A subpopulation of mushroom body intrinsic neurons is generated by protocerebral neuroblasts in the tobacco hornworm moth, Manduca sexta (Sphingidae, Lepidoptera)

Subpopulations of Kenyon cells, the intrinsic neurons of the insect mushroom bodies, are typically sequentially generated by dedicated neuroblasts that begin proliferating during embryogenesis. When present, Class III Kenyon cells are thought to be the first born population of neurons by virtue of the location of their cell somata, farthest from the position of the mushroom body neuroblasts. In the adult tobacco hornworm moth Manduca sexta, the axons of Class III Kenyon cells form a separate Y tract and dorsal and ventral lobelet; surprisingly, these distinctive structures are absent from the larval Manduca mushroom bodies. BrdU labeling and immunohistochemical staining reveal that Class III Kenyon cells are in fact born in the mid-larval through adult stages. The peripheral position of their cell bodies is due to their genesis from two previously undescribed protocerebral neuroblasts distinct from the mushroom body neuroblasts that generate the other Kenyon cell types. These findings challenge the notion that all Kenyon cells are produced solely by the mushroom body neuroblasts, and may explain why Class III Kenyon cells are found sporadically across the insects, suggesting that when present, they may arise through de novo recruitment of neuroblasts outside of the mushroom bodies. In addition, lifelong neurogenesis by both the Class III neuroblasts and the mushroom body neuroblasts was observed, raising the possibility that adult neurogenesis may play a role in mushroom body function in Manduca.     

Development and evolution of the insect mushroom bodies: towards the understanding of conserved developmental mechanisms in a higher brain center

The insect mushroom bodies are prominent higher order neuropils consisting of thousands of approximately parallel projecting intrinsic neurons arising from the minute basophilic perikarya of globuli cells. Early studies described these structures as centers for intelligence and other higher functions; at present, the mushroom bodies are regarded as important models for the neural basis of learning and memory. The insect mushroom bodies share a similar general morphology, and the same basic sequence of developmental events is observed across a wide range of insect taxa. Globuli cell progenitors arise in the embryo and proliferate throughout the greater part of juvenile development. Discrete morphological and functional subpopulations of globuli cells (or Kenyon cells, as they are called in insects) are sequentially produced at distinct periods of development. Kenyon cell somata are arranged by age around the center of proliferation, as are their processes in the mushroom body neuropil. Other aspects of mushroom body development are more variable from species to species, such as the origin of specific Kenyon cell populations and neuropil substructures, as well as the timing and pace of the general developmental sequence.     

Developmental organization of the mushroom bodies of Thermobia domestica (Zygentoma, Lepismatidae): insights into mushroom body evolution from a basal insect

The mushroom bodies of the insect brain are sensory integration centers best studied for their role in learning and memory. Studies of mushroom body structure and development in neopteran insects have revealed conserved morphogenetic mechanisms. The sequential production of morphologically distinct intrinsic neuron (Kenyon cell) subpopulations by mushroom body neuroblasts and the integration of newborn neurons via a discrete ingrowth tract results in an age-based organization of modular subunits in the primary output neuropil of the mushroom bodies, the lobes. To determine whether these may represent ancestral characteristics, the present account assesses mushroom body organization and development in the basal wingless insect Thermobia domestica. In this insect, a single calyx supplied by the progeny of two neuroblast clusters, and three perpendicularly oriented lobes are readily identifiable. The lobes are subdivided into 15 globular subdivisions (Trauben). Lifelong neurogenesis is observed, with axons of newborn Kenyon cells entering the lobes via an ingrowth core. The Trauben do not appear progressively during development, indicating that they do not represent the ramifications of sequentially produced subpopulations of Kenyon cells. Instead, a single Kenyon cell population produces highly branched axons that supply all lobe subdivisions. This suggests that although the ground plan for neopteran mushroom bodies existed in early insects, the organization of modular subunits composed of separate Kenyon cell subpopulations is a later innovation. Similarities between the calyx of Thermobia and the highly derived fruit fly Drosophila melanogaster also suggest a correlation between calyx morphology and Kenyon cell number.     

