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.     

Visual inputs to the mushroom body calyces of the whirligig beetle Dineutus sublineatus: modality switching in an insect

The mushroom bodies are prominent lobed centers in the forebrain, or protocerebrum, of most insects. Previous studies on mushroom bodies have focused on higher olfactory processing, including olfactory-based learning and memory. Anatomical studies provide strong support that in terrestrial insects with mushroom bodies, the primary input region, or calyces, are predominantly supplied by olfactory projection neurons from the antennal lobe glomeruli. In aquatic species that generally lack antennal lobes, the calyces are vestigial or absent. Here we report an exception to this in the whirligig beetle Dineutus sublineatus (Coleoptera: Gyrinidae). This aquatic species lives on water and is equipped with two separate pairs of compound eyes, one pair viewing above and one viewing below the water surface. As in other aquatic insects, the whirligig beetle lacks antennal lobes, but unlike other aquatic insects its mushroom bodies possess robust calyces. Golgi impregnations and fluorescent tracer injections revealed that the calyces are exclusively supplied by visual neurons from the medulla of the dorsal eye optic lobes. No other sensory inputs reach the calyces, thereby showing a complete switch of calyx modality from olfaction to vision. Potential functions of the mushroom bodies of D. sublineatus are discussed in the context of the behavioral ecology of whirligig beetles.     

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.     

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.     

Subdivisions of hymenopteran mushroom body calyces by their afferent supply.

The mushroom bodies are regions in the insect brain involved in processing complex multimodal information. They are composed of many parallel sets of intrinsic neurons that receive input from and transfer output to extrinsic neurons that connect the mushroom bodies with the surrounding neuropils. Mushroom bodies are particularly large in social Hymenoptera and are thought to be involved in the control of conspicuous orientation, learning, and memory capabilities of these insects. The present account compares the organization of sensory input to the mushroom body's calyx in different Hymenoptera. Tracer and conventional neuronal staining procedures reveal the following anatomic characteristics: The calyx comprises three subdivisions, the lip, collar, and basal ring. The lip receives antennal lobe afferents, and these olfactory input neurons can terminate in two or more segregated zones within the lip. The collar receives visual afferents that are bilateral with equal representation of both eyes in each calyx. Visual inputs provide two to three layers of processes in the collar subdivision. The basal ring is subdivided into two modality-specific zones, one receiving visual, the other antennal lobe input. Some overlap of modality exists between calycal subdivisions and within the basal ring, and the degree of segregation of sensory input within the calyx is species-specific. The data suggest that the many parallel channels of intrinsic neurons may each process different aspects of sensory input information.     

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

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

Development-dependent and -independent ubiquitin expression in divisions of the cockroach mushroom body

It has been proposed that the alpha and beta divisions of the mushroom bodies support intermediate and long-term memory whereas the gamma lobes support short-term memory. Here we investigate developmentally dependent versus developmentally independent alterations of mushroom body structure with special emphasis on its lobes. We show that in the cockroach mushroom bodies there are two types of plastic remodeling. One is developmental, in which episodic addition of new circuitry to the alpha and beta lobes is accomplished by newly born Kenyon cells. The second is revealed as a persistent alteration of structure within the gamma lobe. In the alpha/beta lobes, newly generated Kenyon cell axons extend glutamate-immunoreactive collaterals across layers of the axons of mature Kenyon cells. At specific times in each developmental episode (instar) these collaterals express ubiquitin, undergo localized degeneration, and are scavenged by glial cells. In contrast, the mature Kenyon cells that comprise the gamma lobe express detectable ubiquitin throughout each developmental episode. This pattern of ubiquitin expression suggests that the gamma lobe circuitry undergoes continuous modification independent of development.     

Representation of the brain's superior protocerebrum of the flesh fly, Neobellieria bullata, in the central body

The central complex of the insect brain is a system of midline neuropils involved in transforming sensory information into behavioral outputs. Genetic studies focusing on nerve cells supplying the central complex from the protocerebrum propose that such neurons play key roles in circuits involved in learning the distinction of visual cues during operant conditioning. To better identify the possible sites of such circuits we used Bodian and anti-synapsin staining to resolve divisions of the superior protocerebrum into discrete neuropils. Here we show that in the fly Neobellieria bullata, the superior protocerebrum is composed of at least five clearly defined regions that correspond to those identified in Drosophila melanogaster. Intracellular dye fills and Golgi impregnations resolve "tangential neurons" that have intricate systems of branches in two of these regions. The branches are elaborate, decorated with specializations indicative of pre- and postsynaptic sites. The tangentially arranged terminals of these neurons extend across characteristic levels of the central complex's fan-shaped body. In this and another blowfly species, we identify an asymmetric pair of neuropils situated deep in the fan-shaped body, called the asymmetric bodies because of their likely homology with similar elements in Drosophila. One of the pair of bodies receives collaterals from symmetric arrangements of tangential neuron terminals. Cobalt injections reveal that the superior protocerebrum is richly supplied with local interneurons that are likely participants in microcircuitry associated with the distal processes of tangential neurons. Understanding the morphologies and arrangements of these and other neurons is essential for correctly interpreting functional attributes of the central complex.     

