCK and has been ascribed to the inner collar
zone. Tracing Golgi-impregnated axons shows that the
outer collar zone is represented in the lowest part of this
division, corresponding to immunostained strata 9, 10. It
VERTICAL AND GAMMA LOBES IN HONEY BEES
is suggested that these represent the segregation of encrusted and spiny Kenyon cells. Spiny Kenyon cells of the
intermediate collar zone end at the same levels as immunostained strata 7 and 8. A layer labeled incerta sedis (Fig.
7B) suggests a sixth species of Kenyon cell in the collar’s
intermediate zone that has escaped identification. The lip
division has three immunoreactive strata. It is suggested
that these correspond to a segregation amongst the morphological types of class I Kenyon cell identified in the lip.
Gamma lobe: its supply by class II
(clawed) Kenyon cells
The BR, Co, and Li divisions lie above the gamma lobe,
itself divided into three immunoreactive strata (Fig. 7B):
(1) a broad stratum immunopositive to anti-GCCK; (2) a
narrow immunonegative stratum; and (3) a broad stratum
weakly immunopositive to anti-FMRFamide. These strata
are supplied by the axons of clawed Kenyon cells from the
whole calyx.
Clawed Kenyon cells have been described for the mushroom body of the fly Musca domestica (Strausfeld, 1976)
and were identified in the honey bee by Mobbs (1982; loc.
cit. Fig. 28a– c). Clawed Kenyon cells have also been described from Drosophila melanogaster (Lee et al., 1999),
Acheta domesticus (Schürmann, 1970), and Periplaneta
americana (Strausfeld and Li, 1999b). In the honey bee,
clawed Kenyon cells have been identified within the lip
(Figs. 2A, 4A), collar (Fig. 3B,F,G), and basal ring (Fig.
5E–G).
The axons of clawed Kenyon cells lie closest to the inner
wall of the calyx neuropil (Fig. 2A). Ventrally, at the base
of each calyx, clawed Kenyon cell axons collect into four to
six broad swathes. In the pedunculus, these swathes align
and merge to provide six to eight sheets of axons flanked
by two narrower bundles (Fig. 8A). This curtain of axons
plunges obliquely caudally through the vertical lobe into
the gamma lobe (Fig. 8B; see also Fig. 1H). The axons of
clawed Kenyon cells are smooth along most of their length,
suggesting that they do not provide synapses until they
reach the gamma lobe where their terminals give rise to
varicosities and short stubby collaterals. This morphology
distinguishes them from the axons of class I Kenyon cells,
which give rise to spines and varicosities throughout their
projection in the vertical and medial lobes.
Representation of the neck and basal ring
in the gamma lobe by basal ring
intrinsic neurons
The gamma lobe receives a second supply of axon-like
processes. These processes are supplied by a newly identified class of intrinsic neurons (called basal ring intrinsics) that represent the basal ring and the necks of the two
pedunculi. Each process is two to three times the diameter
of a clawed Kenyon cell axon (compare Fig. 8C,D). When
stained en masse from their origin at the pedunculus neck,
these processes follow the same trajectories into the
gamma lobe as axons of clawed Kenyon cells (Fig. 8B).
Basal ring intrinsic neurons supply the calyces from a
cluster of approximately 20 large cell bodies (18 –20 ␮m
diameter) that are distant from the mushroom bodies,
located within the ventrolateral protocerebral rind between the protocerebrum and lobula (Fig. 9A). Each cell
body provides a wide-diameter neurite that extends obliquely centrally and through the protocerebral neuropils
17
to reach the front of the calyces, just beneath where their
basal rings join their respective pedunculus necks. Beneath the calyces, the neurites expand to approximately
four times their original diameter to give rise to branched
processes that extend tangentially within the pedunculus
neck (Figs. 9A, 10C). These processes send ascending varicose branches into the outer basal ring zones and into the
deep layer of the inner basal ring zone (Figs. 9A, 10A,C).
Figure 9A shows a camera lucida reconstruction of these
distinctive sets of processes arising from the swollen neurites of two intrinsic neurons. In this example, the two
cells innervate parts of the neck of the lateral calyx, and
supply processes to half the basal ring neuropil of the
medial calyx. However, some basal ring intrinsic neurons
supply the whole basal ring of one calyx and the neck of
the other calyx. Other cells can supply both of these levels
in a single calyx. Figure 10A illustrates four basal ring
intrinsic neurons, and Figure 10C, a single neuron that
provides processes to the neck and ascending branches to
the basal ring of the medial calyx. Irrespective of these
typical variations in branching pattern, the whole population of basal ring intrinsic neurons together provides a
uniform bilayered system of branches within the basal
ring and pedunculus neck.
Each neuron provides between 8 and 30 axon-like processes, which are decorated with short branches and
pinhead-like spines down much of their length (Figs. 9A,
10A–C). Entirely omitting the medial lobe (Fig. 10B),
these processes terminate in the gamma lobe as tangles of
smooth but slightly swollen endings. Thus, like the axons
of clawed Kenyon cells, these processes are restricted to
the pedunculus and vertical lobe. Unlike the axons of
clawed Kenyon cells, their pin-like specializations suggest
that they have functional contacts along most of their
length.
Mass impregnations of basal ring intrinsic neurons illustrate the curtain-like arrangements of their “axons.”
These closely match the axon projections of class II
Kenyon cells (compare Fig. 8A,B). The present study,
thus, resolves a puzzling feature of Bodian and ethyl
gallate-stained mushroom bodies, which always show an
architectural entity (Fig. 10D), composed of vaults and
columns, that was thought to represent that portion of the
pedunculus in which Kenyon cell axons bifurcated into the
two lobes. However, the columns extending from just beneath where the two pedunculus necks converge (boxed in
Fig. 10D) correspond to bundles of axons of the basal ring
intrinsic cells (boxed in Fig. 10A) that plunge through
the vertical lobe to reach the gamma lobe. In certain
hymenopterans, such as the ant Pogonomyrmex, Bodian
preparations clearly reveal these processes as converging
onto the dendrites of efferent neurons that are unique to
the gamma lobe (Fig. 10D). Other structures seen in reduced silver sections, but which have previously defied
explanation, also correspond to elements of basal ring
intrinsic cells. These structures include the shallow
v-shaped and intensely argyrophilic bands that extend
across the basal ring (bracketed in Fig. 10D), which represent the most distal processes of these intrinsic neurons
(arrowed bracket in Fig. 10C).
