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.     

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.     

Topographically distinct visual and olfactory inputs to the mushroom body in the Swallowtail butterfly,Papilio xuthus

Papilio butterflies depend highly on visual information in their flower‐foraging behavior. The retina of Papilio xuthus has been studied well, whereas the visual system in the brain is poorly understood. By investigating outputs from the optic lobe to the central brain, we found that the mushroom body of P. xuthus receives prominent direct inputs from the optic lobe in addition to olfactory inputs. The mushroom body consists of three components: the calyx, the pedunculus, and the lobes. The calyx is further subdivided into two cup‐shaped primary calyces and an accessory calyx. Each primary calyx consists of three concentric subareas, the inner zone, the outer zone, and the rim of the outer zone. Dextran injections into the optic lobe, the calyx, or the antennal lobe revealed three visual inputs and one olfactory input into the calyx. The visual inputs originate from the medulla, the lobula, and a newly identified neuropil, the ventral lobe of the lobula. All visual inputs first innervate the accessory calyx, and the two lobula inputs further spread their processes through the inner zone and the rim of the outer zone of the primary calyces. Visual inputs from the medulla and the ventral lobe of the lobula collect light information from ventral eye regions, suggesting a role in visual target detection rather than sky compass orientation. In contrast to visual inputs, olfactory inputs innervate only the calycal outer zone. The multisensory inputs to the mushroom bodies in P. xuthus are probably related to their flower‐foraging behavior. J. Comp. Neurol. 523:162–182, 2015. © 2014 Wiley Periodicals, Inc.     

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.     

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.     

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.     

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.     

F-actin at identified synapses in the mushroom body neuropil of the insect brain

The distribution of f-actin stained by fluorescent phalloidin was investigated in the brain of several insect species, with a special focus on the mushroom body. For localizing f-actin in identified neurons and at synapses, additional staining with fluorescent dextrans and anti-synapsin I immunostaining was employed. Intense f-actin staining was consistently found in synaptic complexes of the mushroom body calyces (calycal microglomeruli [MG]). These MG contain a central core of presynaptic boutons, predominantly belonging to deutocerebral cholinergic excitatory projection neurons, which are surrounded by a shell of numerous Kenyon cell (KC) dendritic tips. In the cricket Gryllus bimaculatus, high-resolution confocal laser scanning imaging revealed colocalization of f-actin with KC dendritic spine parts within MG. Although presynaptic boutons appear to be mainly devoid of f-actin-phalloidin fluorescence, there appears to be an accumulation of f-actin in KC dendritic spines synaptically contacting the boutons. Electron microscopy of boutons and dextran-stained KC dendrites revealed their pre- and postsynaptic sites, with KCs being strictly postsynaptic elements. Their subsynaptic membrane appositions are considered to be associated with f-actin. Focal accumulation of f-actin in the dendritic tips of KCs was found to be a general feature of MG, with either spheroidal or indented boutons of different sizes, as encountered in the mushroom bodies of the cricket, honey bee, ant, and fruit fly. The structural similarities of calycal MG and f-actin accumulation in KC dendrites with cerebellar microglomeruli are considered comparatively. The accumulation of f-actin in KC dendrites is discussed in view of mushroom body plasticity and its potential role in learning and memory formation.     

Neuronal assemblies of the Drosophila mushroom body

The mushroom body (MB) of the insect brain has important roles in odor learning and memory and in diverse other brain functions. To elucidate the anatomical basis underlying its function, we studied how the MB of Drosophila is organized by its intrinsic and extrinsic neurons. We screened for the GAL4 enhancer-trap strains that label specific subsets of these neurons and identified seven subtypes of Kenyon cells and three other intrinsic neuron types. Laminar organization of the Kenyon cell axons divides the pedunculus into at least five concentric strata. The alpha', beta', alpha, and beta lobes are each divided into three strata, whereas the gamma lobe appears more homogeneous. The outermost stratum of the alpha/beta lobes is specifically connected with a small, protruded subregion of the calyx, the accessory calyx, which does not receive direct olfactory input. As for the MB extrinsic neurons (MBENs), we found three types of antennal lobe projection neurons, among which two are novel. In addition, we resolved 17 other types of MBENs that arborize in the calyx, lobes, and pedunculus. Lobe-associated MBENs arborize in only specific areas of the lobes, being restricted along their longitudinal axes, forming two to five segmented zones in each lobe. The laminar arrangement of the Kenyon cell axons and segmented organization of the MBENs together divide the lobes into smaller synaptic units, possibly facilitating characteristic interaction between intrinsic and extrinsic neurons in each unit for different functional activities along the longitudinal lobe axes and between lobes. Structural differences between lobes are also discussed.     