CHARACTERIZATION OF NICOTINE ACETYLCHOLINE RECEPTOR SUBUNITS IN THE COCKROACH Periplaneta americana MUSHROOM BODIES REVEALS A STRONG EXPRESSION OF β1 SUBUNIT: INVOLVEMENT IN NICOTINE‐INDUCED CURRENTS

Nicotinic acetylcholine receptors are ligand‐gated ion channels expressed in many insect structures, such as mushroom bodies, in which they play a central role. We have recently demonstrated using electrophysiological recordings that different native nicotinic receptors are expressed in cockroach mushroom bodies Kenyon cells. In the present study, we demonstrated that eight genes coding for cockroach nicotinic acetylcholine receptor subunits are expressed in the mushroom bodies. Quantitative real‐time polymerase chain reaction (PCR) experiments demonstrated that β1 subunit was the most expressed in the mushroom bodies. Moreover, antisense oligonucleotides performed against β1 subunit revealed that inhibition of β1 expression strongly decreases nicotine‐induced currents amplitudes. Moreover, co‐application with 0.5 μM α‐bungarotoxin completely inhibited nicotine currents whereas 10 μM d‐tubocurarine had a partial effect demonstrating that β1‐containing neuronal nicotinic acetylcholine receptor subtypes could be sensitive to the nicotinic acetylcholine receptor antagonist α‐bungarotoxin.     

Evolution of insect mushroom bodies: old clues,new insights

The mushroom bodies are a morphologically diverse sensory integration and learning and memory center in the brains of various invertebrate species, of which those of insects are the best described. Insect mushroom bodies are composed of numerous tiny intrinsic neurons (Kenyon cells) that form calyces with their dendrites and a pedunculus and lobes with their axons. The identities of conserved Kenyon cell subpopulations and the correlations between morphological and functional specializations of the mushroom bodies are just beginning to be elucidated, providing insight into mechanisms of mushroom body evolution. Comparisons of mushroom body organization in different insect lineages reveal trends in the evolution of subcompartments correlated with the elaboration, reduction, acquisition or loss of Kenyon cell subpopulations. Furthermore, these changes often appear correlated with variation in type and strength of afferent input and in behavioral ecology. These and other features of mushroom body organization suggest a striking convergence with mammalian cortex, with Kenyon cell subpopulations displaying evolutionary modularity in a manner reminiscent of cortical areas.     

Mcm3 replicative helicase mutation impairs neuroblast proliferation and memory in Drosophila

In the developing Drosophila brain, a small number of neural progenitor cells (neuroblasts) generate in a co‐ordinated manner a high variety of neuronal cells by integration of temporal, spatial and cell‐intrinsic information. In this study, we performed the molecular and phenotypic characterization of a structural brain mutant called small mushroom bodies (smu), which was isolated in a screen for mutants with altered brain structure. Focusing on the mushroom body neuroblast lineages we show that failure of neuroblasts to generate the normal number of mushroom body neurons (Kenyon cells) is the major cause of the smu phenotype. In particular, the premature loss of mushroom body neuroblasts caused a pronounced effect on the number of late‐born Kenyon cells. Neuroblasts showed no obvious defects in processes controlling asymmetric cell division, but generated less ganglion mother cells. Cloning of smu uncovered a single amino acid substitution in an evolutionarily conserved protein interaction domain of the Minichromosome maintenance 3 (Mcm3) protein. Mcm3 is part of the multimeric Cdc45/Mcm/GINS (CMG) complex, which functions as a helicase during DNA replication. We propose that at least in the case of mushroom body neuroblasts, timely replication is not only required for continuous proliferation but also for their survival. The absence of Kenyon cells in smu reduced learning and early phases of conditioned olfactory memory. Corresponding to the absence of late‐born Kenyon cells projecting to α′/β′ and α/β lobes, smu is profoundly defective in later phases of persistent memory.     

