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.     

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.     

Some cetoniinae (Coleoptera,Scarabaeidae) have structurally different medial and lateral calyces of mushroom bodies

Structural differences between the medial and lateral calyces of mushroom bodies in insects are described for the first time. In two cetoniine scarab beetles, Cetonia aurata and Oxythyrea funesta, the lateral calyces are subdivided into two portions showing a different neuropil structure. This feature is not reflected in the structure of the pedunculus and lobes, as well as in the relative neuropil volume occupied by transformed lateral calyx as compared with unmodified lateral calyx of related scarab beetles. The lateral calyx modification is considered to be related to changes in dendritic arborizations of central Kenyon cells. The subdivision of lateral calyx occurs only in adults and was not observed in larvae.     

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.     

Neural networks in the mushroom bodies of the honeybee

The Kenyon cells (K cells) or intrinsic neurones of the honeybee's mushroom bodies are organised as a series of arrays. In the calyces the arrays form concentric rings that are represented by rectilinear layers in the α and β lobes. The inputs to the calyces have been revealed by intraneuropilar cobalt injection into the optic and antennal lobes. Neurones from the medulla project to the collar neuropil of the calyx while the relay neurones of the antennal lobe project to the lip neuropil of the calyx. Extrinsic neurones of unknown polarity penetrating the α and β lobes have branching patterns that reflect the layered pattern of the intrinsic neurones. The study illustrates the feasibility of producing a fine grain map of the optic lobe and antennal lobe inputs to the mushroom bodies. It is suggested that the map could be produced by making cobalt injections into individual identified antennal glomeruli and at known sites in the medulla retinotopic mosaic.     

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.     

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.     

Tritocerebral tract input to the insect mushroom bodies

Insect mushroom bodies, best known for their role in olfactory processing, also receive sensory input from other modalities. In crickets and grasshoppers, a tritocerebral tract containing afferents from palp mechanosensory and gustatory centers innervates the accessory calyx. The accessory calyx is uniquely composed of Class III Kenyon cells, and was shown by immunohistochemistry to be present sporadically across several insect orders. Neuronal tracers applied to the source of tritocerebral tract axons in several species of insects demonstrated that tritocerebral tract innervation of the mushroom bodies targeted the accessory calyx when present, the primary calyces when an accessory calyx was not present, or both. These results suggest that tritocerebral tract input to the mushroom bodies is likely ubiquitous, reflecting the importance of gustation for insect behavior. The scattered phylogenetic distribution of Class III Kenyon cells is also proposed to represent an example of generative homology, in which the developmental program for forming a structure is retained in all members of a lineage, but the program is not "run" in all branches.     

Diversity in neuroblast number forming mushroom bodies of the higher dipterans (Insecta,Diptera, Brachycera Cyclorrhapha)

Neurogenesis in mushroom bodies is studied in 12 species of the higher dipterans. A significant difference in the number of neuroblasts forming mushroom bodies is found. In the majority of species studied, Kenyon cells are formed by four solitary neuroblasts. Among six calliphorid species, the number of neuroblasts increases up to 10–15 (mean 12.6) in each mushroom body in Calliphora vicina only. In young pupae of Muscina stabulans and M. livida, four polyneuroblastic prolipherative centers occur instead of solitary neuroblasts. These centers disintegrate later into numerous solitary neuroblasts. A hypothesis on the origin of the polyneuroblastic structure of mushroom bodies found in C. vicina and, earlier, in Musca domestica, is proposed.     

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.     

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.     

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.     

Early development of the Drosophila mushroom bodies, brain centres for associative learning and memory

We have studied the formation of Drosophila mushroom bodies using enhancer detector techniques to visualize specific components of these complex intrinsic brain structures. During embryogenesis, neuronal proliferation begins in four mushroom body neuroblasts and the major axonal pathways of the mushroom bodies are pioneered. During larval development, neuronal proliferation continues and further axonal projections in the pedunculus and lobes are formed in a highly structured manner characterized by spatial heterogeneity of reporter gene expression. Enhancer detector analysis identifies many genomic locations that are specifically activated in mushroom body intrinsic neurons (Kenyon cells) during the transition from embryonic to postembryonic development and during metamorphosis.     

A novel,unusual (at least for beetles) mode of Kenyon cell production in the diving beetle Cybister lateralimarginalis Deg. (Coleoptera: Dytiscidae)

Kenyon cell production in the mushroom bodies of Cybister lateralimarginalis is a peculiar process. It has been found that each proliferative center contains one giant neuroblast, which divides unequally, and its smaller daughter cell becomes the 2nd order neuroblast dividing unequally as well. The smaller daughter cell of this neuroblast becomes a ganglion mother cell. The latter, as usual, divides equally producing two Kenyon cells.     

