Organization of olfactory and multimodal afferent neurons supplying the calyx and pedunculus of the cockroach mushroom bodies.

The mushroom bodies of neopteran insects are considered to be higher olfactory centers because their calyces receive abundant collaterals of projection neurons from the antennal lobes. However, intracellular recordings of mushroom body efferent neurons demonstrate that they respond to multimodal stimuli, implying that the mushroom bodies receive a variety of sensory cues. The present account describes new features of the organization of afferent neurons supplying the calyces of the cockroach Periplaneta americana. Afferent terminals segment the calyces into discrete zones, I, II, III, and IIIA, which receive afferents from 1) two discrete populations of sexually isomorphic olfactory glomeruli, 2) two types of male-specific olfactory glomeruli, 3) the optic lobes, and 4) multimodal interneurons that originate in protocerebral neuropils. In addition, intracellular recordings and dye fills show that at least four morphologically distinct GABAergic elements link many regions of the protocerebrum to the calyces. A new type of touch-sensitive centrifugal neuron has been identified terminating in the pedunculus. The dendrites of this afferent reside in satellite neuropil, beneath the mushroom body's medial lobe, which is supplied by collaterals from medial lobe efferent neurons and by terminals from the central complex. The role of this centrifugal cell in odorant sampling is considered. Golgi impregnation identifies other afferents in proximal regions of the calyx (zone IIIA) that also originate from satellite neuropils, suggesting major reafference from the medial lobes channeled through this region. The relevance of multimodal supply to the calyx in odorant discrimination is discussed as are comparisons between mushroom body organization in this phylogenetically basal neopteran and other taxa.     

Multimodal efferent and recurrent neurons in the medial lobes of cockroach mushroom bodies

Previous electrophysiological studies of cockroach mushroom bodies demonstrated the sensitivity of efferent neurons to multimodal stimuli. The present account describes the morphology and physiology of several types of efferent neurons with dendrites in the medial lobes. In general, efferent neurons respond to a variety of modalities in a context-specific manner, responding to specific combinations or specific sequences of multimodal stimuli. Efferent neurons that show endogenous activity have dendritic specializations that extend to laminae of Kenyon cell axons equipped with many synaptic vesicles, termed "dark" laminae. Efferent neurons that are active only during stimulation have dendritic specializations that branch mainly among Kenyon cell axons having few vesicles and forming the "pale" laminae. A new category of "recurrent" efferent neuron has been identified that provides feedback or feedforward connections between different parts of the mushroom body. Some of these neurons are immunopositive to antibodies raised against the inhibitory transmitter gamma-aminobutyric acid. Feedback pathways to the calyces arise from satellite neuropils adjacent to the medial lobes, which receive axon collaterals of efferent neurons. Efferent neurons are uniquely identifiable. Each morphological type occurs at the same location in the mushroom bodies of different individuals. Medial lobe efferent neurons terminate in the lateral protocerebrum among the endings of antennal lobe projection neurons. It is suggested that information about the sensory context of olfactory (or other) stimuli is relayed by efferent neurons to the lateral protocerebrum where it is integrated with information about odors relayed by antennal lobe projection neurons.     