Neck intrinsic neurons
Although it might seem impossible that more neural
processes could fit into an already densely packed system
of Kenyon cell dendrites, axons, and basal ring fibers,
18
N.J. STRAUSFELD
Fig. 8. Projections of gamma lobe intrinsic neurons. A: Mass impregnation of clawed Kenyon cell axons to the gamma lobe. B: Mass
impregnation of basal ring intrinsic neuron “axons” to the gamma lobe
(in all four panels, the margin between the gamma lobe and vertical
lobe is shown arrowed with dashed lines). C: Cross-section of the
gamma ⫹ vertical lobe showing relative locations of clawed Kenyon
cell axons (II) and axons from the lip (Li) and collar (Co). D: Crosssections of axons from basal ring intrinsic neurons shown at the same
level as C. Scale bar ⫽ 50 ␮m in A (applies to A–D).
there is yet another species of intrinsic neuron that adds
to the crowd. These cells, which are illustrated in Figure
9B, derive from a cluster of 8 –10 medium-sized cell bodies
that lie beneath the outer half of the lateral calyx. Each
cell body provides a stout neurite that extends anteriorly
beneath the two calyces, where to each it gives rise to an
elaborate system of processes associated with their pedunculus necks and the basal rings.
Each neurite provides one system of collaterals that
then supply many slender processes extending tangentially within a deep layer of the inner basal ring zone. The
collaterals providing these tangential elements them-
Fig. 9. “Exotic” intrinsic neurons. A: Camera lucida reconstruction
of two basal ring intrinsic neurons showing their spiny axons ending
in the gamma lobe (arrows). Other processes invade the neck (Ne) and
basal ring neuropils (see text). B: Camera lucida reconstruction of
four neck (Ne) intrinsic neurons with dendrite-like processes in the
neck and upper layer of the inner basal ring zone (BR) and recurrent
terminal-like processes in outer basal ring zones. Arrows indicate
branches of four axons that extend forward along the lateral margin
of the gamma lobe. L Ca, lateral calyx; M, medial lobe. Scale bar ⫽ 50
␮m in A (applies to A,B).
Fig. 10. Basal ring intrinsic neurons. A: Neurites provide a layer
of varicose processes to the basal ring (BR) and filamentous and spiny
processes to the pedunculus neck (Ne). These give rise to axon-like
extensions that have short smooth segments (asterisk) that converge
to provide columns of spiny axons (box). B: These columns project
through vertical lobe divisions (BR, collar [Co], lip [Li]) to the gamma
lobe (␥) but not to the medial lobe (also termed ␤). C: Varicose
processes extend into parts of the neck as well as the basal ring,
suggesting complex synaptic relationships at these levels. Forward
projecting “axons” give rise to short branches and spines (arrows).
D: Columnar structure (boxed) seen in the pedunculus and vertical
lobe in a species of ant (Pogonomyrmex) correspond to the axons of
basal ring intrinsic neurons (see box in A). Also, dark argyrophilic
bands in the pedunculus necks (arrowed bracket) correspond to the
outer processes of basal ring intrinsic neurons (arrowed bracket in C).
This reduced silver preparation also shows the type 5 gamma lobe
efferent, with its wide-diameter axon (eff). Scale bars ⫽ 50 ␮m in A
(applies to A,B), 50 ␮m in C, 20 ␮m in D.
VERTICAL AND GAMMA LOBES IN HONEY BEES
21
Fig. 11. Structural features of the gamma lobe. A–C: Glial cell
distribution. A: Rhodamine dextran staining of glial cells. B: Bodian
reduced silver cross-section of the vertical lobe. C: Negative image of
fusion of two successive Bodian-stained sections in which nuclei of
glial somata have been selectively false colored. Note the higher
density of glial cells in the gamma lobe and around the lateral margin
of the vertical lobe, except for its BR division. D–F: Four types of
endings within the gamma lobe exemplify the lobe’s supply by a
variety of protocerebral afferent neurons. The depth of the gamma
lobe (65–75 ␮m, depending on brain size) is indicated by brackets.
selves originate from larger branches of the neurite, from
which arise two further systems of processes. The first of
these comprise ramifying branches equipped with varicose swellings and spines. These branches extend from the
sides of each pedunculus neck into the neck where they
intersect arrays of Kenyon cell axons (Fig. 9B). The second
system of processes consists of large recurrent collaterals
that ascend from the main neurites to invade the lateral
zones of the basal ring, where they contribute to an arabesque of tuberous specializations suggestive of presynaptic specializations (Fig. 9B).
These elaborate nerve cells are disposed to derive
their inputs from Kenyon cell axons within the pedunculus necks. Their more distal arrangements suggest
that they provide recurrent terminals back into the
basal ring zones of the same calyces. However, because
the neurites extending between the two calyces are
large, it is possible that these intrinsic neurons serve to
synchronize activity of both calyces. Each neck intrinsic
neuron also sends a single slender process that extends
from between the lateral and medial calyces forward
alongside the lateral edge of the vertical lobe. These
axons and their endings follow the trajectory of one of
the ascending axons from the type 4 gamma lobe efferent neuron (see below).
Gamma lobe architecture is distinct from
the divisions of the vertical lobe
The gamma lobe is distinguished not only by its supply
from the calyces. It is also distinct from the vertical lobe by
virtue of its glial cell arrangements and by the arrangements of afferent terminals and efferent neuron dendrites
that reciprocally link the gamma lobe with characteristic
regions of the protocerebrum.
Glial cell profiles have previously been revealed in the
vertical lobe by using ethyl gallate or lectin staining
(Hähnlein et al., 1996). Selective staining of glial cells and
their processes is also revealed by locally flooding extracellular space with low molecular weight rhodamine dextran. A striking difference between the gamma lobe and
22
N.J. STRAUSFELD
Fig. 12. Gamma lobe afferent neurons. A: Beaded presynaptic specializations within layer 1 of the
gamma lobe. Ascending processes follow the descending trajectories of the bundles of axons from gamma
lobe intrinsic neurons (see Fig. 9A). B: Dense varicose terminals within layers 2, 3 of the gamma lobe.
Arrows in A,B indicate upper limit of the gamma lobe. Scale bar ⫽ 50 ␮m in A (applies to A,B).
divisions of the vertical lobe is the density of gamma lobe
glial processes, which form three distinct strata corresponding to the three levels revealed by immunocytology
(Figs. 7, 11A). In contrast, the vertical lobe has sparse
glial processes that delineate its three main divisions.
Numerous glial cell bodies (Fig. 11B,C) line the lower
margins of the gamma lobe and each side of the lip and
collar divisions of the vertical lobe. Numerous glial processes occur with axons belonging to afferent neurons
entering the gamma lobe and with axons of gamma lobe
efferent neurons extending around the lobe’s lateral margin.