Fate of neuroblast progeny during postembryonic development of mushroom bodies in the house cricket,Acheta domesticus

Mushroom bodies represent the main sensory integrative center of the insect brain and probably play a major role in the adaptation of behavioral responses to the environment. Taking into account the continuous neurogenesis of cricket mushroom bodies, we investigated ontogenesis of this brain structure. Using BrdU labeling, we examined the fate of neuroblast progeny during the postembryonic development. Preimaginal Kenyon cells survived throughout larval and imaginal moults and persisted during adulthood. Our results indicate that the location of labelled Kenyon cells in the cortex of the adult cricket mainly depends upon the period when they were produced during development. The present data demonstrate that cricket mushroom bodies grow from the inside out and that, at any developmental stage, the center of the cortex contains the youngest Kenyon cells. This study also allowed us to observe the occurrence of quiescent neuroblasts. Kenyon cell death during postembryonic and adult life seems to be reduced. Although preimaginal Kenyon cells largely contribute to adult mushroom body structure, a permanent remodeling of the mushroom body occurs throughout the whole insect life due to the persistence of neurogenesis in the house cricket. Further studies are needed to understand the functional significance of these findings.     

Modulatory action of acetylcholine on the Na-dependent action potentials in Kenyon cells isolated from the mushroom body of the cricket brain

Kenyon cells, intrinsic neurons of the insect mushroom body, have been assumed to be a site of conditioning stimulus (CS) and unconditioned stimulus (US) association in olfactory learning and memory. Acetylcholine (ACh) has been implicated to be a neurotransmitter mediating CS reception in Kenyon cells, causing rapid membrane depolarization via nicotinic ACh receptors. However, the long-term effects of ACh on the membrane excitability of Kenyon cells are not fully understood. In this study, we examined the effects of ACh on Na⁺ dependent action potentials (Na⁺ spikes) elicited by depolarizing current injection and on net membrane currents under the voltage clamp condition in Kenyon cells isolated from the mushroom body of the cricket Gryllus bimaculatus. Current-clamp studies using amphotericin B perforated-patch recordings showed that freshly dispersed cricket Kenyon cells could produce repetitive Na⁺ spikes in response to prolonged depolarizing current injection. Bath application of ACh increased both the instantaneous frequency and the amplitudes of Na⁺ spikes. This excitatory action of ACh on Kenyon cells is attenuated by the pre-treatment of the cells with the muscarinic receptor antagonists, atropine and scopolamine, but not by the nicotinic receptor antagonist mecamylamine. Voltage-clamp studies further showed that bath application of ACh caused an increase in net inward currents that are sensitive to TTX, whereas outward currents were decreased by this treatment. These results indicate that in order to mediate CS, ACh may modulate the firing properties of Na⁺ spikes of Kenyon cells through muscarinic receptor activation, thus increasing Na conductance and decreasing K conductance.     

Mushroom bodies in the brain of carrion beetles (Coleoptera, Silphidae)

Mushroom bodies are developed similarly in all the carrion beetles studied, and the transition from necrophagy to predation in some species does not affect considerably their level of development. The Kenyon cells form two small groups in each brain hemisphere and are subdivided into central and peripheral cells. Two closely located inputs of the Kenyon cell processes enter a single, pillow-shaped calyx region. The pedunculus comprises two shafts along a considerable part of its length, which fuse in the lobes. In the degree of development, the mushroom bodies of carrion beetles resemble those of basal lamellicorn and longhorned beetles.     

Novel Middle-Type Kenyon Cells in the Honeybee Brain Revealed by Area-Preferential Gene Expression Analysis