Parallel organization in honey bee mushroom bodies by peptidergic Kenyon cells

Antisera against the neuromodulatory peptides, Phe-Met-Arg-Phe-NH(2)-amide (FMRFamide) and gastrin cholecystokinin, demonstrate that the mushroom bodies of honey bees are subdivided longitudinally into strata. Three-dimensional reconstructions demonstrate that these strata project in parallel through the entire pedunculus and through the medial and vertical lobes. Immunostaining reveals clusters of immunoreactive cell bodies within the calyx cups and immunoreactive bundles of axons that line the inside of the calyx cup and lead to strata. Together, these features reveal that immunoreactive strata are composed of Kenyon cell axons rather than extrinsic elements, as suggested previously by some authors. Sorting amongst Kenyon cell axons into their appropriate strata already begins in the calyx before these axons enter the pedunculus. The three main concentric divisions of each calyx (the lip, collar, and basal ring) are divided further into immunoreactive and immunonegative zones. The lip neuropil is divided into two discrete zones, the collar neuropil is divided into five zones, and the basal ring neuropil is divided into four zones. Earlier studies proposed that the lip, collar, and basal ring are represented by three broad bands in the lobes: axons from adjacent Kenyon cell dendrites in the calyces are adjacent in the lobes even after their polar arrangements in the calyces have been transformed to rectilinear arrangements in the lobes. The universality of this arrangement is not supported by the present results. Although immunoreactive zones are found in all three calycal regions, immunoreactive strata in the lobes occur mainly in the two bands that were ascribed previously to the collar and the basal ring. In the lobes, immunoreactive strata are visited by the dendrites of efferent neurons that carry information from the mushroom bodies to other parts of the brain. Morphologically and chemically distinct subdivisions through the pedunculus and lobes of honey bees are comparable to longitudinal subdivisions demonstrated in the mushroom bodies of other insects, such as the cockroach Periplaneta americana. The functional and evolutionary significance of the results is discussed.     

Neuronal organization of the hemiellipsoid body of the land hermit crab, Coenobita clypeatus: correspondence with the mushroom body ground pattern

Malacostracan crustaceans and dicondylic insects possess large second-order olfactory neuropils called, respectively, hemiellipsoid bodies and mushroom bodies. Because these centers look very different in the two groups of arthropods, it has been debated whether these second-order sensory neuropils are homologous or whether they have evolved independently. Here we describe the results of neuroanatomical observations and experiments that resolve the neuronal organization of the hemiellipsoid body in the terrestrial Caribbean hermit crab, Coenobita clypeatus, and compare this organization with the mushroom body of an insect, the cockroach Periplaneta americana. Comparisons of the morphology, ultrastructure, and immunoreactivity of the hemiellipsoid body of C. clypeatus and the mushroom body of the cockroach P. americana reveal in both a layered motif provided by rectilinear arrangements of extrinsic and intrinsic neurons as well as a microglomerular organization. Furthermore, antibodies raised against DC0, the major catalytic subunit of protein kinase A, specifically label both the crustacean hemiellipsoid bodies and insect mushroom bodies. In crustaceans lacking eyestalks, where the entire brain is contained within the head, this antibody selectively labels hemiellipsoid bodies, the superior part of which approximates a mushroom body's calyx in having large numbers of microglomeruli. We propose that these multiple correspondences indicate homology of the crustacean hemiellipsoid body and insect mushroom body and discuss the implications of this with respect to the phylogenetic history of arthropods. We conclude that crustaceans, insects, and other groups of arthropods share an ancestral neuronal ground pattern that is specific to their second-order olfactory centers.     

Fine structural organization of the hemiellipsoid body of the land hermit crab, Coenobita clypeatus

Electron microscopical observations of the hemiellipsoid bodies of the land hermit crab Coenobita clypeatus resolve microglomerular synaptic complexes that are comparable to those observed in the calyces of insect mushroom bodies and which characterize olfactory inputs onto intrinsic neurons. In an adult hermit crab, intrinsic neurons and one class of efferent neurons originate from neuronal somata of globuli cells covering the hemiellipsoid bodies. Counts of their nucleoli show that about 120,000 globuli cells supply each hemiellipsoid body in an adult hermit crab. This number is comparable to the number of globuli cells supplying mushroom bodies of certain insects, such as honey bees and cockroaches. Counts of axons in tracts leading from the olfactory lobes to the hemiellipsoid bodies resolve 20,000 afferent axons, however, an order of magnitude greater than known for any insect. These afferent axons provide numerous swollen varicosities, each presynaptic to many small profiles, and thus comparable to the microglomeruli that characterize insect mushroom body calyces. Also, common to mushroom bodies and hemiellipsoid bodies are arrangements of intrinsic neurons, afferent neurons containing dense core vesicles, and systems of serial synaptic complexes that relate to postsynaptic profiles of efferent neurons. Together, the ultrastructural organization of the hemiellipsoid bodies of C. clypeatus supports the proposition that this center may share a common origin with the insect mushroom body despite obvious divergent evolution of overall shape.     