Gamma lobe afferent neurons
An often repeated statement in the literature is that the
calyces are the primary input regions of the mushroom
bodies and the lobes the primary output regions (Menzel
et al., 1994, 2001). However, this is unlikely to be correct
because of the phylogenetic precedence in paleopteran
insects, such as odonates and thysanurans. In these taxa,
mushroom bodies lack calyces that are supplied by firstorder sensory interneurons. Nevertheless their mushroom
bodies have large vertical and medial lobes that are provided by many thousands of axon-like intrinsic neurons
(Strausfeld et al., 1998). The same is seen in some aquatic
neopteran insects that are secondarily anosmic and lack
antennal lobes and calyces. Thus, there are many taxa
that possess mushroom body lobes, formed by intrinsic
neuron axons that lack distal dendritic trees. Interestingly, the fibroarchitecture of lobes in aquatic species is
barely distinguishable from the lobes of closely related
terrestrial species that posses calyces (Strausfeld et al.,
1998). Because calyxless mushroom bodies do not operate
in an afferent vacuum, they must receive their primary
(and phylogenetically oldest) inputs to their lobes.
It is not surprising, therefore, that neopteran insects
such as the honey bee have lobes that are extravagantly
invaded by the terminals of afferent neurons that have
their dendritic trees in various regions of the protocere-
brum. Thus, as shown by electron microscopy (Schürmann, 1970; Strausfeld and Li, 1999b), the lobes support
complex synaptic interactions between afferents, Kenyon
cell processes, and dendrites of efferent neurons. This
finding suggests that, instead of the calyces being the
mushroom bodies’ main input neuropil, the role of presynaptic afferents onto class I Kenyon cells in the calyces
might be to modulate local circuits that are provided by
Kenyon cell axons interposed between afferent endings
and efferent dendrites in the lobes.
The gamma lobe is no exception to this organization.
The densely packed arbors of at least 20 afferent neurons,
but possibly twice that number, supply the gamma lobe
from the protocerebrum. Examples of these diverse endings are shown in Fig. 11D–F, Fig. 12A,B, and Fig. 13B,E.
Most of these neurons derive from clusters of cell bodies
lying behind the antennal lobes, flanking the esophageal
foramen (Fig. 12A). Others have cell bodies that lie superficially in the anterior protocerebral rind (Fig. 12B). Their
dendrites occupy a variety of protocerebral regions, including regions lateral to the gamma lobe (Fig. 12A) and
immediately beneath it (Fig. 12B). Afferent terminals reflect the layering of the gamma lobe. They form dense
tangles of narrow rhizome-like processes (Fig. 11D), tufts
of beaded or varicose endings (Fig. 11E), or layers of varicose endings, as shown in Figs. 11F and 13B. These
arrangements match either the layers of intrinsic neuron
terminals from the calyces (compare Fig. 11D with Fig.
8C), or they reflect the main branches of gamma lobe
efferent neurons, or their dendritic fields (compare Figs.
11E and 12A with 13A; Figs. 12B and 13E with 14C).
Some gamma lobe afferents provide elaborate systems of
climbing fibers that appear to follow back the vaulted
trajectories of axon bundles from clawed Kenyon cells and
basal ring intrinsic neurons (compare Fig. 12A and 8A,B).
Gamma lobe efferent neurons
Reduced silver sections (Fig. 10D) illustrate the basic
relationship between gamma lobe intrinsic neurons and
VERTICAL AND GAMMA LOBES IN HONEY BEES
Fig. 13. Structural features of the gamma lobe. Lateral is to the
left in all panels except C. The basal ring (BR), collar (Co), and lip (Li)
divisions of the vertical lobe and the gamma (␥) lobe are bracketed in
A–F. A: Dendritic tree of a type 5 gamma lobe efferent neuron immediately beneath the unistratified tree of a recurrent (feedback) neuron
within the lip division. B: A bundle of axons entering the lower
margin of the gamma lobe provides layered varicose afferent processes at the level of the type 5 gamma efferent. C: Stratified efferent
dendritic tree associated with the lip, collar, and lowermost strata
(3-5, Fig. 7B) of the basal ring division. D: Type 1 gamma efferents,
23
lying deep in the gamma lobe, shown with a unistratified tree of a
recurrent efferent neuron that links the lip division of the vertical lobe
to the lip zones of the calyces. E: Gamma lobe afferent terminals at
the level of the type 3 efferents. F: Broad dendritic tree through the lip
division (see Fig. 15). G,H: The vertical/gamma lobe showing the
match between afferent and efferent neurons and immunoreactive
strata: anti-FMRFamide in G, and anti-GCCK in H. I: Bodian-stained
vertical/gamma lobe showing large argyrophilic cross-sections (arrows) of gamma lobe efferent neurons. Width of gamma lobe brackets ⫽ 65–75 ␮m.
Fig. 14. Morphologies of gamma lobe efferent neurons. A: Comparison of type 5 (left) and type 1a,b (right) efferents to show their
convergent terminal domains in the inferior lateral protocerebrum
(shaded areas). Note the bistratified arrangement of type 1 dendritic
trees (arrows). The bracket indicates the width of gamma lobe. ACT,
antennocerebral tract. B: Types 1a,b are compared here with the type
4 gamma efferent (right) to show their different relationships with
inferior protocerebral neuropils. However, both send axon collaterals
around the vertical lobe into concentric neuropils of the medial pro-
tocerebrum. The type 4 gamma efferent also provides a system of axon
terminals in the superior lateral protocerebrum, near the lateral
extension of the antennocerebral tract, shown in cross-section (shaded
area). C: Type 3 (left) and type 2 (right) gamma efferent neurons
comparing their terminal domains. Like the type 1a,b and type 4
efferents, their dendritic trees derive from contralateral cell bodies.
Like type 4, types 2 and 3 provide groups of axon terminals in the
superior protocerebrum, near the lateral extension of the antennocerebral tract. Scale bars ⫽ 50 ␮m in A–C.
VERTICAL AND GAMMA LOBES IN HONEY BEES
gamma lobe outputs. Axon-like processes of intrinsic neurons (clawed Kenyon cells and basal ring intrinsic neurons) all terminate amongst a set of prominent efferent
neurons, here numbered type 1–5 from the tip of the lobe
(Fig. 14). The large-diameter axon of each of these neurons
provides discrete terminal domains consisting of axon collaterals and their branches, which are decorated with
beaded swellings (types 1a,b) or with large and irregular
varicosities (types 2–5). Their domains occupy characteristic regions of neuropil in the frontal and lateromedial
protocerebrum.