The mushroom bodies (a higher center) of the honeybee (Apis mellifera L) brain were considered to comprise three types of intrinsic neurons, including large- and small-type Kenyon cells that have distinct gene expression profiles. Although previous neural activity mapping using the immediate early gene kakusei suggested that small-type Kenyon cells are mainly active in forager brains, the precise Kenyon cell types that are active in the forager brain remain to be elucidated. We searched for novel gene(s) that are expressed in an area-preferential manner in the honeybee brain. By identifying and analyzing expression of a gene that we termed mKast (middle-type Kenyon cell-preferential arrestin-related protein), we discovered novel ‘middle-type Kenyon cells’ that are sandwiched between large- and small-type Kenyon cells and have a gene expression profile almost complementary to those of large– and small-type Kenyon cells. Expression analysis of kakusei revealed that both small-type Kenyon cells and some middle-type Kenyon cells are active in the forager brains, suggesting their possible involvement in information processing during the foraging flight. mKast expression began after the differentiation of small- and large-type Kenyon cells during metamorphosis, suggesting that middle-type Kenyon cells differentiate by modifying some characteristics of large– and/or small-type Kenyon cells. Interestingly, CaMKII and mKast, marker genes for large– and middle-type Kenyon cells, respectively, were preferentially expressed in a distinct set of optic lobe (a visual center) neurons. Our findings suggested that it is not simply the Kenyon cell-preferential gene expression profiles, rather, a ‘clustering’ of neurons with similar gene expression profiles as particular Kenyon cell types that characterize the honeybee mushroom body structure.     

Not all dytiscidae have poorly developed mushroom bodies: The enigma of Cybister lateralimarginalis

The majority of diving beetles studied has completely differentiated but poorly developed mushroom bodies. The Kenyon cells are not numerous, the calyces are small, and the pedunculi and lobes have a simple structure. New Kenyon cells are produced by few solitary neuroblasts. Cybister lateralimarginalis makes an amazing exception. Its mushroom bodies are strongly developed and comprise numerous Kenyon cells, large calyces, and a peduncular apparatus of a complicated structure. The Kenyon cells are produced in polyneuroblast proliferative centers. The grounds of such strong development of the mushroom body in Cybister remain unknown.     

Learning channels. Cellular physiology of odor processing neurons within the honeybee brain

To understand the cellular mechanisms of olfactory learning in the honeybee brain we study the physiology of identified neurons within the olfactory pathway. Here, we review data on the voltage-sensitive and ligand-gated ionic currents of mushroom body Kenyon cells and antennal lobe neurons in vitro and in situ. Both cell types generate action potentials in vitro, but have different voltage-sensitive K+ currents. They express nicotinic acetylcholine receptors and ionotropic GABA receptors, representing the major transmitter systems in the insect olfactory system. Our data are interpreted with respect to learning-dependent plasticity in the honeybee brain.     

Cultured Giant Fiber Lobe of Squid Expresses Three Distinct Potassium Channel Activities in Selective Combinations

Neurons from the giant fiber lobe (GFL) of squid Loligo bleekeri were dissociated and cultured. The ionic currents were recorded using whole-cell patch clamp methods. The sodium current and the noninactivating potassium current like those elicited by the giant axon were among the currents expressed in axonal bulbs and bulblike structures upon dissociation. Meanwhile axonless cell bodies did not elicit such currents. Axonless cell bodies and some bulblike structures elicited two kinds of inactivating potassium currents, the slow- and the fast-inactivating current, which differed in their inactivation kinetics and pharmacology. Within 24 hr of plating, the current composition remained the same. While the noninactivating current was not sensitive to 4-aminopyridine, the two inactivating currents were sensitive, the slow-inactivating current being more sensitive. Selective combinations of the sodium current and the three potassium currents expressed in different structures of the acutely dissociated GFL could have resulted from cellular control of synthesis and transportation of the channel proteins to the somatic and the axonal membrane. The sodium current and the noninactivating potassium current could be recorded from some axonless cell bodies maintained in culture for over three days, indicating that the separation of the giant axon from its somata could result in the transportation of the channels normally expressed on the giant axon membrane to the somatic membrane. Received: 24 October 1995/Revised: 5 March 1996     

20-Hydroxyecdysone inhibits the mitotic activity of neuronal precursors in the developing mushroom bodies of the honeybee,Apis mellifera

