Metamorphic development of locusta-tachykinin immunoreactive neurons of the antennal lobes of the beetle Tenebrio molitor and the effect of fenvalerate application

The effects of the widely used neurotoxic pyrethroid insecticides on neuronal development or plasticity are unclear. To expand knowledge about the influence of the pyrethroid fenvalerate on neuronal development, metamorphic remodelling of the primary olfactory neuropil of the beetle Tenebrio molitor has been studied. The antennal lobe is subdivided into distinct glomeruli before metamorphosis. This is in contrast to that which occurs in other well-studied holometabolous insects such as the moth Manduca sexta and the honeybee. As an indicator of antennal lobe interneurons, locusta-tachykinin immunoreactive neurons have been used. They project into the antennal lobes and form tufted arbors in larval and adult stages within glomeruli throughout the neuropil. These glomerular structures are invaded by glomerular sensory afferent axons and are surrounded by processes of glia cells. With pupation, the glomerulization is lost and no locusta-tachykinin or substance P immunoreactivity is visible in the antennal lobe. The immunoreactivity reappears during metamorphosis, starting with diffusely branched arbors that later become tufted. Application of the neurotoxic insecticide fenvalerate at pupation in sublethal concentrations resulted in a loss or reduction of glomerular pattern formation by neurons and glia cells during metamorphosis. Labelling of antennal sensory axons revealed that the olfactory neuropil was not deafferented, and also that the sensory axons were not organized into a normal glomerular pattern. In addition to the morphological differences, fenvalerate treatment caused locusta-tachykinin immunoreactivity to reappear prematurely during metamorphosis. Possible reasons for fenvalerate-induced alterations in antennal lobe development and their implications for normal development are discussed.     

Development of the labial pit organ glomerulus in the antennal lobe of the moth Manduca sexta: the role of afferent projections in the formation of identifiable olfactory glomeruli.

Iterated neuropil modules called glomeruli are characteristic of primary olfactory centers in both vertebrates and invertebrates. To gain insight into the developmental mechanisms underlying the formation of such structured, organized neuropil, we have examined the development of an identified glomerulus in the olfactory (antennal) lobe of the moth Manduca sexta. The labial pit organ glomerulus (LPOG) receives bilateral sensory projections from the labial pit organs in the labial palps of the mouthparts, while other glomeruli in the antennal lobe receive unilateral projections from the antenna. Here, we chronicle the development of the LPOG under normal and perturbed conditions. Our findings suggest that the sensory axons of the labial pit organ, like those of the antenna, induce and shape growth of interneuronal arborizations, but specific features of interneuronal arborizations such as the relative position of glomerular arborizations within the antennal lobe are independent of both classes of afferent innervation. Labial pit organ axons and antennal axons exhibit a high degree of specificity for their respective target regions, independent of the presence or absence of the other class of afferent axon or the route taken to the antennal lobe. Specification of glomerular position is intrinsic to the antennal lobe rather than a consequence of competition between afferent axons.     

Physiological mismatching between neurons innervating olfactory glomeruli in a moth

The primary olfactory centres of most vertebrates and most neopteran insects are characterized by the presence of spherical neuropils, glomeruli, where synaptic interactions between olfactory receptor neurons and second-order neurons take place. In the neopteran insect taxa investigated so far, receptor neurons of a specific physiological identity target one glomerulus and thus bestow a functional identity on the glomerulus. In moths, input from pheromone-specific receptor neurons is received in a male-specific structure of the antennal lobe, called the macroglomerular complex (MGC), which consists of a number of specialized glomeruli. Each glomerulus of the complex receives a set of peripheral sensory afferents that encode one of several compounds involved in sexual communication. The complex is also innervated by dendritic branches of antennal lobe output neurons called projection neurons, which transfer information from the antennal lobe to higher centres of the brain. A hypothesis stemming from earlier work on moths claims that the receptor neuron innervation pattern of the MGC should be reflected in the pattern of dendrites of projection neurons invading the different MGC glomeruli. In this study we show that in the noctuid moth Trichoplusia ni, as in several other noctuid moth species, this hypothesis does not hold. The degree of matching between axon terminals of receptor neurons and the dendritic branches of identified projection neurons that express similar physiological specificity is very low.     