Figures 13A,D and 14 illustrate efferents of the gamma
lobe. These are compared with other efferent neurons in or
shared by the Li division adjacent to the gamma lobe
(dendrites at level Li in Fig. 13A; dendrites in C; at level
Li in D; and dendrites in F). Figure 15A,B shows reconstructions of two Li division efferents. Gamma lobe efferents have stratified (Fig. 13A,D) or diffuse arrangements
of dendrites (Fig. 14B, right, C). The dendritic trees of
gamma lobe efferent neurons are the densest in the mushroom bodies and, with the exception of the Pe-1 neuron
(see Rybak and Menzel, 1998), arise from the largest diameter trunks and axons. Their dendritic trees are also in
register with the terminal fields of afferents arriving at
the gamma lobe from protocerebral regions. This finding is
exemplified in Figure 13A,B, which show, respectively, an
efferent dendritic tree in the upper stratum of the gamma
lobe and a bundle of afferent endings that invades the
same domain. Likewise the pair of efferent neurons shown
in Figure 13D, which betray the layered organization of
the gamma lobe, match the domains of afferent endings
shown in Figure 11D,F.
Five morphological types of gamma efferent neurons
can be identified, four of which are arranged successively
from the tip of the gamma lobe inward. Their major trunks
characteristically penetrate the gamma lobe through its
lower margin as do some efferents of the Li division (Fig.
15A,B). This finding contrasts with recurrent efferent
neurons of the Li and Co divisions, whose axons extend
from the lateral margin of the vertical lobe (Fig. 13A,D), or
efferents from the Co division (Fig. 13C).
Type 1 gamma efferent neurons occur as a pair in each
gamma lobe (type 1a,b, Figs. 13D, 14B, left) and reside at
the lobe’s tip. Their dendritic trees, which consist of many
straight, extremely thin branches, equipped with minute
thorns, derive from an axon that hugs the front margin of
the lobe and extends medially around it (Fig. 14B, left).
The two axons branch many times, providing collaterals
that fan out toward the brain’s midline. These branches
are decorated with small oval or irregular swellings suggestive of presynaptic sites. Collaterals also invade a volume of inferior medial protocerebral neuropil, which additionally recruits axon collaterals from two other gamma
lobe outputs, the type 4 (Fig. 14B, right) and type 5 efferent neurons (Figs. 13A, 14A, left).
The next in the series of efferent neurons are the types
2 and 3 efferents, shown in Figure 14C. Their dendritic
trees consist of thousands of twig-like processes that extend from recurved dendritic trunks that converge to stout
(10- to 12-␮m diameter) axons. Although there are subtle
differences in the dendritic branching patterns of these
cells (compare Fig. 14C, left and right), these alone do not
justify classifying the neurons as separate types. However, they are distinguished on the basis of their axonal
targets. Axon collaterals of the type 2 neuron (Fig. 14C,
25
right) extend laterally from the gamma lobe, branching
into regions of the frontal-lateral protocerebrum, approximately 50 ␮m behind the upper border of the anterior
optic tubercle. These terminal domains overlap, but are
distinct from, the terminal domains of the type 3 gamma
efferent, which has two separate fields in the frontal protocerebrum, one abutting the anterior optic tubercle. The
type 2 and 3 neurons each send an axon into the superior
protocerebrum where each provides short branches
around and amongst axons of antennal lobe projection
neurons that extend to the lateral protocerebrum by
means of the lateral projection of the medial antennocerebral tract. The type 2 and 3 efferent neurons show subtle
but diagnostic differences at this level also (Fig. 14C).
The fourth species of gamma lobe efferent, and the last
in the distoproximal stack of efferent dendritic trees, has
a similarly dense appearance due to its composition of
many thousands of postsynaptic “twigs.” The border of the
tree appears to have an indented outline (Fig. 14B, right),
which is due to the dendrites bunching amongst incoming
bundles of clawed Kenyon cell axons. The type 3 efferent
also has a similar but less ragged outline. This is due to its
relatively forward location in the lobe, where most axon
bundles from the calyx have already spread out laterally.
The axon of the type 4 efferent sends one collateral
medially around the vertical lobe to invade medial protocerebral neuropils. Like the axons of type 2 and 3 efferent
neurons, the type 4 neuron sends an unbranched axon
around the lateral edge of the vertical lobe as far as the
margin of the superior protocerebrum. There, it terminates as a crown of processes that extend into the lateral
extension of the inner antennocerebral tract. Its thick
main axon provides many varicose collaterals into the
medial inferior and lateral inferior protocerebrum (Fig.
14B, right). Although these terminal domains are separate from those of type 2 and 3 neurons, with these other
types of gamma efferents, the type 4 neuron contributes
presynaptic inputs to frontal inferior medial and inferior
lateral neuropils. These areas of the protocerebrum are
distinct from areas receiving vertical lobe output neurons
(see Fig. 15A,B).
The dendrites of a fifth species of gamma efferent (type
5) occupy stratum 1 of the gamma lobe (Figs. 13A, 14A,
left). The dendrites reside above those of the type 2 efferent, slightly overlapping its dendritic branches at the
same position along the lobe. The axon trajectory of this
cell is unique in that it crosses the brain’s midline. The
axon provides mainly unilateral collaterals that supply
the inferior medial protocerebrum overlapping some of the
axon collaterals from the pair of type 1 efferents (Fig. 14A,
right). The type 5 neuron also provides a second axonal
branch that extends around the lateral edge of the vertical
lobe where its subsequent tributaries coincide with those
belonging to forward projecting axons of the neck intrinsic
neurons (compare Fig. 14A, left with Fig. 9B).
Except for the type 5 gamma efferent, whose ipsilateral
cell body is located in the rind beneath the vertical lobe,
the other gamma lobe efferent neurons originate from
contralateral cell bodies lying superficially in frontal rind
of the medial protocerebrum.
As mentioned above, these gamma lobe efferents differ
markedly from outputs from the vertical lobe’s Li, Co, and
BR divisions, whose efferent neurons each has its own
characteristic target area. For example, efferents from the
Li division (Fig. 13F,H) mainly terminate in regions of the
Fig. 15. Lip division efferent neurons have characteristic terminals in the protocerebrum. Efferent neurons are classified according
to their location in the lobe, their dendritic morphology, and their
destinations. These examples show two lip efferents that target different neuropils within the superior lateral (A) and superior medial
(B) protocerebrum (see also Fig. 1C). The arrowed shaded area in A
denotes the terminal domain of the cell in B. L Ca, lateral calyx; ACT,
antennocerebral tract; A Lo, antennal lobe. Scale bar ⫽ 50 ␮m in A
(applies to A,B).
VERTICAL AND GAMMA LOBES IN HONEY BEES
27
Fig. 16. Is the Pe-1 associated with the gamma lobe or its intrinsic
supply? Afferent columns and the Pe-1 dendritic tree are compared.