Mushroom bodies of vespid wasps

Mushroom bodies are higher centers in the brains of insects. Studies on honey bees and species of ants suggest that these centers are particularly prominent in social insects. The present study confirms the presence of large mushroom bodies in five subfamilies of vespid wasps, while at the same time showing significant departures from the mushroom body organization that typifies bees and ants. Although the basic organizational plan of the insect mushroom body into calyces, peduncle, and lobes is maintained, as is the arrangement of axons of intrinsic neurons, the size and arrangements of the vespid mushroom body lobes differ markedly from those known from other Hymenoptera. Furthermore, considerable variation is found both between and within vespid subfamilies. The present results are discussed with respect to current hypotheses about functional attributes of mushroom bodies and the phylogeny of the Vespidae.     

Segregation of visual input to the mushroom bodies in the honeybee (Apis mellifera)

Insect mushroom bodies are brain regions that receive multisensory input and are thought to play an important role in learning and memory. In most neopteran insects, the mushroom bodies receive direct olfactory input. In addition, the calyces of Hymenoptera receive substantial direct input from the optic lobes. We describe visual inputs to the calyces of the mushroom bodies of the honeybee Apis mellifera, the neurons' dendritic fields in the optic lobes, the medulla and lobula, and the organization of their terminals in the calyces. Medulla neurons terminate in the collar region of the calyx, where they segregate into five layers that receive alternating input from the dorsal or ventral medulla, respectively. A sixth, innermost layer of the collar receives input from lobula neurons. In the basal ring region of the calyx, medulla neuron terminals are restricted to a small, distal part. Lobula neurons are more prominent in the basal ring, where they terminate in its outer half. Although the collar and basal ring layers generally receive segregated input from both optic neuropils, some overlap occurs at the borders of the layers. At least three different types of mushroom body input neurons originate from the medulla: (a) neurons with narrow dendritic fields mainly restricted to the vicinity of the medulla's serpentine layer and found throughout the medulla; (b) neurons restricted to the ventral half of the medulla and featuring long columnar dendritic branches in the outer medulla; and (c) a group of neurons whose dendrites are restricted to the most ventral part of the medulla and whose axons form the anterior inferior optic tract. Most medulla neurons (groups a and b) send their axons via the anterior superior optic tract to the mushroom bodies. Neurons connecting the lobula with the mushroom bodies have their dendrites in a defined dorsal part of the lobula. Their axons form a third tract to the mushroom bodies, here referred to as the lobula tract. Our findings match the anatomy of intrinsic mushroom body neurons (Strausfeld, 2002) and together indicate that the mushroom bodies may be composed of many more functional subsystems than previously suggested.     

Ground plan of the insect mushroom body: Functional and evolutionary implications

In most insects with olfactory glomeruli, each side of the brain possesses a mushroom body equipped with calyces supplied by olfactory projection neurons. Kenyon cells providing dendrites to the calyces supply a pedunculus and lobes divided into subdivisions supplying outputs to other brain areas. It is with reference to these components that most functional studies are interpreted. However, mushroom body structures are diverse, adapted to different ecologies, and likely to serve various functions. In insects whose derived life styles preclude the detection of airborne odorants, there is a loss of the antennal lobes and attenuation or loss of the calyces. Such taxa retain mushroom body lobes that are as elaborate as those of mushroom bodies equipped with calyces. Antennal lobe loss and calycal regression also typify taxa with short nonfeeding adults, in which olfaction is redundant. Examples are cicadas and mayflies, the latter representing the most basal lineage of winged insects. Mushroom bodies of another basal taxon, the Odonata, possess a remnant calyx that may reflect the visual ecology of this group. That mushroom bodies persist in brains of secondarily anosmic insects suggests that they play roles in higher functions other than olfaction. Mushroom bodies are not ubiquitous: the most basal living insects, the wingless Archaeognatha, possess glomerular antennal lobes but lack mushroom bodies, suggesting that the ability to process airborne odorants preceded the acquisition of mushroom bodies. Archaeognathan brains are like those of higher malacostracans, which lack mushroom bodies but have elaborate olfactory centers laterally in the brain. J. Comp. Neurol. 513:265–291, 2009. © 2009 Wiley‐Liss, Inc.     