Development of the labial pit organ glomerulus in the antennal lobe of the moth Manduca sexta: The role of afferent projections in the formation of identifiable olfactory glomeruli

Iterated neuropil modules called glomeruli are characteristic of primary olfactory centers in both vertebrates and invertebrates. To gain insight into the developmental mechanisms underlying the formation of such structured, organized neuropil, we have examined the development of an identified glomerulus in the olfactory (antennal) lobe of the moth Manduca sexta. The labial pit organ glomerulus (LPOG) receives bilateral sensory projections from the labial pit organs in the labial palps of the mouthparts, while other glomeruli in the antennal lobe receive unilateral projections from the antenna. Here, we chronicle the development of the LPOG under normal and perturbed conditions. Our findings suggest that the sensory axons of the labial pit organ, like those of the antenna, induce and shape growth of interneuronal arborizations, but specific features of interneuronal arborizations such as the relative position of glomerular arborizations within the antennal lobe are independent of both classes of afferent innervation. Labial pit organ axons and antennal axons exhibit a high degree of specificity for their respective target regions, independent of the presence or absence of the other class of afferent axon or the route taken to the antennal lobe. Specification of glomerular position is intrinsic to the antennal lobe rather than a consequence of competition between afferent axons. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 28–44, 1999     

A neuroanatomical study on the organization of the central antennal pathways in insects

1. Single unimodal (olfactory) or multimodal (olfactory and mechanosensory) neurons in the antennal lobe of the deutocerebrum of the American cockroach were characterized functionally by microelectrode recording, and their morphological types and positions in the brain were established by dye injection. Thus individual, physiologically identified neurons of known shape could be mapped in reference to the areas of soma groups, glomeruli, tracts and their projection regions in the brain. 2. All of these neurons send processes to deutocerebral glomeruli, i.e., the regions in which the axons of antennal sensory cells terminate. Output neurons have axons that leave the deutocerebrum whereas local interneurons are anaxonic. 3. An output neuron innervates only one glomerulus, sending its axon into the calyces of the corpora pedunculata (CP) in the protocerebrum, where by multiple branching they reach many CP neurons. The same axons send collaterals into the lateral lobe of the protocerebrum. Because of this arrangement, each deutocerebral glomerulus is represented individually and separately in the two projection regions. The fine structure of the endings of the deutocerebral axons in the protocerebrum is described. In the CP calyces they form microglomeruli with typical divergent connectivity. 4. A local interneuron innervates many glomeruli without sending processes to other parts of the brain. 5. Unimodal olfactory and multimodal neurons can be either output neurons or local interneurons; multimodal information is sent to the protocerebrum directly, in parallel with the unimodal information. 6. At least one glomerulus--the macroglomerulus of the male deutocerebrum--is specialized so as to provide an exclusive topographic representation of certain olfactory stimuli not represented elsewhere (female sexual pheromone).     

Antennal lobe projection destinations of Helicoverpa zea male olfactory receptor neurons responsive to heliothine sex pheromone components

We used single sensillum recordings to define male Helicoverpa zea olfactory receptor neuron physiology followed by cobalt staining to trace the axons to destination glomeruli of the antennal lobe. Receptor neurons in type A sensilla that respond to the major pheromone component, (Z)-11-hexadecenal, projected axons to the cumulus of the macroglomerular complex (MGC). In approximately 40% of these sensilla a second receptor neuron was stained that projected consistently to a specific glomerulus residing in a previously unrecognized glomerular complex with six other glomeruli stationed immediately posterior to the MGC. Cobalt staining corroborated by calcium imaging showed that receptor neurons in type C sensilla sensitive to (Z)-9-hexadecenal projected to the dorsomedial posterior glomerulus of the MGC, whereas the co-compartmentalized antagonist-sensitive neurons projected to the dorsomedial anterior glomerulus. We also discovered that the olfactory receptor neurons in type B sensilla exhibit the same axonal projections as those in type C sensilla. Thus, it seems that type B sensilla are anatomically type C with regard to the projection destinations of the two receptor neurons, but physiologically one of the receptor neurons is now unresponsive to everything except (Z)-9-tetradecenal, and the other responds to none of the pheromone-related odorants tested.     