Stratification and synaptogenesis in the mushroom body of the honeybee,Apis mellifera

Stratification is a basic anatomical feature of central brain in both vertebrates and many invertebrates. The aim of this study was to investigate the relationship between stratification and synaptogenesis in the developing mushroom bodies of the honeybee. During metamorphosis, the vertical lobe of mushroom body shows progressive stratification with three thick primary strata and more secondary strata and laminae. Three primary strata are formed at the metamorphic stage P1, before the youngest generation of the mushroom body intrinsic neurons, Kenyon cells, is produced. Thus, the primary strata within the lobe are unlikely to represent three major subpopulations of the Kenyon cells sequentially produced in the mushroom bodies. Formation of laminae starts at the stage P2 and culminates at the end of metamorphosis. The laminae appear within the lobe rather than being added sequentially from the ingrowth stratum. Alternating dark and light lamina (lamina doublets) are formed in the vertical lobe in late metamorphosis (stages P6–P9), but they are not visible in adults. The pattern of stratification is not continuous along the vertical lobe at the same developmental stage, and resorting of axons of the Kenyon cells is likely to occur within dark laminae. In the developing vertical lobe, dark laminae show lower synaptic density and exhibit an ultra structure that is indicative for a delay in synaptogenesis relative to the primary strata. A local transient block of synaptogenesis within the dark laminae may provide correct targeting of Kenyon cells by extrinsic mushroom body neurons. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

A network capable of reading the temporal codes in the olfactory discrimination of insects]

Recent work on the insect olfactory system has shown that its mushroom bodies (one of its major components) are involved in the fine discrimination of odours and that the temporal organisation of spike discharges plays a fundamental role. We propose here a model of a network that is able to decode the temporal patterns which characterise an odour. This model has three fundamental properties that seem to exist in all mushroom bodies of insects studied so far: a) long lasting inhibitions with rebounds, able to facilitate delayed spike generation; b) synaptic plasticity, which allows the network to learn to recognise temporal patterns; c) above all a large interconnection, which allows this network to recognise intervals of various duration. This model thus appears suited to identify combinations of temporal patterns in the dendrites of Kenyon cells (the principal cells in the calyces of the mushroom bodies). Moreover, the mushroom bodies integrate multimodal inputs, suggesting that the detection of temporal patterns may be extended to the detection of a complex environment, combining in particular olfactive and visual inputs.     

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.     

On structural-functional organization of dragonfly mushroom bodies and some general considerations about purpose of these formations

Anatomy as well as (for the first time) the fine structure have been studied of the mushroom bodies located in protocerebrum of the supraesophageal ganglion of dragonflies—the most ancient flying insects on Earth. Used in the work are larvae of the last age (prior to winging), in which the mushroom body structure has already been completely formed and corresponds to that in imago. The total organization of the dragonfly mushroom bodies has been established to be more primitive than that of other insects studied so far. This involves both the number of interneurons (Kenyon cells) present in the mushroom bodies and the character of anaptic connections formed by these cells. There is confirmed the absence in dragonflies of the mushroom body calyces that in opinion of some authors are obligatory input gates into these structures. Peculiarities of the neuropil structure in the area of the absent calyces are studied in detail. For the first time in insects there are revealed the direct (without additional synaptic switching) pathways forming the afferent input from optic lobes into the mushroom body calyx area. Also detected are the direct pathways going from the mushroom bodies to the abdominal chain (efferent output). A possible functional significance of these findings as well as the general role of mushroom bodied in control of some forms of insect behavior are discussed.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 40, No. 6, 2004, pp. 495–507     

The brain of the horseshoe crab (Limulus polyphemus) II. Architecture of the corpora pendunculata

The corpora pedunculata, or mushroom bodies, of the horseshoe crab, Limulus polyphemus, form a bulbous ventral hemisphere composed of two internal lobes that are highly branched like a cauliflower. This organ is clothed with a deep layer of small association neurons called globuli or Kenyon cells. In an animal that is 50 mm in width, they number 3.7 × 10⁶, a value that rises to about 1 × 10⁸ in an adult (250 mm width). The neuropil of each corpus pedunculatum converges from its peripheral lobules toward several major peduncles, which are in communication with the protocerebral neuropil by a narrow stalk containing about 5000 fibers in a 50 mm animal. The numerical relations suggest that presumptive second-order chemosensory fibers enter the corpora pedunculata and synapse divergently onto Kenyon cells. The axons of Kenyon cells, in turn, converge onto efferent fibers that leave through the stalk.     

Male-associated polypeptide (MAP) expression in different compartments of the reproductive system of the mussel Mytilus galloprovincialis: immunocytochemical and Western blot study

The dissociation and maintenance in culture of cells derived from the mushroom bodies of adult crickets (Acheta domesticus) are described. This primary culture was developed in order to investigate maturation and differentiation of mushroom-body cells including Kenyon cells, the major intrinsic interneurons of mushroom bodies, which have been shown to be involved in learning and memory in insects. Three distinct cell types were observed, all identified as neural cells on the basis of their size, morphology and immunocytochemical staining with horseradish peroxidase. These cells appear to correspond to the three cell types observed in vivo: Kenyon cells, ganglion mother cells and neuroblasts. Some cells showed neurite growth, usually with long unipolar processes, occasionally with either bipolar or, more rarely, multipolar processes. Neuronal cell bodies readily formed seals with patch pipettes, allowing stable, whole-cell, patch-clamp electrophysiological recordings. Depolarization of the cell under voltage-clamp resulted in at least two types of outwardly directed potassium currents: a delayed rectifier-type of current that was sensitive to tetraethylammonium, and a cadmium-sensitive current with rapid inactivation. Neither type of current was affected by quinidine, a blocker of potassium currents recorded from pupal honeybee Kenyon cells. Other ionic currents, which have yet to be characterized, were also observed. Received: 30 October 1996 / Accepted: 11 July 1997