Representation of the calyces in the medial and vertical lobes of cockroach mushroom bodies.

Previous studies of honey bee and cockroach mushroom bodies have proposed that afferent terminals and intrinsic neurons (Kenyon cells) in the calyces are arranged according to polar coordinates. It has been suggested that there is a transformation by Kenyon cell axons of the polar arrangements of their dendrites in the calyces to laminar arrangements of their terminals in the lobes. Findings presented here show that cellular organization in the calyx of an evolutionarily basal neopteran, Periplaneta americana, is instead rectilinear, as it is in the lobes. It is shown that each calyx is divided into two halves (hemicalyces), each supplied by its own set of Kenyon cells. Each calyx is separately represented in the medial lobe where the dendritic trees of some efferent neurons receive inputs from one calyx only. Kenyon cell dendrites are arranged as narrow elongated fields, organized as rows in each hemicalyx. Dendritic fields arise from 14 to 16 sheets of Kenyon cell axons stacked on top of each other lining the inner surface of the calyx cup. A sheet consists of approximately 60 small bundles, each containing 5-15 axons that converge from the rim of the calyx to its neck. Each sheet contributes to a pair oflaminae, one dark one pale, called a doublet, that extends through the mushroom body. Dark laminae contain Kenyon cell axons packed with synaptic vesicles. Axons in pale laminae are sparsely equipped with vesicles. By analogy with photoreceptors, and with reference to field potential recordings, it is speculated that dark laminae are continuously active, being modulated by odor stimuli, whereas pale laminae are intermittently activated. Timm's silver staining and immunocytology reveal a second type of longitudinal division of the lobes. Five layers extend through the pedunculus and lobes, each composed of subsets of doublets. Four layers represent zones of afferent endings in the calyces. A fifth (the y layer) represents a specific type of Kenyon cell. It is concluded that the mushroom bodies comprise two independent modular systems, doublets and layers. Developmental studies show that new doublets are added at each instar to layers that are already present early in second instar nymphs. There are profound similarities between the mushroom bodies of Periplaneta, an evolutionarily basal taxon, and those of Drosophila melanogaster and the honey bee.     

Distribution of serotonin-containing neurons and their pathways in the supraoesophageal ganglion of the cockroach Periplaneta americana (L.) as revealed by immunocytochemistry

The distribution of serotonin (5-HT)-containing neurons in the supraoesophageal (cerebral) ganglion of the cockroach Periplaneta americana was studied using immunocytochemistry and the formaldehyde histofluorescence method ( Klemm , '83). In this material immunocytochemistry was more sensitive than the formaldehyde histofluorescence procedure. A relatively small number of 5-HT-immunoreactive cell bodies (220-280) were found. For the first time, their processes could be followed. They highly arborize and innervate many brain regions. Three patterns of monoamine innervation have been demonstrated: (1) 5-HT and catecholamine fibres ( Klemm , '83) occurring in the same region (e.g., outer lateral protocerebral neuropil, stratum caudale , mushroom body, fan-shaped body, olfactory lobe), but having certain differences with respect to the organization of their projection fields; (2) 5-HT fibres innervating a region lacking catecholamine-containing fibres (pons); and (3) catecholamine neurons innervating a region lacking 5-HT fibres (ellipsoid body). In the mushroom body only the extrinsic neurons contain 5-HT immunoreactivity. They form a commissural fibre system linking the left- and right-hand mushroom bodies and other brain regions. The pons is part of a 5-HT-neuron fibre system innervating many areas including the mushroom bodies. The present study demonstrates novel, complex, and widely distributed connections within the insect brain.     