Glomerular targets of Heliothis subflexa male olfactory receptor neurons housed within long trichoid sensilla

We used single-sensillum recordings to characterize male Heliothis subflexa antennal olfactory receptor neuron physiology in response to compounds related to their sex pheromone. The recordings were then followed by cobalt staining in order to trace the neurons' axons to their glomerular destinations in the antennal lobe. Receptor neurons responding to the major pheromone component, (Z)-11-hexadecenal, in the first type of sensillum, type-A, projected axons to the cumulus of the macroglomerular complex (MGC). In approximately 40% of the type-A sensilla, a colocalized receptor neuron was stained that projected consistently to the posterior complex 1 (PCx1), a specific glomerulus in an 8-glomerulus complex that we call the Posterior Complex (PCx). We found that receptor neurons residing in type-B sensilla and responding to a secondary pheromone component, (Z)-9-hexadecenal, send their axons to the dorsal medial glomerulus of the MGC. As in the type-A sensilla, we found a cocompartmentalized neuron within type-B sensilla that sends its axon to a different glomerulus of the PCx4. One neuron in type-C sensilla tuned to a third pheromone component, (Z)-11-hexadecenol, and a colocalized neuron responding to (Z)-11-hexadecenyl acetate projected their axons to the anteromedial and ventromedial glomeruli of the MGC, respectively.     

Glomerulus development in the absence of a set of mitral-like neurons in the insect olfactory lobe

Mitral cells are the first neurons in the mammalian olfactory bulb to synapse with olfactory receptor axons during glomerulus development, and in an invertebrate, the moth Manduca sexta, mitral-like neurons overlap very early with olfactory receptor axons as they begin to form protoglomeruli. The possibility for early interaction between receptor neurons and mitral-like neurons led us to ask whether such an interaction plays an essential role in glomerulus development. In the current study in the moth, we surgically removed a major class of these mitral-like neurons before glomeruli began to form and asked: (a) Is the formation of the array of olfactory glomeruli triggered by an interaction of the first-arriving receptor axons with the dendrites of mitral-like neurons? (b) At the level of individual glomeruli, must the mitral-like dendrites be in place either to maintain receptor axons in a glomerular arrangement, or to guide later-growing dendrites of other types into the developing glomeruli? Our results indicate that even without the participation of this group of mitral-like neurons, the array of sexually isomorphic ordinary glomeruli forms and the basic substructure of individual glomeruli develops apparently normally. We conclude that the mitral-like neurons in Manduca are not essential for the formation of ordinary olfactory glomeruli during development. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 41–52, 1998     

Distinct Projections of Two Populations of Olfactory Receptor Axons in the Antennal Lobe of the Sphinx Moth Manduca sexta

The central projections of olfactory receptor cells associatedwith two distinct types of antennal sensilla in the sphinx mothManduca sexta were revealed by anterograde staining. In bothsexes, receptor axons that arise from sexually isomorphic, type-IItrichoid sensilla (and possibly some basiconic sensilla) projectto the spheroidal glomeruli in the ipsilateral antennal lobe.Each axon terminates in one glomerulus. Axons from a limitedregion of the antenna project to glomeruli throughout the lobe,arguing against strict topographic mapping of antennal receptorcells onto the array of glomeruli. Axons of sex-pheromone-selectivereceptor cells in the male-specific type-I trichoid sensillaproject exclusively to the sexually dimorphic macroglomerularcomplex (MGC). Axons from sensilla on the dorsal surface ofthe antenna are biased toward the medial MGC and those fromventral sensilla, toward the lateral MGC. Some receptor-cellaxons branch before reaching the MGC, but their terminals arealways confined to one of the two main glomerular divisionsof the MGC, the cumulus and toroid. These findings confirm thatprimary-afferent information about pheromonal and non-pheromonalodors is segregated in the antennal lobe and suggest that thereis a functional correspondence between particular olfactoryreceptor cells and specific glomeruli. Chem. Senses 20: 313–323,1995.     