The mushroom bodies (MBs) within the brain of the honeybee, Apis mellifera, are prominent paired neuropil structures consisting of a lateral and a median subunit. The intrinsic MB neurons (Kenyon cells) of each of these subunits are generated in four distinct proliferation centers, each associated with a calyx. Previous BrdU studies revealed that neurogenesis of Kenyon cells starts at the first larval stage (L1) by symmetrical cell division of Kenyon precursor cells, and ceases abruptly at a midpupal stage (P5). In the present work, we confirmed these results using the antiphospho histone H3 mitosis marker to label mitotically active cells in a cell culture system, in histological sections, and in whole-mount brain preparations. To elucidate whether the steroid hormone ecdysone plays a role in the termination of Kenyon cell neurogenesis, we manipulated the hormone titer by injecting 20-hydroxyecdysone (20E) into animals of those pupal stages (P0/1, P3, P4) in which neurogenesis of Kenyon cells was still extensive. The effects of 20E were evaluated by determining the number of mitotically active cells in confocal microscopic images of squash preparations of the MB proliferation centers. In all pupal stages studied, 20E caused a reduction of mitotic activity, indicating its involvement in the cessation of Kenyon cell neurogenesis.     

Study of nicotinic acetylcholine receptors on cultured antennal lobe neurones from adult honeybee brains

In insects, acetylcholine (ACh) is the main neurotransmitter, and nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission. In the honeybee, nAChRs are expressed in diverse structures including the primary olfactory centres of the brain, the antennal lobes (AL) and the mushroom bodies. Whole-cell, voltage-clamp recordings were used to characterize the nAChRs present on cultured AL cells from adult honeybee, Apis mellifera. In 90% of the cells, applications of ACh induced fast inward currents that desensitized slowly. The classical nicotinic agonists nicotine and imidacloprid elicited respectively 45 and 43% of the maximum ACh-induced currents. The ACh-elicited currents were blocked by nicotinic antagonists methyllycaconitine, dihydroxy-β-erythroidine and α-bungarotoxin. The nAChRs on adult AL cells are cation permeable channels. Our data indicate the existence of functional nAChRs on adult AL cells that differ from nAChRs on pupal Kenyon cells from mushroom bodies by their pharmacological profile and ionic permeability, suggesting that these receptors could be implicated in different functions.     

Acetylcholine, GABA, glutamate and NO as putative transmitters indicated by immunocytochemistry in the olfactory mushroom body system of the insect brain

The distribution of glutamate, GABA and ChAT and of NADPH-diaphorase was immunocytochemically and histochemically investigated in the mushroom bodies of the cricket (Gryllus bimaculatus) and of the fruitfly (Drosophila melanogaster). Glutamate and NO are considered as putative transmitters of mushroom body Kenyon cell types. In the input area (calyces) of the mushroom bodies of Drosophila, the majority of olfactory projection neurons is stained with antibodies against ChAT. In addition, small GABA-immunoreactive presynaptic fibres of extrinsic neurons occur intermingled with the ChAT-immunoreactive elements in the calyces, and occupy distinct compartments in the stalk and lobes. Complex synaptic connectivity of putatively cholinergic and GABAergic extrinsic neurons and of Keyon cell dendrites within the calycal glomeruli of mushroom bodies is discussed.     

Neurons with dopamine-like immunoreactivity target mushroom body Kenyon cell somata in the brain of some hymenopteran insects

The mushroom bodies of the insect brain are centers for olfactory and multimodal information processing and they are involved in associative olfactory learning. They are comprised of numerous (340,000 in the bee brain), small (3–8 μm soma diameter) local interneurons, the Kenyon cells. In the brain of honeybees (Apis mellifera) of all castes (worker bees, drones and queens), wasps (Vespula germanica) and hornets (Vespa crabro) immunostaining revealed fibers with dopamine-like immunoreactivity projecting from the pedunculus and the lip neuropil of the mushroom bodies into the Kenyon cell perikaryal layer. These fibers terminate with numerous varicosities, mainly around the border between medial and lateral Kenyon cell soma groups. Visualization of immunostained terminals in the transmission electron microscope showed that they directly contact the somata of the Kenyon cells and contain presynaptic elements. The somata of the Kenyon cells are clearly non-immunoreactive. Synaptic contacts at the somata are unusual for the central nervous systems of insects and other arthropods. This finding suggests that the somata of the Kenyon cells of Hymenoptera may serve an integrative role, and not merely a supportive function.     