Connections between the deutocerebrum and the protocerebrum,and neuroanatomy of several classes of deutocerebral projection neurons in the brain of male Periplaneta americana

The topography and neuroanatomy of fibers connecting the deutocerebrum to the protocerebrum in the brain of the American cockroach Periplaneta americana were investigated by staining single or multiple deutocerebral neurons with cobalt, Lucifer Yellow, or biocytin. Five tracts are distinguished on the basis of their routes from origins in the antennal lobe to the protocerebral neuropil: the inner antenno-cerebral tract (IACT); antenno-cerebral tracts II, III, and IV (ACT II, III, IV), and the outer antenno-cereral tract (OACT). These tracts are largely composed of the axons of four classes of deutocerebral projection neurons, which have been identified morphologically; the neuronal arborizations in the glomeruli of the antennal lobe and in the protocerebral projection regions have been examined. Projection neurons with processes in the inner antenno-cerebral tract and in the antenno-cerebral tract II each innervate a single glomerulus in the antennal lobe, and both types have terminals in the calyces of the mushroom bodies and in the lateral lobe of the protocerebrum. The axons of pheromone-sensitive projection neurons with dendritic trees in the male-specific macroglomerulus seem to run exclusively in the inner antenno-cerebral tract. Subgroups of these pheromone sensitive neurons differ in relative sensitivity to the two female attractant components as well as in the arborization pattern of their dendrites in the macroglomerulus. The projection neurons of two other classes each innervate many glomeruli in the antennal lobe, those of one class sending their axons into the protocerebrum in the antenno-cerebral tract IV and the other, in the outer antenno-cerebral tract. The neurons of antenno-cerebral tract IV innervate not only the mushroom body calyces and the lateral lobe but also neuropil regions not previously described in the cockroach. Neurons with axons in the outer antenno-cerebral tract have no terminals in the calyces but innervate the lateral lobe and the neuropil surrounding the tract. The morphological findings presented here show that, in addition to the tracts previously documented in the cockroach brain, there are other, presumably olfactory, connections between the deutocerebrum and the protocerebrum. © 1993 Wiley-Liss, Inc.     

A unique mushroom body substructure common to basal cockroaches and to termites

The mushroom bodies of the cockroach Periplaneta americana are made up of intrinsic neurons (class I and class II Kenyon cells) with dendrites in a dorsal calyx and axons that bifurcate into medial and vertical lobes. Here, we describe a substructure of the cockroach mushroom bodies composed of a previously unrecognized class of Kenyon cells with distinct morphologies. The embryonically produced class III Kenyon cells form a separate accessory calyx below the calyx proper. The medial branches of class III Kenyon cell axons form the previously described "gamma bulb," whereas the vertical branches leave the vertical lobe to form a toroidal "lobelet" around the posterior surface. Taking advantage of the morphologically and immunochemically distinct nature of the lobelet, we have attempted to determine the distribution of this unique structure in other insects of the taxon Dictyoptera (cockroaches, mantises, and termites). Our data indicate that the lobelet is present only in basal cockroaches and in termites, supporting existing theories of a close phylogenetic relationship between these groups. Higher termites possess a duplicated lobe structure due to immense elaboration of the processes of class III Kenyon cells. The degree of complexity in the mushroom body lobes of termites agrees with current taxonomic arrangements of the Isoptera based on non-neural morphological and DNA sequence analyses. It thus appears that the evolution of the Dictyoptera has been accompanied by increasing complexity of the mushroom bodies, achieved in part through the further specialization and elaboration of a subset of Kenyon cells.     