Physiological and morphological characterization of honeybee olfactory neurons combining electrophysiology,calcium imaging and confocal microscopy

The insect antennal lobe is the first brain structure to process olfactory information. Like the vertebrate olfactory bulb the antennal lobe is substructured in olfactory glomeruli. In insects, glomeruli can be morphologically identified, and have characteristic olfactory response profiles. Local neurons interconnect glomeruli, and output (projection) neurons project to higher-order brain centres. The relationship between their elaborate morphology and their physiology is not understood. We recorded electrophysiologically from antennal lobe neurons, and iontophoretically injected a calcium-sensitive dye. We then measured their spatio-temporal calcium responses to a variety of odours. Finally, we confocally reconstructed the neurons, and identified the innervated glomeruli. An increase or decrease in spiking frequency corresponded to an intracellular calcium increase or decrease in the cell. While intracellular recordings generally lasted between 10 and 30 min, calcium imaging was stable for up to 2 h, allowing a more detailed physiological analysis. The responses indicate that heterogeneous local neurons get input in the glomerulus in which they branch most strongly. In many cases, the physiological response properties of the cells corresponded to the known response profile of the innervated glomerulus. In other words, the large variety of response profiles generally found when comparing antennal lobe neurons is reduced to a more predictable response profile when the innervated glomerulus is known.Abbreviations ACT antenno-cerebralis-tract - AL antennal lobe - AP action potential - l-ACT lateral ACT - LN local neuron - LPL lateral protocerebral lobe - m-ACT medial ACT - MB mushroom body - OSN olfactory sensory neuron - PN projection neuron - T1 tract 1 of the antennal nerve     

Central projections of olfactory receptor neurons from single antennal and palpal sensilla in mosquitoes

In insects, olfactory receptor neurons (ORNs) are located in cuticular sensilla, that are present on the antennae and on the maxillary palps. Their axons project into spherical neuropil, the glomeruli, which are characteristic structures in the primary olfactory center throughout the animal kingdom. ORNs in insects often respond specifically to single odor compounds. The projection patterns of these neurons within the primary olfactory center, the antennal lobe, are, however, largely unknown.We developed a method to stain central projections of intact receptor neurons known to respond to host odor compounds in the malaria mosquito, Anopheles gambiae. Terminal arborizations from ORNs from antennal sensilla had only a few branches apparently restricted to a single glomerulus. Axonal arborizations of the different neurons originating from the same sensillum did not overlap.ORNs originating from maxillary palp sensilla all projected into a dorso-medial area in both the ipsi- and contralateral antennal lobe, which received in no case axon terminals from antennal receptor neurons. Staining of maxillary palp receptor neurons in a second mosquito species (Aedes aegypti) revealed unilateral arborizations in an area at a similar position as in An. gambiae.     

Olfactory activation patterns in the antennal lobe of the sphinx moth,<Emphasis Type="Italic"> Manduca sexta</Emphasis>

The sphinx moth Manduca sexta is a well-studied insect with regard to central olfactory functions. Until now, the innervation patterns of olfactory receptor neurons into the array of olfactory glomeruli in the antennal lobe have, however, been unclear. Using optical imaging to visualize calcium dynamics within the antennal lobe we demonstrate specific patterns elicited by sex pheromone components and plant-derived odours. These patterns mainly reflect receptor neuron activity. Within the male-specific macroglomerular complex the two major pheromone components evoke stereotyped activity in either of two macroglomerular complex glomeruli. Based on previous knowledge of output neuron specificity, our results suggest a matching of information between input and output in the macroglomerular complex. Plant odours evoked activity in the sexually isomorphic glomeruli. Two major results were obtained: (1). terpenes and aromatic compounds activate different clusters of glomeruli with only minor overlapping, and (2). the position of certain key glomeruli is fixed in both males and females, which suggests that host-plant related odorants are processed in a similar way in both sexes.     

