Aspects of the embryology and neural development of the American lobster.

It is feasible to study the anatomical, physiological, and biochemical properties of identifiable neurons in lobster embryos. To exploit fully the advantages of this preparation and to lay the foundation for single-cell studies, our recent goals have been to 1) establish a quantitative staging system for embryos, 2) document in detail the lobster's embryonic development, 3) determine when uniquely identifiable neurons first acquire their transmitter phenotypes, and 4) identify particular neurons that may serve developmental functions. Behavioral, anatomical, morphometric, and immunocytochemical studies have led to a detailed characterization of the growth and maturation of lobster embryos and to the adoption of a percent-staging system based upon the eye index of Perkins (Fish. Bull., 70:95-99, 1972). It is clear from these studies that the lobster nauplius molts at approximately 12% embryonic development (E12%) into a metanauplius, which subsequently undergoes a complete molt cycle within the egg. This molt cycle climaxes with the emergence of the first-stage larva shortly after hatching. Serotonin and proctolin, neurohormones widely distributed in the lobster nervous system, appear at different times in development. Serotonin immunoreactive neurons begin to appear at approximately E10%, with the adult complement being established by E50%. In contrast, proctolin immunoreactive neurons appear later and attain their full complement over a protracted period including larval and juvenile stages. The development of serotonergic deutocerebral neurons and their targets, the olfactory and accessory lobes in the brain, are also examined. The olfactory lobes are forming by E10% and have acquired their glomerular organization by E50%, whereas the formation of the accessory lobes is delayed; the early rudiments of the accessory lobes are seen by E50%, and glomeruli do not form until the second larval stage.     

Serotonin depletion by 5,7-dihydroxytryptamine alters deutocerebral development in the lobster,Homarus americanus

The olfactory and accessory lobes constitute prominent histological structures within the larval and mature lobster deutocerebrum, and both are associated with a dense innervation from paired serotonergic nerve cells, the dorsal giant neurons (DGNs). During development, the cell bodies of the DGNs are the first central somata to express serotonin (5-HT), and the onset of their 5-HT immunoreactivity coincides with the beginning of accessory lobe formation. In contrast, the olfactory lobe anlagen emerge much earlier and grow in the apparent absence of serotonin. The role of serotonergic input for the development of these brain structures was investigated in lobster embryos after serotonin had been depleted pharmacologically with the neurotoxin 5,7-dihydroxytryptamine. A ∼90% reduction of serotonin was confirmed in eggs using high-performance liquid chromatography with electrochemical detection. Morphometric analyses suggested that serotonin depletion dramatically slowed the growth of olfactory and accessory lobes, although glomeruli differentiated at the normal time in both areas. The toxin exhibited a high degree of specificity for serotonergic neurons and associated target regions, and serotonin depletion persisted for at least 2 months following treatment. The goal of future experiments is to determine which of the cell types that innervate the olfactory and accessory lobes are affected by toxin treatment, thereby resulting in the retarded growth of these areas. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 357–373, 1997     

Aglomerular hemipteran antennal lobes--basic neuroanatomy of a small nose

We have compared the basic organization of the primary olfactory centre, the antennal lobe (AL), in 4 hemipteran species representing the 2 major lineages in this order. The Homoptera were represented by the psyllid Trioza apicalis and its aphid relatives the grain aphid Sitobion avenae Fabricius and the rose-grain aphid Metopolophium dirhodum Walker, whereas the Heteroptera were represented by the pentatomid stink bug Euschistus heros Fabricius. The olfactory systems of psyllids and aphids are generally very small, with low numbers of afferents in comparison to other insect groups, and the smallest described so far belongs to T. apicalis, comprising less than 50 olfactory receptor neurons (ORNs). Originally, we tried to estimate numbers of olfactory glomeruli in the AL of T. apicalis, which in insects generally correspond closely to the number of different types of ORNs. Neither immunocytochemical staining nor anterograde staining of ORNs revealed any glomerular structures in the ALs of T. apicalis or the 2 aphids that were included for comparison. In contrast, the ALs of the pentatomid stink bug E. heros displayed numerous distinct and well-delineated glomeruli, showing that aglomerular ALs are not typical of all insects within the order Hemiptera. Glomeruli are hallmark features of olfactory lobes in many different phyla, and the absence of glomerular structures in psyllids and aphids appears to be unique in insects that depend on olfactory orientation.     

Axons of olfactory receptor cells of transsexually grafted antennae induce development of sexually dimorphic glomeruli in Manduca sexta

The influence of olfactory receptor cell (ORC) axons from transsexually grafted antennae on the development of glomeruli in the antennal lobes (ALs), the primary olfactory centers, was studied in the moth Manduca sexta. Normally during metamorphic adult development, the pheromone-specific macroglomerular complex (MGC) forms only in the ALs of males, whereas two lateral female-specific glomeruli (LFGs) develop exclusively in females. A female AL innervated by ORC axons from a grafted male antenna developed an MGC with three glomeruli, like the MGC of a normal male AL. Conversely, a male AL innervated by ORC axons from a grafted female antenna lacked the MGC but exhibited LFGs. ORC axons from grafted male antenna terminated in the MGC-specific target area, even in cases when the antennal nerve (AN) entered the AL via an abnormal route. Within ectopic neuromas formed by ANs that had become misrouted and failed to enter the brain, male-specific axons were not organized and formed terminal branches in many areas. The results suggest the presence of guidance cues within the AL for male-specific ORC axons. Depending on the sex of the antennal innervation, glial borders formed in a pattern characteristic of the MGC or LFGs. The sex-specific number of projection neurons (PNs) in the medial group of AL neurons remained unaffected by the antennal graft, but significant changes occurred in the organization of PN arborizations. In gynandromorphic females, LFG-specific PNs extended processes into the induced MGC, whereas in gynandromorphic males, PNs became restricted to the LFGs. The results indicate that male-and female-specific ORC axons play important roles in determining the position, anatomical features, and innervation of sexually dimorphic glomeruli.     