A simple mushroom body in an African scarabid beetle

This account describes novel mushroom body organization in a coleopteran insect, the African fruit chafer Pachnoda marginata. Each of its prominent mushroom bodies possesses a pair of simple calyces comprising two populations of Kenyon cells, the dendrites of which are organized into a central and an annular zone. Kenyon cells of the central zone extend their dendrites downward and toward the perimeter of the calyx. Their axon-like processes in the pedunculus are densely packed to make up a distinctive shaft of neuropil. Toward the front of the brain, the shafts, one from each calyx, bifurcate to provide a pair of subdivisions in the medial and vertical lobes. Dendrites of Kenyon cells supplying the annular zone extend from the calyx perimeter toward its center. Axons from the annular zones of both calyces together provide a sleeve of axons that ensheaths the two shafts. Sleeve axons bifurcate to provide a second pair of divisions in each of the lobes. These arrangements provide each lobe with a discrete representation of the two Kenyon cell populations of the two calyces. Kenyon cells supplying the central zone have dendritic morphologies reminiscent of class II clawed Kenyon cells that supply the gamma lobes in other taxa. Kenyon cells supplying axons to the sleeve are suggestive of class III Kenyon cell morphologies described from cockroaches and termites. Elaborate intrinsic neurons, comparable to exotic intrinsic neurons in the honey bee gamma lobes, have processes that interact with shaft axons. The present observations suggest that mushroom bodies of Pachnoda represent either a basal organization entirely lacking class I Kenyon cells or an evolutionary modification in which there is no clear morphological distinction of class I and II Kenyon cells. In either case, cellular organization in Pachnoda's mushroom body is simple compared with that of other taxa.     

Distribution of dendrites of descending neurons and its implications for the basic organization of the cockroach brain

To determine precisely the brain areas from which descending neurons (DNs) originate, we examined the distribution of somata and dendrites of DNs in the cockroach brain by retrogradely filling their axons from the cervical connective. At least 235 pairs of somata of DNs were stained, and most of these were grouped into 22 clusters. Their dendrites were distributed in most brain areas, including lateral and medial protocerebra, which are major termination areas of output neurons of the mushroom body, but not in the optic and antennal lobes, the mushroom body, the central complex, or the posteroventral part of the lateral horn. The last area is the termination area of major types of olfactory projection neurons from the antennal lobe, i.e., uni- and macroglomerular projection neurons, so these neurons have no direct connections with DNs. The distribution of axon terminals of ascending neurons overlaps with that of DN dendrites. We propose, based on these findings, that there are numerous parallel processing streams from cephalic sensory areas to thoracic locomotory centers, many of which are via premotor brain areas from which DNs originate. In addition, outputs from the mushroom body, central complex, and posteroventral part of the lateral horn converge on some of the premotor areas, presumably to modulate the activity of some sensorimotor pathways. We propose, based on our results and documented findings, that many parallel processing streams function in various forms of reflexive and relatively stereotyped behaviors, whereas indirect pathways govern some forms of experience-dependent modification of behavior.     

Distribution of dendrites of descending neurons and its implications for the basic organization of the cockroach brain

To determine precisely the brain areas from which descending neurons (DNs) originate, we examined the distribution of somata and dendrites of DNs in the cockroach brain by retrogradely filling their axons from the cervical connective. At least 235 pairs of somata of DNs were stained, and most of these were grouped into 22 clusters. Their dendrites were distributed in most brain areas, including lateral and medial protocerebral, which are major termination areas of output neurons of the mushroom body, but not in the optic and antennal lobes, the mushroom body, the central complex, or the posteroventral part of the lateral horn. The last area is the termination area of major types of olfactory projection neurons from the antennal lobe, i.e., uni- and macroglomerular projection neurons, so these neurons have no direct connections with DNs. The distribution of axon terminals of ascending neurons overlaps with that of DN dendrites. We propose, based on these findings, that there are numerous parallel processing streams from cephalic sensory areas to thoracic locomotory centers, many of which are via premotor brain areas from which DNs originate. In addition, outputs from the mushroom body, central complex, and posteroventral part of the lateral horn converge on some of the premotor areas, presumably to modulate the activity of some sensorimotor pathways. We propose, based on our results and documented findings, that many parallel processing streams function in various forms of reflexive and relatively stereotyped behaviors, whereas indirect pathways govern some forms of experience-dependent modification of behavior.     