Columnar groups of afferent climbing fibers (left) originate from a
dendritic tree in the inferior medial protocerebrum (I M Pr), from
which originate other afferent neurons supplying the gamma lobe (see
Fig. 12). The spacing (indicated by arrows) of these columnar afferents matches that of the columnar dendrites of the Pe-1 efferent
neuron (right). Together, these columnar ensembles are similar to the
spacing amongst groups of class II Kenyon cell axons and the processes of basal ring intrinsic neurons extending obliquely across the
vertical lobe and into the gamma lobe (see Fig. 8A,B). These arrangements suggest a possible relationship between the Pe-1 neuron and
intrinsic neurons supplying the gamma lobe. ACT, antennocerebral
tract; L Pr, lateral protocerebrum. Scale bar ⫽ 50 ␮m.
superior lateral and medial protocerebrum. However, as
shown in Figure 15A,B, each type of Li division efferent
has its own characteristic terminal domain. Recurrent
efferent neurons (as defined by Gronenberg, 1987;
Grünewald, 1999), which have uni- or multistratified dendrites in the Li, Co, and BR divisions, send axons back to
the calyces where they end in corresponding calycal zones.
One other type of efferent neuron from the vertical lobe
requires special consideration. This is the unique neuron
Pe-1, which was first described by Mauelshagen (1993)
and then by Rybak and Menzel (1998). Pe-1 possesses a
large tristratified dendritic tree that extends across the
entire vertical lobe. The main branches of this tree are
collected into five to six columnar groups, such that there
are discontinuities across its strata. Although classified by
Mauelshagen (1993) as an efferent neuron of the pedunculus (thus its notation Pe), Pe-1’s dendrites in the lobe
originate from a wide flattened trunk that penetrates the
front of the gamma lobe, some 200 ␮m forward of the
pedunculus and immediately behind the type 4 gamma
lobe efferent neuron. Pe-1 has a layered dendritic organization whose processes are arranged as three strata. Each
arises from bundles of upright dendritic stems that divide
into smaller and approximately parallel branchlets.
Pe-1 originates circuitously from an ipsilateral cell body
lying superficially and medially within the frontal rind of
the protocerebrum. Its cell body fiber crosses the brain’s
midline and then thickens, extending some distance
alongside the inner (medial) edge of the contralateral vertical lobe. There it provides several short dendrites into
the adjacent protocerebral neuropil. The thickened neurite next projects back across the brain’s midline to reach
the lower perimeter of the gamma lobe. During its course
across the protocerebrum, the neurite provides several
dendritic branches into the inferior medial protocerebrum.
Thus, although originating from a cell body that is on the
same side of the brain as its dendritic tree, the Pe-1
neuron first provides several spiny and tapering dendritic
processes that presumably receive inputs from sites in the
contralateral protocerebrum immediately adjacent to the
contralateral mushroom body.
The size of this neuron’s axon, and its entry point into
the lobe, is similar to gamma lobe efferents. But is it
functionally distinct from the gamma lobe? Its first layer
of dendrites appears to reside within the lip division of the
vertical lobe. However, spacing amongst Pe-1’s bundled
ascending dendritic branches is reminiscent of that of
descending bundles of axons belonging to basal ring intrinsic neurons and clawed Kenyon cells (compare Fig. 16,
right, and Figs. 8A,B, 9A). A functional relationship between Pe-1 and the gamma lobe is further suggested by an
afferent neuron, which provides a system of climbing fibers that enwraps the bundled ascending dendritic
branches of Pe-1 (Fig. 16, left). The dendrites of this afferent occur in protocerebral regions receiving terminal
branches of gamma lobe efferent neurons.
28
N.J. STRAUSFELD
DISCUSSION
The present account demonstrates that the honey bee
mushroom body is equipped with a gamma lobe. This lobe
is distinct from other components in that it is supplied by
its own subset of intrinsic neurons, receives its own distinct afferents, and provides a system of efferent neurons
that target characteristic regions of the protocerebrum. Its
clawed Kenyon cells are the first to differentiate and, thus,
correspond to clawed Kenyon cells in other taxa. The following sections discuss earlier studies and how the
present results differ from them. The discussion also considers reasons for homologizing the honey bee gamma
lobe, which is part of the vertical lobe, with the gamma
lobe of the fruit fly, which is part of the medial lobe.
Discrepancies with previous studies
The identification of a gamma lobe in the honey bee
provides a crucial difference between Mobbs’s 1982 account and the present study. Mobbs (1982, 1985) identified three major anatomical divisions across what he
termed the alpha and beta lobes (vertical and medial
lobes; see Fig. 1A), which he further divided into six (sic,
seven) narrower bands (numbered 1– 6: Mobbs, 1982; loc.
cit. Figs. 10, 49). Mobbs assigned reduced silver-stained
axons from the calyx lips to bands 4, 5, and 6 in the lower
third of the alpha lobe. He assigned axons from the calyx
collars to band 3, and axons from the basal ring zones to
bands 1 and 2 in the upper quarter of the alpha lobe. The
present results support only the relationship between the
basal ring and Mobbs’s bands 1 and 2.
These discrepancies are significant because much emphasis has been placed on the honey bee as a model
system for learning and memory research (see Menzel et
al., 1994, 2001). These studies have often relied on
Mobbs’s 1982 account, which until now has been the only
detailed study of the relation between the lobes and calyces. However, reduced silver techniques are inadequate
for tracing single axons (Strausfeld, 1976), as was strongly
cautioned by Mobbs in his 1985 account of the honey bee
brain.
Thus, studies predicated on Mobbs’s original 1982 description require critical reassessment. Such studies include observations of feedback neurons (Gronenberg,
1987; Grünewald, 1999), the immunocytological localization of neuromodulators (Bicker et al., 1985; Schäfer et al.,
1988; Bicker, 1991; Strausfeld et al., 2000: the last reassessed here), and second messenger components (Menzel
et al., 1994; Müller, 1997, 1999). For example, an antibody
directed against phosphokinase A (PKA) most intensely
stains the lower third of the vertical lobe (Müller, 1997),
which was interpreted to represent the calyx lip zone and,
thus by inference, to be an olfactory component of the lobe.
However, it is shown here that the PKA-stained division
corresponds to the gamma lobe, supplied by class II
Kenyon cells representing the entire calyx.
Descriptions of the reciprocal relationships between the
lobes and zones in the calyces also refer to Mobbs’s (1982)
distinction of six bands dividing the vertical lobe and their
assumed relationships with the calyces (Grünewald, 1999;
loc. cit. Fig. 2B). Reciprocal connections are provided by
“feedback” neurons (Gronenberg, 1987), which have been
described as having dendrites arborizing through the middle division of the lobes (Mobbs’s band 3), supposedly
representing the collar zone (Bicker et al., 1985; Gronen-
berg, 1987; Grünewald, 1999). However, the present study
shows that this middle division is composed of two discrete
components, the Li and Co divisions, which are supplied,
respectively, by the lip and collar. Feedback neurons
whose dendrites are restricted to the Li division supply
recurrent terminals into the lip zone. Feedback neurons
whose dendrites are situated in the Co division of the
vertical lobe provide terminals in the calyx collar zone.