Developmental origin of wiring specificity in the olfactory system of Drosophila

In both insects and mammals, olfactory receptor neurons (ORNs) expressing specific olfactory receptors converge their axons onto specific glomeruli, creating a spatial map in the brain. We have previously shown that second order projection neurons (PNs) in Drosophila are prespecified by lineage and birth order to send their dendrites to one of approximately 50 glomeruli in the antennal lobe. How can a given class of ORN axons match up with a given class of PN dendrites? Here, we examine the cellular and developmental events that lead to this wiring specificity. We find that, before ORN axon arrival, PN dendrites have already created a prototypic map that resembles the adult glomerular map, by virtue of their selective dendritic localization. Positional cues that create this prototypic dendritic map do not appear to be either from the residual larval olfactory system or from glial processes within the antennal lobe. We propose instead that this prototypic map might originate from both patterning information external to the developing antennal lobe and interactions among PN dendrites.     

The sox gene Dichaete is expressed in local interneurons and functions in development of the Drosophila adult olfactory circuit

In insects, the primary sites of integration for olfactory sensory input are the glomeruli in the antennal lobes. Here, axons of olfactory receptor neurons synapse with dendrites of the projection neurons that relay olfactory input to higher brain centers, such as the mushroom bodies and lateral horn. Interactions between olfactory receptor neurons and projection neurons are modulated by excitatory and inhibitory input from a group of local interneurons. While significant insight has been gleaned into the differentiation of olfactory receptor and projection neurons, much less is known about the development and function of the local interneurons. We have found that Dichaete, a conserved Sox HMG box gene, is strongly expressed in a cluster of LAAL cells located adjacent to each antennal lobe in the adult brain. Within these clusters, Dichaete protein expression is detected in both cholinergic and GABAergic local interneurons. In contrast, Dichaete expression is not detected in mature or developing projection neurons, or developing olfactory receptor neurons. Analysis of novel viable Dichaete mutant alleles revealed misrouting of specific projection neuron dendrites and axons, and alterations in glomeruli organization. These results suggest noncell autonomous functions of Dichaete in projection neuron differentiation as well as a potential role for Dichaete‐expressing local interneurons in development of the adult olfactory circuitry. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Glomerular interactions in olfactory processing channels of the antennal lobes

An open question in olfactory coding is the extent of interglomerular connectivity: do olfactory glomeruli and their neurons regulate the odorant responses of neurons innervating other glomeruli? In the olfactory system of the moth Manduca sexta, the response properties of different types of antennal olfactory receptor cells are known. Likewise, a subset of antennal lobe glomeruli has been functionally characterized and the olfactory tuning of their innervating neurons identified. This provides a unique opportunity to determine functional interactions between glomeruli of known input, specifically, (1) glomeruli processing plant odors and (2) glomeruli activated by antennal stimulation with pheromone components of conspecific females. Several studies describe reciprocal inhibitory effects between different types of pheromone-responsive projection neurons suggesting lateral inhibitory interactions between pheromone component-selective glomerular neural circuits. Furthermore, antennal lobe projection neurons that respond to host plant volatiles and innervate single, ordinary glomeruli are inhibited during antennal stimulation with the female’s sex pheromone. The studies demonstrate the existence of lateral inhibitory effects in response to behaviorally significant odorant stimuli and irrespective of glomerular location in the antennal lobe. Inhibitory interactions are present within and between olfactory subsystems (pheromonal and non-pheromonal subsystems), potentially to enhance contrast and strengthen odorant discrimination.     

