Metamorphosis and adult development of the mushroom bodies of the red flour beetle, Tribolium castaneum.

The insect mushroom bodies play important roles in a number of higher processing functions such as sensory integration, higher level olfactory processing, and spatial and associative learning and memory. These functions have been established through studies in a handful of tractable model systems, of which only the fruit fly Drosophila melanogaster has been readily amenable to genetic manipulations. The red flour beetle Tribolium castaneum has a sequenced genome and has been subject to the development of molecular tools for the ready manipulation of gene expression; however, little is known about the development and organization of the mushroom bodies of this insect. The present account bridges this gap by demonstrating that the organization of the Tribolium mushroom bodies is strikingly like that of the fruit fly, with the significant exception that the timeline of neurogenesis is shifted so that the last population of Kenyon cells is born entirely after adult eclosion. Tribolium Kenyon cells are generated by two large neuroblasts per hemisphere and segregate into an early-born delta lobe subpopulation followed by clear homologs of the Drosophila gamma, alpha'/beta' and alpha/beta lobe subpopulations, with the larval-born cohorts undergoing dendritic reorganization during metamorphosis. BrdU labeling and immunohistochemical staining also reveal that a proportion of individual Tribolium have variable numbers of mushroom body neuroblasts. If heritable, this variation represents a unique opportunity for further studies of the genetic control of brain region size through the control of neuroblast number and cell cycle dynamics.

Development of laminar organization in the mushroom bodies of the cockroach: Kenyon cell proliferation, outgrowth, and maturation.

The mushroom bodies of the insect brain are lobed integration centers made up of tens of thousands of parallel-projecting axons of intrinsic (Kenyon) cells. Most of the axons in the medial and vertical lobes of adult cockroach mushroom bodies derive from class I Kenyon cells and are organized into regular, alternating pairs (doublets) of pale and dark laminae. Organization of Kenyon cell axons into the adult pattern of laminae occurs gradually over the course of nymphal development. Newly hatched nymphs possess tiny mushroom bodies with lobes containing a posterior lamina of ingrowing axons, followed by a single doublet, which is flanked anteriorly by a gamma layer composed of class II Kenyon cells. Golgi impregnations show that throughout nymphal development, regardless of the number of doublets present, the most posterior lamina serves as the "ingrowth lamina" for axons of newborn Kenyon cells. Axons of the ingrowth lamina are taurine- and synaptotagmin-immunonegative. They produce fine growth cone tipped filaments and long perpendicularly oriented collaterals along their length. The maturation of these Kenyon cells and the formation of a new lamina are marked by the loss of filaments and collaterals, as well as the onset of taurine and synaptotagmin expression. Class I Kenyon cells thus show plasticity in both morphology and transmitter expression during development. In a hemimetabolous insect such as the cockroach, juvenile stages are morphologically and behaviorally similar to the adult. The mushroom bodies of these insects must be functional from hatching onward, while thousands of new neurons are added to the existing structure. The observed developmental plasticity may serve as a mechanism by which extensive postembryonic development of the mushroom bodies can occur without disrupting function. This contrasts with the more evolutionarily derived holometabolous insects, such as the honey bee and the fruit fly, in which nervous system development is accomplished in a behaviorally simple larval stage and a quiescent pupal stage.

Taurine-, aspartate- and glutamate-like immunoreactivity identifies chemically distinct subdivisions of Kenyon cells in the cockroach mushroom body.

The lobes of the mushroom bodies of the cockroach Periplaneta americana consist of longitudinal modules called laminae. These comprise repeating arrangements of Kenyon cell axons, which like their dendrites and perikarya have an affinity to one of three antisera: to taurine, aspartate, or glutamate. Taurine-immunopositive laminae alternate with immunonegative ones. Aspartate-immunopositive Kenyon cell axons are distributed across the lobes. However, smaller leaf-like ensembles of axons that reveal particularly high affinities to anti-aspartate are embedded within taurine-positive laminae and occur in the immunonegative laminae between them. Together, these arrangements reveal a complex architecture of repeating subunits whose different levels of immunoreactivity correspond to broader immunoreactive layers identified by sera against the neuromodulator FMRFamide. Throughout development and in the adult, the most posterior lamina is glutamate immunopositive. Its axons arise from the most recently born Kenyon cells that in the adult retain their juvenile character, sending a dense system of collaterals to the front of the lobes. Glutamate-positive processes intersect aspartate- and taurine-immunopositive laminae and are disposed such that they might play important roles in synaptogenesis or synapse modification. Glutamate immunoreactivity is not seen in older, mature axons, indicating that Kenyon cells show plasticity of neurotransmitter phenotype during development. Aspartate may be a universal transmitter substance throughout the lobes. High levels of taurine immunoreactivity occur in broad laminae containing the high concentrations of synaptic vesicles.

Experience- and age-related outgrowth of intrinsic neurons in the mushroom bodies of the adult worker honeybee.

A worker honeybee performs tasks within the hive for approximately the first 3 weeks of adult life. After this time, it becomes a forager, flying repeatedly to collect food outside of the hive for the remainder of its 5-6 week life. Previous studies have shown that foragers have an increased volume of neuropil associated with the mushroom bodies, a brain region involved in learning, memory, and sensory integration. We report here that growth of the mushroom body neuropil in adult bees occurs throughout adult life and continues after bees begin to forage. Studies using Golgi impregnation asked whether the growth of the collar region of the mushroom body neuropil was a result of growth of the dendritic processes of the mushroom body intrinsic neurons, the Kenyon cells. Branching and length of dendrites in the collar region of the calyces were strongly correlated with worker age, but when age-matched bees were directly compared, those with foraging experience had longer, more branched dendrites than bees that had foraged less or not at all. The density of Kenyon cell dendritic spines remained constant regardless of age or behavioral state. Older and more experienced foragers therefore have a greater total number of dendritic spines in the mushroom body neuropil. Our findings indicate that, under natural conditions, the cytoarchitectural complexity of neurons in the mushroom bodies of adult honeybees increases as a function of increasing age, but that foraging experience promotes additional dendritic branching and growth.