Postembryonic development of γ-aminobutyric acid-like Immunoreactivity in the brain of the sphinx moth Manduca sexta

We have investigated the distribution of immunocytochemical staining for the neurotransmitter γ-aminobutyric acid (GABA) in the brain of the sphinx moth Manduca sexta during larval, pupal, and adult development. In the larval brain, about 300 neurons are GABA-immunoreactive. All neuropil areas except the mushroom bodies and central complex show intense immunostaining. Only minor changes in the pattern of immunoreactivity occur during larval development. During metamorphosis, changes in immunostaining occur in two phases. Beginning in wandering fifth-instar larvae (stage W2), immunoreactivity appears in numerous neurons of the central body and optic lobe and becomes more intense during early pupal stages. At the same time, GABA-like immunoreactivity disappears in most neuropil areas of the brain and becomes faint in many immunoreactive somata. Neurons with arborizations in the ventrolateral protocerebrum, however, continue to exhibit intense immunostaining during this period, and strongly immunolabeled fibers connect these areas with the ventral nerve cord. The second phase of transformation begins around pupal stage P5/P6, when faint immunostaining appears in many previously nonimmunoreactive somata and most neuropil areas of the brain. In subsequent stages (P8–P10), this immunoreactivity disappears again in most somata, but in certain cell groups, it becomes more intense and gradually develops to the adult pattern. Most larval GABA-immunoreactive neurons appear to survive through metamorphosis into the adult. Neurons in the midbrain that acquire GABA-like immunoreactivity during metamorphosis usually lie adjacent to larval immunostained neurons, suggesting common lineages. The onsets of the two developmental phases of GABA-like immunoreactivity correlate with sharp rises in hemolymph titers of ecdysteroid hormones, suggesting a role for ecdysteroids in the regulation of GABA synthesis. We hypothesize that the disappearance of GABA in many areas of the brain starting 2 days prior to pupation dramatically alters its functional circuitry and thus may account for profound changes in the behavior of the animal. © 1994 Wiley-Liss, Inc.     

A multiterminal stretch receptor, chordotonal organ, and hair plate at the wing-hinge of Manduca sexta: unravelling the mystery of the noctuid moth ear B cell.

The present study aims to shed light on the evolutionary origin of the B cell, a sensory element of unknown function in the noctuid moth ear. Peripheral projections of the metathoracic nerve IIIN1b1, homologue of the noctuid moth tympanic nerve, are described in the atympanate moth Manduca sexta on the basis of dissections with the aid of Janus Green B, and intracellular tracer dyes Lucifer yellow and cobalt lysine. A large multiterminal (Type II) neurone, attaching to membranous cuticle ventral to the hind wing axillary cord, was discovered. This cell appears to be homologous to the B cell in the noctuid moth ear. Recordings from the IIIN1b1 nerve in M. sexta reveal a continuous train of large, uniform spikes, presumed to originate from the multiterminal cell. This unit increases its rate of firing in response to hind wing elevation, suggesting that it functions as a stretch receptor monitoring wing movements during flight. Also identified in the tympanic nerve homologue, and closely associated with the multiterminal cell, were a chordotonal organ and hair plate. The chordotonal organ consists of a proximal scolopidial region and a distal strand that attaches to the sclerotized epimeron slightly medial to the multiterminal cell. This simple chordotonal organ, having three uniterminal (Type I) sensory cells, is homologous to the auditory cells of the noctuid moth ear. The significance of these receptors as proprioceptors in M. sexta, and as evolutionary precursors to the noctuid moth ear, is 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.     

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.     

A map of octopaminergic neurons in the Drosophila brain

The biogenic amine octopamine modulates diverse behaviors in invertebrates. At the single neuron level, the mode of action is well understood in the peripheral nervous system owing to its simple structure and accessibility. For elucidating the role of individual octopaminergic neurons in the modulation of complex behaviors, a detailed analysis of the connectivity in the central nervous system is required. Here we present a comprehensive anatomical map of candidate octopaminergic neurons in the adult Drosophila brain: including the supra‐ and subesophageal ganglia. Application of the Flp‐out technique enabled visualization of 27 types of individual octopaminergic neurons. Based on their morphology and distribution of genetic markers, we found that most octopaminergic neurons project to multiple brain structures with a clear separation of dendritic and presynaptic regions. Whereas their major dendrites are confined to specific brain regions, each cell type targets different, yet defined, neuropils distributed throughout the central nervous system. This would allow them to constitute combinatorial modules assigned to the modulation of distinct neuronal processes. The map may provide an anatomical framework for the functional constitution of the octopaminergic system. It also serves as a model for the single‐cell organization of a particular neurotransmitter in the brain. J. Comp. Neurol. 513:643–667, 2009. © 2009 Wiley‐Liss, Inc.     