Thus, feedback neurons terminate in those calycal zones
supplying class I Kenyon cell axons to their dendrites in
the vertical lobe. Many feedback neurons have bistratified
dendrites in the lobes. For example, feedback neurons
with dendrites in one stratum of the BR division and one
stratum of the Li division have terminals in the calyx lips
and the corresponding outer zone of the basal ring. Feedback neurons with dendrites in the Li and Co divisions of
the vertical lobe have terminals in the lip and collar of the
calyces. There is no compelling evidence for proposing that
feedback neurons transfer information between layers in
the vertical lobe representing one sensory modality to a
zone in the calyces representing a different modality, as
has been proposed by Menzel et al. (1994) and Grünewald
(1999; loc. cit. Fig. 11).
Studies on efferent neurons have also unwittingly misinterpreted the lobes. Although providing valuable information about cell body positions, Rybak and Menzel’s
1993 study on vertical lobe organization offers problems in
interpretation. The dendritic trees of two efferent neurons
called the A5-1 and A5-2 neurons (Rybak and Menzel,
1993; loc. cit. Fig. 11) were ascribed to Kenyon cell projections from the lip. However, the dendrites illustrated are
restricted to the gamma lobe, which is supplied by clawed
Kenyon cells and basal ring intrinsic neurons. The dendritic tree of one of these neurons (A5-1: Rybak and Menzel, 1993; loc. cit. Fig. 8a) resembles the present type 5
gamma lobe efferent (Figs. 13A, 14A), although the extremely slender processes of the illustrated A5-1 approximate the trajectory and collateral fields from the much
stouter type 3 gamma efferent neuron described here.
Rybak and Menzel’s A5-2 neuron (1993; loc. cit. Fig. 8b)
superficially resembles the dendritic tree of the type 2
gamma efferent described here, but the A5-2 axon and its
branches are much slenderer. Processes from both cells,
described as looping around the lobe, have not been seen
in Golgi impregnations. One explanation for this discrepancy is that these processes are artifacts due to the
method used by Rybak and Menzel (1993) to fill neurons.
This included, before precipitating with cobalt sulfide, a
lengthy incubation of cobalt-injected brains in Ringer solution, which can result in the displacement of cobalt ions
into nearby elements. Several processes ascribed to the
A5-1 and A5-2 neurons probably belong to processes of
other nerve cells. An alternative explanation for why none
of the efferent neurons in the present study correspond
completely to the A5-1 and A5-2 neurons would be that the
Golgi method is a stochastic method and stained a different subset of efferents than did cobalt injection. Nevertheless, the positions of A5-1 and A5-2 cell bodies, which are
depicted as contralateral and superficial, correspond to
two of the five types of gamma lobe efferents described
here.
Novel intrinsic neurons
Two new classes of neurons invading the calyces are
described here for the first time: the basal ring and neck
VERTICAL AND GAMMA LOBES IN HONEY BEES
intrinsic cells (Fig. 9). Their baroque arborizations in the
lower layer of the inner basal ring zone, as well as
amongst Kenyon cell axons of the neck, suggest that these
parts of the mushroom bodies deserve much closer scrutiny. Although not described here, certain feedback neurons provide dense groups of terminal specializations
within the basal ring and at the exit of some bundles of
Kenyon cell axons from the calyx.
The basal ring intrinsic cells provide a dense cascade of
parallel fibers, the appearance of which is reminiscent of
Kenyon cell axons, although they obtain twice their diameter. These axon-like processes accompany the axons of
clawed Kenyon cells into the gamma lobe where they end
in its two lower strata. Thus, the gamma lobe is supplied
by two parallel sets of axons. Those of clawed Kenyon cells
represent the entire calyx; those of basal ring intrinsic
neurons are mainly associated with the basal ring and
neck. The axons from basal ring intrinsic neurons might
provide additional information from modalities not represented elsewhere in the calyx, or they might receive information from the outgoing axons of class I Kenyon cells.
Thus, together, the clawed Kenyon cells and basal ring
intrinsic neurons may monitor all activity of class I
Kenyon cells anywhere within the calyx neuropil.
Neck intrinsic neurons have dendrite-like processes in
the basal ring and within the neck, amongst axons of
outgoing Kenyon cells. These neurons also have many
varicose, possibly presynaptic processes that ascend into
the neuropils of the basal ring. Together, these two novel
types of neurons confer an organization to the basal ring
that is distinct and much more complex than that of either
the collar or lip. The shapes and dispositions of their
arborizations suggest that these two species of neurons
participate in interactions amongst outgoing axons of
Kenyon cells in the pedunculus necks.
Cross-taxonomic comparisons: memory,
mushroom body development, and the origin
of the gamma lobes
Three studies provide evidence that specific parts of the
mushroom bodies might separately support short- and
long-term memory. The first, by Erber et al. (1980) on the
honey bee, demonstrated that focal cooling of the vertical
(“␣”) lobes reversibly blocks short-term olfactory memory.
The second, by Zars et al. (2000) on Drosophila, demonstrated the importance of the gamma lobe by using mutants that were deficient in type 1 calcium-calmodulin
dependent adenylyl cyclase, which is encoded by a “memory” gene, called rutabaga (rut; Han et al., 1992; Levin et
al., 1992). The deletion of this gene also abolishes shortterm memory. Expression of wild-type rut DNA in only
those GAL 4 lines that include expression in the gamma
lobe in an adenylyl cyclase-deficient brain rescues shortterm olfactory memory (Zars et al., 2000).
This result supports previous studies showing that
ubiquitous second messenger systems may play crucial
roles in learning and memory. In particular the enzyme
cyclic adenosine 3⬘5⬘-monophosphate phosphodiesterase
(cAMP-PDE) appears to be abundant in the mushroom
bodies and illustrations of the localization of antibodies
against cAMP-PDE (Nighorn et al., 1991) suggest that
cAMP-PDE is expressed at higher levels in the gamma
lobe than in other subdivisions. A deficiency of this enzyme, which degrades cAMP, leads to a deficit in olfactory
29
conditioning, as in the mutant dunce (dnc; Dudai et al.,
1976; Tully and Quinn, 1985).