Organization of Kenyon cells in subdivisions of the mushroom bodies of a lepidopteran insect

The mushroom bodies are paired structures in the insect brain involved in complex functions such as memory formation, sensory integration, and context recognition. In many insects these centers are elaborate, sometimes comprising several hundred thousand neurons. The present account describes the mushroom bodies of Spodoptera littoralis, a moth extensively used for studies of olfactory processing and conditioning. The mushroom bodies of Spodoptera consist of only about 4,000 large-diameter Kenyon cells. However, these neurons are recognizably similar to morphological classes of Kenyon cells identified in honey bees, Drosophila, and cockroaches. The spodopteran mushroom body is equipped with three major divisions of its vertical and medial lobe, one of which, the gamma lobe, is supplied by clawed class II Kenyon cells as in other described taxa. Of special interest is the presence of a discrete tract (the Y tract) of axons leading from the calyx, separate from the pedunculus, that innervates lobelets above and beneath the medial lobe, close to the latter's origin from the pedunculus. This tract is comparable to tracts and resultant lobelets identified in cockroaches and termites. The article discusses possible functional roles of the spodopteran mushroom body against the background of olfactory behaviors described from this taxon and discusses the possible functional relevance of mushroom body structure, emphasizing similarities and dissimilarities with mushroom bodies of other species, in particular the fruit fly, Drosophila melanogaster.     

Substance P antibody reveals homologous neurons with axon terminals among somata in the crayfish and crab brain.

In the search for particular neurons that stain selectively and can be identified, the cerebral ganglia (brains) of the crayfish Cherax destructor and the crab Leptograpsus variegatus were immunocytochemically treated with a monoclonal antibody raised against substance P. Four large neurons in the cerebral ganglion of the crayfish and crab label selectively with a monoclonal antibody raised against substance P. Two of the large neurons have their cell bodies in the protocerebrum and two in the deutocerebrum in both animals. Each protocerebral cell in both animals projects through the ipsilateral and contralateral olfactory lobes to end among the lateral cell somata of the olfactory lobe and not in the neuropile. Electron micrographs show the presence of synapses within the cell somata area and on the cell somata themselves. Each deutocerebral cell in both animals projects only ipsilaterally and ends within the neuropile of the olfactory lobes. The immunoreactivity to substance P antibody and the shapes and the unique projections of the four cells suggest that they are homologous in the two species. Synaptic connections between axons and cell somata are rare in the arthropods but have been found on the Kenyon cells of the mushroom bodies of Limulus. This raises questions about homologies between the crustacean olfactory lobe and the mushroom bodies of Limulus and insects.     

Mushroom body neuronal remodelling is necessary for short-term but not for long-term courtship memory in Drosophila