Morphology of feedback neurons in the mushroom body of the honeybee,Apis mellifera

The anatomy of γ-aminobutyric acid (GABA)-immunoreactive, recurrent feedback neurons in the mushroom body (MB) of the honeybee, Apis mellifera, was investigated by using intraneuropilar injections of cobalt ions and light microscopic techniques. Each MB contains approximately 110 GABA-immunoreactive neurons, and approximately 50% of them are feedback neurons, i.e., they connect the MB output regions—the α-lobe, β-lobe, and pedunculus—with its input regions—the calyces. Their somata are located in the lateral protocerebral lobe, and their primary neurites project medially and bifurcate near the α-lobe. In the α-lobe feedback neurons form narrow banded, horizontal arborizations in the dorsal and median α-lobe; each cell innervates a certain α-lobe layer. The neurons form additional branches in the pedunculus and the β-lobe. All calycal subcompartments—the lip, collar, and basal ring—are innervated by feedback neurons. However, individual feedback neurons innervate exclusively a certain subcompartment in both the median and lateral calyx. Due to the arrangement of intrinsic Kenyon cells, each calycal subcompartment is connected to its specific, corresponding layer in the α-lobe. Feedback neurons interconnect the α-lobe and the calyces in either a corresponding or a noncorresponding fashion. With respect to their branching pattern in the α-lobe, the basal ring and the collar neuropil receive input from feedback neurons innervating the corresponding dorsal and median α-lobe layers. By contrast, the lip region, which receives olfactory antennal input, is innervated by feedback neurons with arborizations in a noncorresponding dorsal α-lobe layer. J. Comp. Neurol. 404:114–126, 1999. © 1999 Wiley-Liss, Inc.     

Different classes of input and output neurons reveal new features in microglomeruli of the adult Drosophila mushroom body calyx

To investigate how sensory information is processed, transformed, and stored within an olfactory system, we examined the anatomy of the input region, the calyx, of the mushroom bodies of Drosophila melanogaster. These paired structures are important for various behaviors, including olfactory learning and memory. Cells in the input neuropil, the calyx, are organized into an array of microglomeruli each comprising the large synaptic bouton of a projection neuron (PN) from the antennal lobe surrounded by tiny postsynaptic neurites from intrinsic Kenyon cells. Extrinsic neurons of the mushroom body also contribute to the organization of microglomeruli. We employed a combination of genetic reporters to identify single cells in the Drosophila calyx by light microscopy and compared these with cell shapes, synapses, and circuits derived from serial-section electron microscopy. We identified three morphological types of PN boutons, unilobed, clustered, and elongated; defined three ultrastructural types, with clear- or dense-core vesicles and those with a dark cytoplasm having both; reconstructed diverse dendritic specializations of Kenyon cells; and identified Kenyon cell presynaptic sites upon extrinsic neurons. We also report new features of calyx synaptic organization, in particular extensive serial synapses that link calycal extrinsic neurons into a local network, and the numerical proportions of synaptic contacts between calycal neurons. All PN bouton types had more ribbon than nonribbon synapses, dark boutons particularly so, and ribbon synapses were larger and with more postsynaptic elements (2-14) than nonribbon (1-10). The numbers of elements were in direct proportion to presynaptic membrane area. Extrinsic neurons exclusively had ribbon synapses.     

Motoneurons, DUM cells, and sensory neurons in an insect thoracic ganglion: a tracing study in the stick insect Carausius morosus

Anatomical features of leg motoneurons, dorsal unpaired median (DUM) cells, and sensory neurons in stick insect mesothoracic ganglia were examined using fluorescent dye backfills of lateral nerves. Structures were analyzed in whole-mounts of ganglia and transverse sections. Numbers of motoneurons and details of their structure by far exceed previously published data. The general neuroanatomical layout of motoneurons matches the general orthopteran pattern. Cell bodies of excitatory motoneurons form clusters in the lateral cortex, dendrites branch mainly in the dorsal neuropil. We identified nine DUM cells, six of which have axons in nerve nl5. Most sensory fibers terminate in the ventral association center (VAC). Twenty-three small cell bodies located close to the soma of the fast extensor tibiae motoneuron likely belong to strand receptors. Labeled structures are compared with previously published data from stick insects and other orthopterous insects.     

Anatomical basis of sun compass navigation I: the general layout of the monarch butterfly brain

Each fall, eastern North American monarch butterflies (Danaus plexippus) use a time-compensated sun compass to migrate to their overwintering grounds in central Mexico. The sun compass mechanism involves the neural integration of skylight cues with timing information from circadian clocks to maintain a constant heading. The neuronal substrates for the necessary interactions between compass neurons in the central complex, a prominent structure of the central brain, and circadian clocks are largely unknown. To begin to unravel these neural substrates, we performed 3D reconstructions of all neuropils of the monarch brain based on anti-synapsin labeling. Our work characterizes 21 well-defined neuropils (19 paired, 2 unpaired), as well as all synaptic regions between the more classically defined neuropils. We also studied the internal organization of all major neuropils on brain sections, using immunocytochemical stainings against synapsin, serotonin, and γ-aminobutyric acid. Special emphasis was placed on describing the neuroarchitecture of sun-compass-related brain regions and outlining their homologies to other migratory species. In addition to finding many general anatomical similarities to other insects, interspecies comparison also revealed several features that appear unique to the monarch brain. These distinctive features were especially apparent in the visual system and the mushroom body. Overall, we provide a comprehensive analysis of the brain anatomy of the monarch butterfly that will ultimately aid our understanding of the neuronal processes governing animal migration.     