A third study, which was on long-term memory, showed
that in those 5% of alpha lobe absent mutants (ala) for
which the vertical lobes are absent so is their ability to
establish long-term memory, although the absence of vertical lobes does not impair short-term memory acquisition
(Pascual and Préat, 2001).
If short- and long-term memory are supported by two
distinct parts of the mushroom bodies in Drosophila,
might they be supported by homologous regions of the
mushroom bodies in honey bees? Can the location of shortterm memory in the vertical lobe, as suggested by the
study of Erber et al. (1980), now be explained by the
existence of a region of the vertical lobe that is homologous
to the gamma lobe of Drosophila? And, might acquired
changes in the Pe-1 neuron’s responses to associative
stimuli be ascribable to elements of the honey bee gamma
lobe?
Despite their superficial differences in structure and the
vast differences in Kenyon cell number, the mushroom
bodies of honey bees and fruit flies share much in common.
In both taxa, the mushroom bodies are quadripartite
structures. The honey bee mushroom body has two cuplike calyces, each composed of two apparently identical
halves. In Drosophila, each mushroom body has a cap-like
calyx, which although it appears to be a single neuropil, is
phylogenetically derived from the basal neopteran condition of two calyces, which in Diptera have become fused
and condensed. As in the two separate calyces of the honey
bee, the two fused calyces of Drosophila are divided into
four identical components. Each of these arises from one of
four embryonic mushroom body neuroblasts (Ito et al.,
1997), which originate early in embryogenesis, being
amongst the first protocerebral progenitors to delaminate
from the procephalic ectoderm (Noveen et al., 2000). Later
in embryogenesis, the four neuroblasts divide to form four
identical groups of ganglion mother cells that eventually
provide four clusters of approximately 750 progeny each.
Each cluster provides equally the same proportions of
Kenyon cell types (Ito et al., 1997), of which three different
morphological types of Kenyon cell have been positively
identified to segregate to three divisions of the medial and
vertical lobes (Lee et al., 1999).
Although there are approximately two orders of magnitude more Kenyon cells in the honey bee than in the fruit
fly, the sequence, although not the timing, of Kenyon cell
development is similar. The four neuroblasts of the fruit
fly mushroom body generate approximately 3,000 Kenyon
cells (Technau, 1984) in 18 days, whereas at eclosion, the
worker bee mushroom body has acquired in only 10 days
at least 340,000 Kenyon cells (Witthöft, 1967). To produce
so many in so short a time, the mushroom body’s neuroblasts first divide symmetrically to provide approximately
2,000 descendants, which then produce the required number of intrinsic neurons (Farris et al., 1999). Four clusters
of mushroom body neuroblasts have been identified in the
honey bee larva at hatching, although it is not yet known
whether these derive from four original embryonic neuroblasts as in Drosophila (Farris et al., 1999). In the honey
bee, the four neuroblast clusters give rise to discrete populations of Kenyon cell perikarya that differ from each
other with respect to their cell body morphology and their
locations relative to the middle of the calyx (Farris et al.,
1999).
30
Developmental studies using bromodeoxyuridine incorporation to trace cell lineages have shown that the first
Kenyon cells to differentiate in the honey bee eventually
reside outside the calyx (Farris et al., 1999), as they do in
cockroaches (Farris and Strausfeld, 2001). Counts of these
cell bodies from ethyl gallate preparations suggest that at
least 14,000 such cells are associated with each calyx.
Golgi impregnations show that these neurons can invade
any part of the calyx where they have characteristic clawshaped dendrites. In the adult, their unbranched axons
contribute to that part of the vertical lobe here defined as
the gamma lobe.
In Drosophila, there is also a defined sequence of
Kenyon cell development (Lee et al., 1999). The earliest
Kenyon cells to differentiate supply axons to the gamma
(␥) division of the medial lobe, and in the larva also to a
corresponding division of the vertical lobe. These neurons,
which in the imago invade only the gamma lobe, have
stout claw-like dendritic specializations in the calyces.
Kenyon cells generated next supply axons to the ␤⬘ division of the medial lobe and axon collaterals to the ␣⬘
division of the vertical lobe. The last born Kenyon cells
supply the ␤ and ␣ divisions of the two lobes (Lee et al.,
1999).
Thus, Kenyon cells can be classified according to their
date of birth and the position of their cell bodies with
respect to the proliferation zone within the calyx. In Drosophila, Periplaneta, and Apis, Kenyon cells can be classified according to their axonal projections and dendritic
morphologies. In all three taxa, the earliest developing
Kenyon cells supply axons to the first and developmentally oldest division of the mushroom bodies, the gamma
lobe. In all three taxa, cell bodies of Kenyon cells supplying the gamma lobe lie furthest from the proliferation zone
of the calyces. Kenyon cells supplying the basal ring arise
from a central column of perikarya that are the last to
differentiate during postembryonic development (Farris et
al., 1999). Thus, in Apis, as in the cockroach (Farris and
Strausfeld, 2001) and Drosophila (Lee et al., 1999), the
division of the lobes furthest from the gamma lobe is
developmentally the youngest.
One objection that might be raised against comparing
the honey bee with Drosophila and Periplaneta is that the
gamma lobe of Drosophila extends medially, whereas in
the cockroach it is branched, extending alongside the medial and vertical lobes, and in the honey bee it is attached
to the vertical lobe. However, these differences might not
be significant because, irrespective of species, the gamma
lobe may be autonomous with respect to its supply of
Kenyon cells, and the afferents and efferent neurons associated with it (see Ito et al., 1998).
In larval Drosophila, the axons of clawed Kenyon cells
originally contribute to a gamma lobe that has a vertical
and medial branch, as it does in the hemimetabolous
cockroach. But these neurons in Drosophila undergo drastic remodeling during metamorphosis: clawed Kenyon
cells retract their axons and grow out new ones that in the
adult extend only medially (Lee et al., 1999). It will be
interesting to determine whether, in early pupal development of the honey bee, clawed Kenyon cells have bifurcating axons that supply a branched gamma lobe but by adult
emergence their medial branches have been eliminated.
There is already a dramatic precedent for mushroom body
reorganization in Hymenoptera. In polistine wasps, the
mushroom bodies appear almost bee-like up to the mid-
N.J. STRAUSFELD
pupal stage, at which time they begin a complete reorganization. The vertical lobes shorten and swell to lie either
side of the central complex. The medial lobes degenerate
and regrow their axons recurrently up through the pedunculus to finally end behind the calyces (see Ehmer and
Hoy, 2000). In the cockroach, there is no such remodeling,
suggesting that gamma lobe reorganization might be typical only of holometabolous insects.
Could the gamma lobe be phylogenetically ancient? Observations of odonate mushroom bodies do not indicate
any subdivisions within the medial lobe, suggesting that
there may be a uniform type of intrinsic cell supplying it.