Intrinsic neurons of Drosophila mushroom bodies express short neuropeptide F: relations to extrinsic neurons expressing different neurotransmitters

Mushroom bodies constitute prominent paired neuropils in the brain of insects, known to be involved in higher olfactory processing and learning and memory. In Drosophila there are about 2,500 intrinsic mushroom body neurons, Kenyon cells, and a large number of different extrinsic neurons connecting the calyx, peduncle, and lobes to other portions of the brain. The neurotransmitter of the Kenyon cells has not been identified in any insect. Here we show expression of the gene snpf and its neuropeptide products (short neuropeptide F; sNPFs) in larval and adult Drosophila Kenyon cells by means of in situ hybridization and antisera against sequences of the precursor and two of the encoded peptides. Immunocytochemistry displays peptide in intrinsic neuronal processes in most parts of the mushroom body structures, except for a small core in the center of the peduncle and lobes and in the alpha'- and beta'-lobes. Weaker immunolabeling is seen in Kenyon cell bodies and processes in the calyx and initial peduncle and is strongest in the more distal portions of the lobes. We used different antisera and Gal4-driven green fluorescent protein to identify Kenyon cells and different populations of extrinsic neurons defined by their signal substances. Thus, we display neurotransmitter systems converging on Kenyon cells: neurons likely to utilize dopamine, tyramine/octopamine, glutamate, and acetylcholine. Attempts to identify other neurotransmitter components (including vesicular glutamate transporter) in Kenyon cells failed. However, it is likely that the Kenyon cells utilize an additional neurotransmitter, yet to be identified, and that the neuropeptides described here may represent cotransmitters.     

Amelα8 subunit knockdown in the mushroom body vertical lobes impairs olfactory retrieval in the honeybee,Apis mellifera

We studied the involvement of the α8 subunit of nicotinic acetylcholine receptors (nAChRs) in olfactory learning and memory in Apis mellifera. We have previously shown, by injecting different nicotinic antagonists into the bee brain, that pharmacologically different subtypes of nAChRs are important for honeybee memory –α‐bungarotoxin‐sensitive receptors are necessary for memory consolidation and mecamylamine‐sensitive receptors are involved in retrieval processes. Here, we took advantage of the honeybee genome sequencing and the development of a small interfering RNA (siRNA) tool to focus on the role of the α8 subunit, which has been shown to be expressed in brain areas important for olfactory learning, such as the antennal lobes and mushroom bodies. We first demonstrated the efficacy of the siRNA tool by showing a decrease of the α8 protein level at 6 h after brain injection of α8 siRNA. We then tested the general role of this subunit in olfactory conditioning, using brain systemic or localized siRNA injections in the antennal lobes or the calyces and vertical lobes of the mushroom bodies. These injections were performed at either 6 h before the learning acquisition or 6 h before the memory test. The most prominent result was that 6‐h pre‐test injection of siRNA in the mushroom body vertical lobes impaired memory retrieval at 24 and 48 h post‐training. This indicated the importance of cholinergic extrinsic neurons and nAChRs containing the α8 subunit for this process.     

Quantitative analysis of olfactory receptor neuron projections in the antennal lobe of the malaria mosquito, Anopheles gambiae

Mosquitoes are highly dependent on the olfactory sense to find their hosts. How olfactory information concerning host odors is represented and processed in the brain to elicit olfactory guided behavior is not known. We present an exploratory analysis of central projections of olfactory receptor neurons originating from antennal and maxillary palp sensilla known to be involved in the detection of host odors in the malaria mosquito, Anopheles gambiae. We developed computational neuroanatomic methods to determine quantitatively the positions of olfactory receptor neuron terminal arborizations and compare them between brains. These quantitative analyses suggested the existence of five nonoverlapping projection zones within the antennal lobe, with one zone receiving exclusive input from maxillary palp sensilla and two zones each receiving exclusive input from trichoid or grooved-peg antennal sensilla. Projection patterns were not found to depend significantly on the odorants used during the staining procedure. The separate zones receiving input from different sensillum types seemed to represent a functional segregation because olfactory receptor neurons present in the different sensilla differed in their response profiles.     

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.