Glial cells in the developing and adult olfactory lobe of the moth Manduca sexta

The antennal lobe of the moth contains several classes of glial cells that are likely to play functional roles in both the developing and mature lobe. In this study, confocal and electron microscopy were used to examine in detail the morphology of two classes of glial cells, those associated with olfactory receptor axons as they course to their targets in the lobe and those that form borders around the synaptic neuropil of the olfactory glomeruli. The former, the nerve-layer glia, have long processes with multiple expansions that enwrap axon fascicles; the latter, the neuropil glia, constitute two subgroups: complex glia with large cell bodies and branching, vellate arbors; and simple glia, with multiple, mostly unbranched processes with many lamellate expansions along their lengths. The processes of complex glia appear to be closely associated with axon fascicles as they enter the glomeruli, while those of the simple glia surround the glomeruli as part of a multi-lamellar glial envelope, their processes rarely invading the synaptic neuropil of the body of the glomerulus. The full morphological development of antennal-lobe glial cells requires more than two-thirds of metamorphic development. During this period, cells that began as cuboidal or spindle-shaped cells that were extensively dye-coupled to one another gradually assume their adult form and, at least under nonstimulated conditions, greatly reduce their coupling. These changes are only weakly dependent on the presence of olfactory receptor axons. Glial processes are somewhat shorter and less branched in the absence of these axons, but basic structure and degree of dye-coupling are unchanged.     

Multiple factors shape development of olfactory glomeruli: Insights from an insect model system

The antennal system of the moth Manduca sexta is a useful model for studies of the development of olfactory glomeruli, the complex synaptic structures that typically underlie the initial processing of olfactory input in vertebrates and invertebrates. In this review, we summarize cellular events in the construction of glomeruli in Manduca and highlight experiments that reveal factors that influence glomerulus development. By methodically manipulating each of various cell types, both neuronal and glial, that contribute to glomerular architecture, we have found that: olfactory receptor axons lay a template for developing glomeruli, stabilization of the template by glial cells is necessary to permit subsequent steps in development of the glomeruli, and the hormone that regulates adult development causes production of adequate numbers of glial cells. Neither electrical activity nor the presence of a serotonin-containing neuron that persists throughout development is required for a glomerular pattern to develop; these factors might, however, influence the synaptic organization of individual glomeruli. © 1996 John Wiley & Sons, Inc.     

昆虫触角叶的结构

触角叶是昆虫脑内初级嗅觉中心,通过触角神经与触角联系。触角叶主要由嗅觉受体神经元、局域中间神经元、投射神经元和远心神经元构成。这些神经元的形态多样,其形态变化与其功能和昆虫嗅觉行为相关。这些神经元在触角叶内交织形成神经纤维网,在突触联系紧密的地方形成纤维球,纤维球通常排列在触角叶外周。通常,昆虫触角叶内纤维球的数量、大小和位置相对固定,并且几乎每个小球都可以被识别和命名。不同种类、性别和品级的昆虫中,纤维球的数量、大小和排列方式各不相同。触角叶结构神经元组成和纤维球的多样性,与各种昆虫嗅觉行为的特异性相对应。     

Morphological and developmental analysis of peripheral antennal chemosensory sensilla and central olfactory glomeruli in worker castes of Camponotus compressus (Fabricius, 1787)

The antennal lobes of different castes of the ant species Camponotus compressus show a marked diversity in the organization of their olfactory glomeruli. Notably, there is a significant difference in the number and size of glomeruli between the reproductives and the workers and among the different worker castes. In this report, we investigate the notion that these caste-specific differences in glomerular number might be accounted for, at least in part, by the differences in numbers of olfactory sensilla that target the antennal lobe. For this, we examine the number of sensilla on the antennal flagella of all the individual castes of C. compressus. This analysis reveals a striking correlation between sensillar number and the number of antennal glomeruli in a given caste. As a first step in investigating the causal mechanisms that might give raise to this correlation, we carry out an initial characterization of olfactory system development in the minor workers of C. compressus. We analyze the temporal pattern of innervations of the developing antennal lobe by olfactory sensory neuron axons. We document the development of the olfactory glomeruli in the antennal lobe during this process, which occurs during early pupal stages. Our findings provide the basis for future manipulative developmental studies on the role of sensory afferent number in glomerular development of different castes within the same species.