Ontogeny of orientation flight in the honeybee revealed by harmonic radar.

Cognitive ethology focuses on the study of animals under natural conditions to reveal ecologically adapted modes of learning. But biologists can more easily study what an animal learns than how it learns. For example, honeybees take repeated 'orientation' flights before becoming foragers at about three weeks of age. These flights are a prerequisite for successful homing. Little is known about these flights because orienting bees rapidly fly out of the range of human observation. Using harmonic radar, we show for the first time a striking ontogeny to honeybee orientation flights. With increased experience, bees hold trip duration constant but fly faster, so later trips cover a larger area than earlier trips. In addition, each flight is typically restricted to a narrow sector around the hive. Orientation flights provide honeybees with repeated opportunities to view the hive and landscape features from different viewpoints, suggesting that bees learn the local landscape in a progressive fashion. We also show that these changes in orientation flight are related to the number of previous flights taken instead of chronological age, suggesting a learning process adapted to changes in weather conditions, flower availability and the needs of bee colonies.

Larval and pupal development of the mushroom bodies in the honey bee, Apis mellifera.

The mushroom bodies are paired neuropils in the insect brain that act as multimodal sensory integration centers and are involved in learning and memory. Our studies, by using 5-bromo-2-deoxyuridine incorporation and the Feulgen technique, show that immediately before pupation, the brain of the developing honey bee (Apis mellifera) contains approximately 2,000 neuroblasts devoted to the production of the mushroom body intrinsic neurons (Kenyon cells). These neuroblasts are descended from four clusters of 45 or fewer neuroblasts each already present in the newly hatched larva. Subpopulations of Kenyon cells, distinct in cytoarchitecture, position, and immunohistochemical traits, are born at different, but overlapping, periods during the development of the mushroom bodies, with the final complement of these neurons in place by the mid-pupal stage. The mushroom bodies of the adult honey bee have a concentric arrangement of Kenyon cell types, with the outer layers born first and pushed to the periphery by later born neurons that remain nearer the center of proliferation. This concentricity is further reflected in morphologic and immunohistochemical traits of the adult neurons, and is demonstrated clearly by the pattern of expression of Drosophila myocyte enhancer factor 2 (DMEF2)-like immunoreactivity. This is the first comprehensive study of larval and pupal development of the honey bee mushroom bodies. Similarities to patterns of neurogenesis observed in the mushroom bodies of other insects and in the vertebrate cerebral cortex are discussed.

Experience-expectant plasticity in the mushroom bodies of the honeybee.

Worker honeybees (Apis mellifera) were reared in social isolation in complete darkness to assess the effects of experience on growth of the neuropil of the mushroom bodies (MBs) during adult life. Comparison of the volume of the MBs of 1-day-old and 7-day-old bees showed that a significant increase in volume in the MB neuropil occurred during the first week of life in bees reared under these highly deprived conditions. All regions of the MB neuropil experienced a significant increase in volume with the exception of the basal ring. Measurement of titers of juvenile hormone JH) in a subset of bees indicated that, as in previous studies, these rearing conditions induced in some bees the endocrine state of high JH associated with foraging, but there was no correlation between JH titer and volume of MB neuropil. Treatment of another subset of dark-reared bees with the JH analog, methoprene, also had no effect of the growth of the MB neuropil. These results demonstrate that there is a phase of MB neuropil growth early in the adult life of bees that occurs independent of light or any form of social interaction. Together with previous findings showing that an increase in MB neuropil volume begins around the time that orientation flights occur and then continues throughout the phase of life devoted to foraging, these results suggest that growth of the MB neuropil in adult bees may have both experience-expectant and experience-dependent components.

Expansion of the neuropil of the mushroom bodies in male honey bees is coincident with initiation of flight.

The mushroom bodies (MB), the insect brain structures most often associated with learning, have previously been shown to exhibit structural plasticity during the adult behavioral development of female worker and queen honey bees. We now show that comparable morphological changes occur in the brains of male honey bees (drones). The volume of the MB in the brains of drones was estimated from tissue sections using the Cavalieri method. Brains were obtained from six groups of drones that differed in age and flight experience. Circulating levels of juvenile hormone (JH) in these drones were determined by radioimmunoassay (RIA). There was an expansion of the neuropil of the MB that was temporally associated with drone behavioral development, as in female queens and workers. The observed changes in drones were maintained in the presence of low levels of JH, also as in females. These results suggest that expansion of the neuropil of the MB in honey bees is associated with learning the location of the nest, because this learning is the most prominent aspect of behavioral development common to all members (workers, drones, queen) of the honey bee colony.

Preservation of spatial learning in fyn tyrosine kinase knockout mice.

Previous work has shown that knockout mice lacking the fyn tyrosine kinase gene (fyn-/-) are impaired in spatial learning. Here, we have re-examined the spatial learning of fyn-/- mutants in an open field water maze. Unlike wild-type mice, fyn-/- knockouts often floated without moving when placed in the water but could swim adequately when their hind feet were mechanically stimulated. Under these conditions, fyn-/- mice showed significant improvement over trials in locating a hidden platform. On a transfer trial, at the end of training, they spent a disproportionate amount of time swimming in the location of the previously hidden platform. These findings suggest that fyn-/- knockouts are capable of spatial learning, but suffer an impairment that compromises their ability to swim normally.