Ventral nucleus of the lateral lemniscus in guinea pigs: Cytoarchitecture and inputs from the cochlear nucleus

Cytoarchitectonic criteria were used to distinguish three subdivisions of the ventral nucleus of the lateral lemniscus in guinea pigs. Axonal tracing techniques were used to examine the projections from the cochlear nucleus to each subdivision. Based on the cell types they contain and their patterns of input, we distinguished ventral, dorsal, and anterior subdivisions of the ventral nucleus of the lateral lemniscus. All three subdivisions receive bilateral inputs from the cochlear nucleus, with contralateral inputs greatly outnumbering ipsilateral inputs. However, the relative density of the inputs varies: the ventral subdivision receives the densest projection, whereas the anterior subdivision receives the sparsest projection. Further differences are apparent in the morphology of the afferent axons. Following an injection of Phaseolus vulgaris-leucoagglutinin into the ventral cochlear nucleus, most of the axons on the contralateral side and all of the axons on the ipsilateral side are thin. Thick axons are present only in the ventral subdivision contralateral to the injection site. The evidence from both anterograde and retrograde tracing studies suggests that the thick axons originate from octopus cells, whereas the thin axons arise from multipolar cells and spherical bushy cells. The differences in constituent cell types and in patterns of inputs suggest that each of the three subdivisions of the ventral nucleus of the lateral lemniscus makes a distinct contribution to the analysis of acoustic signals. J. Comp. Neurol. 379:363–385, 1997. © 1997 Wiley-Liss, Inc.     

Parasol cells of the hemiellipsoid body in the crayfish Procambarus clarkii: dendritic branching patterns and functional implications

Multimodal, higher-order sensory integration in decapod crustaceans occurs in local interneurons (parasol cells) within a structure in the lateral protocerebrum, the hemiellipsoid body, which is located dorsal to the terminal medulla. The hemiellipsoid body is targeted by projection neuron inputs by means of the olfactory globular tract from bilateral deutocerebral neuropils, the accessory lobes, which receive secondary visual, mechanosensory, and olfactory inputs. Parasol cell dendrites arborize extensively within the two neuropils of the hemiellipsoid body and possibly have some neurites within another neuropil at its base. The two neuropils of the hemiellipsoid body, neuropils I and II, are known to receive asymmetrical inputs from the contralateral and ipsilateral accessory lobes, and our current study addresses the question of the distribution of parasol cells within these two neuropils. Three anatomic methods were used to analyze this distribution: intracellular filling of cells with neurobiotin and visualization of the cells by using either a fluorescent or a peroxidase avidin conjugate, or placement of a fluorescent lipophilic tracer within a lobe of the hemiellipsoid body. All of these methods demonstrated that single parasol cells exclusively arborize within one of the two lobes of the hemiellipsoid body, but not in both lobes. Electrophysiological recordings from pairs of parasol cells with dendrites in the same or different lobes confirm a functional separation between neuropils I and II. Comparisons are made between insect and crustacean systems, emphasizing the inputs to the hemiellipsoid body and the mushroom body and similarities between extrinsic cells in insects and parasol cells in decapod crustaceans.     

Visual perception of bodily interactions in the primary somatosensory cortex

Brain imaging studies in humans have revealed the existence of a visuo-tactile system, which matches observed touch with felt touch. In this system, the primary somatosensory cortex (SI) appears to play a causal role in the visual processing of tactile events. Whether this visuo-tactile mechanism for touch in SI applies to the sight of 'any' touch, or whether it is restricted to the domain of body-related tactile experiences remains unresolved. To address this issue, repetitive transcranial magnetic stimulation (rTMS) was used to determine whether activity in SI is strictly related to the visual processing of human body-part interactions, or is also involved in processing the contact between inanimate objects, or between human body-parts and objects. The results show that rTMS over SI selectively impaired the processing of a contralateral visual stimulus depicting a human body-part being touched by a human agent, while it did not affect the visual perception of contact between objects, or between human body-parts and objects. Correlation analysis shows that this effect was associated with the intensity and embodiment of the observed touched. This result suggests that SI is more suited to represent social touch, contributing to our understanding of the effect of interpersonal tactile interactions between people.