Effects of experience and juvenile hormone on the organization of the mushroom bodies of honey bees

There is an age-related division of labor in the honey bee colony that is regulated by juvenile hormone. After completing metamorphosis, young workers have low titers of juvenile hormone and spend the first several weeks of their adult lives performing tasks within the hive. Older workers, approximately 3 weeks of age, have high titers of juvenile hormone and forage outside the hive for nectar and pollen. We have previously reported that changes in the volume of the mushroom bodies of the honey bee brain are temporally associated with the performance of foraging. The neuropil of the mushroom bodies is increased in volume, whereas the volume occupied by the somata of the Kenyon cells is significantly decreased in foragers relative to younger workers. To study the effect of flight experience and juvenile hormone on these changes within the mushroom bodies, young worker bees were treated with the juvenile hormone analog methoprene but a subset was prevented from foraging (big back bees). Stereological volume estimates revealed that, regardless of foraging experience, bees treated with methoprene had a significantly larger volume of neuropil in the mushroom bodies and a significantly smaller Kenyon cell somal region volume than did 1-day-old bees. The bees treated with methoprene did not differ on these volume estimates from untreated foragers (presumed to have high endogenous levels of juvenile hormone) of the same age sampled from the same colony. Bees prevented from flying and foraging nonetheless received visual stimulation as they gathered at the hive entrance. These results, coupled with a subregional analysis of the neuropil, suggest a potentially important role of visual stimulation, possibly interacting with juvenile hormone, as an organizer of the mushroom bodies. In an independent study, the brains of worker bees in which the transition to foraging was delayed (overaged nurse bees) were also studied. The mushroom bodies of overaged nurse bees had a Kenyon cell somal region volume typical of normal aged nurse bees. However, they displayed a significantly expanded neuropil relative to normal aged nurse bees. Analysis of the big back bees demonstrates that certain aspects of adult brain plasticity associated with foraging can be displayed by worker bees treated with methoprene independent of foraging experience. Analysis of the over-aged nurse bees suggests that the post-metamorphic expansion of the neuropil of the mushroom bodies of worker honey bees is not a result of foraging experience. © 1995 John Wiley & Sons, Inc.     

Expression of two ecdysteroid-regulated genes, Broad-Complex and E75, in the brain and ovary of the honeybee (Apis mellifera L.)

We previously demonstrated that two ecdysteroid-regulated genes, Mblk-1/E93 and E74, are expressed selectively in Kenyon cell subtypes in the mushroom bodies of the honeybee (Apis mellifera L.) brain. To further examine the possible involvement of ecdysteroid-regulated genes in brain function as well as in oogenesis in the honeybee, we isolated cDNAs for two other ecdysteroid-regulated genes, Broad-Complex (BR-C) and E75, and analyzed their expression in the worker brain as well as in the queen abdomen. In situ hybridization revealed that BR-C, like Mblk-1/ E93, is expressed selectively in the large-type Kenyon cells of the mushroom bodies in the worker brain, whereas E75 is expressed in all mushroom body neuron subtypes, suggesting a difference in the mode of response to ecdysteroid among Kenyon cell subtypes. In the queen ovary, both BR-C and E75 are expressed preferentially in the follicle cells that surround egg cells at the late stage, suggesting their role in oogenesis. These results suggest that BR-C and E75 are involved in the regulation of brain function as well as in reproductive physiology in the adult honeybee.     

Taurine in the insect central nervous system

1. Taurine is one of the most abundant free amino acids found in the tissues of insect nervous systems. A brief survey of its immunocytochemical distribution is provided for the brain of worker honeybees.2. The protocerebral mushroom bodies are prominent neuropiles of the insect brain. Immunoreactivity for taurine was compared in the mushroom body intrinsic Kenyon cells of Apis, Drosophila, and Locusta.3. In all three species Kenyon cells expressed immunoreactivity.4. The intensity of the immunoreactivity was, however, graded, depending on the species.5. Recent technical advances in the primary culture of the Kenyon cells of honeybees in a defined taurine-free medium provide the opportunity to investigate the action of taurine in a controlled environment.6. Taurine-like immunoreactivity has been described in the photoreceptor cells of insect and mammalian visual systems. Physiological evidence for similar functions of taurine in mammalian and insect nervous systems is reviewed.