Also, the observation that the gamma lobe develops earliest might reflect an ancient origin, the other lobes possibly evolving only with the acquisition of antennal lobes
and calyces.
Reassessment of mushroom body circuitry
A commonly held view of the mushroom bodies is that of
a neuropil that receives its major inputs in the calyces and
provides outputs from the lobes (Menzel and Müller, 2001;
Menzel, 2001), with Kenyon cells serving as intermediary
pathways between these two levels. This view, proposed
by Vowles (1955) and sustained by many since, may be
misleading.
Although anatomical and physiological studies show
that class I Kenyon cells receive substantial inputs provided in the calyces by collateral terminals of sensory
interneurons (Schürmann, 1971; MacLeod and Laurent,
1996; Gronenberg, 2001; Yusuyama et al., 2002), Kenyon
cells are not necessarily the sole inputs to efferent neurons
that leave the lobes. Electron microscopical evidence
shows that the mushroom body lobes receive abundant
presynaptic profiles from the protocerebrum (Frontali and
Mancini, 1970; Schürmann, 1970; Strausfeld and Li,
1999a). Protocerebral afferents to the lobes have been
described from cockroaches and Drosophila (Li and
Strausfeld, 1997; Ito et al., 1998), and the existence of
afferents to the vertical lobes in honey bees is shown here
from Golgi impregnations (see also, Strausfeld et al.,
2000). Furthermore, because apterygote and paleopteran
insects do not possess calyces that are supplied by sensory
neuropils, their mushroom bodies must be assumed to
receive abundant afferents to the lobes.
It has been proposed that in paleopteran and apterygote
mushroom bodies, as well as those of neopteran taxa, the
axon-like processes of class I Kenyon cells are likely to
contribute to elaborate integrative networks in the lobes
that provide circuits between afferent endings and efferent dendrites (Strausfeld and Li, 1999a; Strausfeld, 2001).
In the honey bee, such networks, each involving a few
thousand Kenyon cell axons, are suggested to constitute
each stratum, itself representing a specific zone of the
calyx. Thus, although afferent supply to the calyces must
be crucial to the integrative actions of the neopteran
mushroom body, any debate about how neurons in the
mushroom body interact functionally should take the following possibilities into consideration: (1) Systems of intrinsic axons provide local circuits amongst afferents and
efferents in the lobes. (2) The role of the afferent supply to
different zones of the calyces might be to modify the activity of these local circuits, thus providing a sensory
context to interactions between afferents and efferents in
the lobes.
VERTICAL AND GAMMA LOBES IN HONEY BEES
31
With these considerations in mind, the honey bee mushroom body can be viewed as a parallel processor whose
partitions in the lobes are likely to show functional independence. Because the gamma lobe is supplied by its own
unique sets of intrinsic neurons representing the whole
calyx, this division may be fundamentally different from
other lobe divisions. Its uniqueness is also suggested by its
outputs, which target regions of the protocerebrum that
are distinct from those targeted by outputs from other
divisions of the mushroom body. And, unlike the Li and Co
divisions of the vertical lobe, the gamma lobe does not
appear to provide efferent feedback to the calyces.
A crucial difference between the gamma lobe and other
divisions of the mushroom body must relate to the dendritic relationships of its class II Kenyon cells. Although
clawed Kenyon cells might receive inputs from sensory
interneurons, the size and orientation of their dendritic
claws suggest other possible relationships, such as with
the terminals of recurrent efferents from the vertical
lobe’s Li and Co divisions (Fig. 17A), or with the branches
and trunks of clusters of class I Kenyon cells supplying the
Li, Co, and BR divisions (Fig. 17B).
The convergence of axons of class II Kenyon cells from
the whole calyx to the gamma lobe suggests that any
sensory modality received by the calyx could modulate
local networks of class II axons in the gamma lobe. This
suggestion would hold true if class II Kenyon cell dendrites are postsynaptic to clusters of class I Kenyon cells
(Fig. 17B), or if class II dendrites monitor the activity of
class I Kenyon cells by means of feedback neurons from
the vertical lobe (Fig. 17A), or if class II dendrites receive
afferent supply directly in the calyces (Fig. 17C). In comparison, class I Kenyon cell networks within, say, the Li
division of the vertical lobe would be unimodally modulated by olfactory information received by their dendrites
in the calyx lip.
The organization of the gamma lobe is of crucial interest
if it is a unique component of the mushroom body supporting short-term memory as proposed by Zars et al. (2000),
and implicit in the account of Erber et al. (1980). Differences between the gamma lobe’s organization and the
organization of the rest of the vertical lobe may provide
insight into how different circuits in the brain serving
Fig. 17. Three proposed arrangements of class II (clawed) Kenyon
cells. In all three models, class II Kenyon cells are proposed to monitor
activity within the entire calyx. A: Clawed Kenyon cells (II K) are
postsynaptic to recurrent efferent (r eff) neurons, originating from the
vertical lobe where they receive inputs from protocerebral afferents
(pr aff) by means of local circuits supplied by the “axons” of class I
Kenyon cells (I K). Class I Kenyon cell “axons” are suggested to
provide local circuits between protocerebral afferents to the vertical
lobe and efferent neurons leaving it for the protocerebrum (pr eff).
Likewise, in the gamma lobe, the “axons” of clawed Kenyon cells
might provide local circuits between protocerebral afferents (pr ␥ aff)
supplying the gamma lobe and efferent neurons (pr ␥ eff) leaving it.
B: Clawed Kenyon cells (II K) are postsynaptic to class I Kenyon cells
(I K), which themselves receive inputs from sensory interneurons (s
aff, in C). In A and B, class II Kenyon cells would indirectly, by means
of recurrent efferent neurons (A), or directly from class I K cells (B)
monitor the activity of class I Kenyon cells. C: Clawed Kenyon cells
are postsynaptic to sensory interneuron afferents (s aff) supplying the
calyx. In this third model, local circuits in the gamma lobe, provided
by class II Kenyon cell “axons,” would be modulated by all sensory
modalities supplying the calyces.
32
N.J. STRAUSFELD
short- and long-term memory store and retrieve information.
ACKNOWLEDGMENTS
I thank Michael Zimmerman, B.Sc., for making exquisite Golgi preparations of the honey bee brain. Dr. Sarah
Farris provided the preparation that yielded the reconstruction in Figure 9B. This study profited from advice
and discussions with Drs. Sarah Farris, Birgit Ehmer, and
Wulfila Gronenberg. Drs. Ehmer and Gronenberg also
generously discussed their unpublished results on afferent organization to the calyces. Lastly, I am grateful, as
always, to Dr. Camilla Strausfeld for critically reading
and editing the manuscript.
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