The remodelling of neurons during their development is considered necessary for their normal function. One fundamental mechanism involved in this remodelling process in both vertebrates and invertebrates is axon pruning. A well-documented case of such neuronal remodelling is the developmental axon pruning of mushroom body γ neurons that occurs during metamorphosis in Drosophila. The γ neurons undergo pruning of larval-specific dendrites and axons at metamorphosis, followed by their regrowth as adult-specific dendrites and axons. We recently revealed a molecular cascade required for this pruning. The nuclear receptor ftz-f1 activates the expression of the steroid hormone receptor EcR-B1, a key component for γ remodelling, and represses expression of Hr39, an ftz-f1 homologous gene. If ectopically expressed in the γ neurons, HR39 inhibits normal pruning, probably by competing with endogenous FTZ-F1, which results in decreased EcR-B1 expression. The mushroom bodies are a bilaterally symmetric structure in the larval and adult brain and are involved in the processing of different types of olfactory memory. How memory is affected in pruning-deficient adult flies that possess larval-stage neuronal circuitry will help to explain the functional role of neuron remodelling. Flies overexpressing Hr39 are viable as adults and make it possible to assess the requirement for wild-type mushroom body pruning in memory. While blocking mushroom body neuron remodelling impaired memory after short-term courtship conditioning, long-term memory was normal. These results show that larval pruning is necessary for adult memory and that expression of courtship short-term memory and long-term memory may be parallel and independent.     

Complete identification of four giant interneurons supplying mushroom body calyces in the cockroach Periplaneta americana

Global inhibition is a fundamental physiological mechanism that has been proposed to shape odor representation in higher‐order olfactory centers. A pair of mushroom bodies (MBs) in insect brains, an analog of the mammalian olfactory cortex, are implicated in multisensory integration and associative memory formation. With the use of single/multiple intracellular recording and staining in the cockroach Periplaneta americana, we succeeded in unambiguous identification of four tightly bundled GABA‐immunoreactive giant interneurons that are presumably involved in global inhibitory control of the MB. These neurons, including three spiking neurons and one nonspiking neuron, possess dendrites in termination fields of MB output neurons and send axon terminals back to MB input sites, calyces, suggesting feedback roles onto the MB. The largest spiking neuron innervates almost exclusively the basal region of calyces, while the two smaller spiking neurons and the second‐largest nonspiking neuron innervate more profusely the peripheral (lip) region of the calyces than the basal region. This subdivision corresponds well to the calycal zonation made by axon terminals of two populations of uniglomerular projection neurons with dendrites in distinct glomerular groups in the antennal lobe. The four giant neurons exhibited excitatory responses to every odor tested in a neuron‐specific fashion, and two of the neurons also exhibited inhibitory responses in some recording sessions. Our results suggest that two parallel olfactory inputs to the MB undergo different forms of inhibitory control by the giant neurons, which may, in turn, be involved in different aspects of odor discrimination, plasticity, and state‐dependent gain control. J. Comp. Neurol. 525:204–230, 2017. © 2016 Wiley Periodicals, Inc.     

A new ascending sensory tract to the calyces of the honeybee mushroom body,the subesophageal-calycal tract

The mushroom bodies of the honeybee are important neuropils for learning and memory. Therefore, knowledge about their input and output connections is essential to understanding how these neuropils function. A newly described input tract to the mushroom body is presented here, which is called the subesophageal-calycal tract (SCT) and connects the subesophageal ganglion with the calyces of the mushroom bodies. The neuronal somata of the SCT neurons lie in one cluster between the lobula of the optic lobe and a neuropil area that is formed from the fusion of the tritocerebrum and the subesophageal ganglion. Within the subesophageal ganglion, the dendritic fibers of SCT neurons overlap with terminals of sensory neurons from the proboscis. Therefore, we conclude that the SCT neurons might process gustatory and mechanosensory information from the proboscis. Individual SCT neurons receive unilateral input within the subesophageal ganglion and may connect to either the ipsilateral or the contralateral mushroom body. On their way to the mushroom bodies, the SCT neuron axons meet the roots of the antennocerebralis tracts (ACTs) and from this point follow the same path as the median ACT neurons for a short distance. Within the calyces, the SCT neurons innervate two separate areas, a small area within the dorsal collar just below the lip and a part of the basal ring. Double-labeling experiments show that the projections of the SCT neurons do not overlap with the projections of the olfactory projection neurons and visual projection neurons from the dorsal medulla. The possible function of the SCT neurons and the relation of the SCT to known input tracts of the mushroom bodies in other insects are discussed.