Structure of cephalic ganglia and their changes in the post-embryonic development of <Emphasis Type="Italic">Calliphora vomitoria</Emphasis> (L.) (Diptera,Calliphoridae)

The central nervous system of Calliphora vomitoria larvae is situated in the metathoracic and the first abdominal segments and is characterized by a high degree of oligomerization. It consists of only two ganglia: the supraoesophageal ganglion, or brain, and one large synganglion, a product of fusion of the suboesophageal ganglion, three thoracic, and all the abdominal ganglia. Weak development of the neuropil of the larval optic and olfactory lobes in the supraoesophageal ganglion is the result of a significant reduction of the head capsule and sensory organs in the larvae. The formation of the imaginal optic lobes begins at the III larval instar. The commissure of the future central body is present in the I instar larva, but formation of the imaginal structure of the central complex proceeds in the 3-day pupae and ends at the late pupal stage. The mushroom bodies are represented in the I instar larvae only by the pedunculi; the calyces can be distinguished in the II instar larvae but the final formation of their structure and the lobes of the imaginal type occurs at the pupal stage. The glomeruli in the deutocerebrum are also formed at a late stage of pupal development. Based on the degree of development of ganglia of the central nervous system, we can conclude that individual development of higher Diptera is characterized by deep de-embryonization.     

The structural organization of glomerular neuropile in the olfactory and accessory lobes of an Australian freshwater crayfish,Cherax destructor

Summary The olfactory and accessory lobes of the crayfish, Cherax destructor contain glomeruli. Light microscope and electron microscope studies show that these glomeruli are the only regions of synaptic activity in the lobes and that at least four separate sets of axons meet within the glomeruli. The olfactory glomeruli are column shaped, complex structures with no large single pre- or postsynaptic elements. The accessory lobe glomeruli follow a more conventional pattern and each has one large axon ending in a terminal arborization where it makes synaptic contact with large numbers of smaller fibres. The large fibre is presynaptic.     

Constancy and variability of identified glomeruli in antennal lobes: computational approach in <Emphasis Type="Italic">Spodoptera littoralis</Emphasis>

The primary olfactory centres share striking similarities across the animal kingdom. The most conspicuous is their subdivision into glomeruli, which are spherical neuropil masses in which synaptic contacts between sensory and central neurons occur. Glomeruli have both an anatomical identity (being invariant in location, size and shape) and a functional identity (each glomerulus receiving afferents from olfactory receptor neurons that express the same olfactory receptor). Identified glomeruli offer a favourable system for analysing quantitatively the constancy and variability of the neuronal circuits, an important issue for understanding their function, development and evolution. The noctuid moth Spodoptera littoralis with its well-studied pheromone communication system has become a model species for olfaction research. We analyse here its glomerular organisation based on ethyl-gallate-stained and synapsin-stained preparations. Although we have confirmed that the majority of glomeruli can be individually identified in various antennal lobes, we have recognised several types of biological variability. Some glomeruli are absent, possibly indicating the lack of the corresponding receptor neuron type or its misrouting during development. The antennal lobes vary in global shape and, consequently, the spatial location of the glomerular changes. Although they do not prevent glomerulus identification when quantitative analysis methods are used, these variations place limits on the straightforward identification of glomeruli in functional studies, e.g. calcium-imaging or single-cell staining, when using conventional three-dimensional maps of individual antennal lobes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users. This work was supported by research grants from INRA (Projet Jeune Equipe and Projet S.P.E.) to S.A. and J.P.R. and from ANR-BBSRC 07 BSYS 006 (Pherosys) to J.P.R. and S.A. and by a PhD grant from INRA Departments M.I.A. and S.P.E. to L.C.     

Constancy and variability of glomerular organization in the antennal lobe of the silkmoth

We investigated the anatomical organization of glomeruli in the antennal lobes (ALs) of male silkmoths. We reconstructed 10 different ALs and established an identification procedure for individual glomeruli by using size, shape, and position relative to anatomical landmarks. Quantitative analysis of these morphological characteristics supported the validity of our identification strategy. The glomerular organization of the ALs was roughly conserved between different ALs. However, we found individual variations that were reproducibly observed. The combination of a digital atlas with other experimental techniques, such as electrophysiology, optical imaging, and genetics, should facilitate a more in-depth analysis of sensory information processing in silkmoth ALs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Tomoki Kazawa and Shigehiro Namiki contributed equally to this work. This research was supported by Grant-in-Aid for Scientific Research from the Japan Ministry of Education, Culture, Sports, Science, and Technology (area no. 454, to R.K.).     

Spatial representation of odours in the antennal lobe of the moth Spodoptera littoralis (Lepidoptera: Noctuidae)

Glomeruli within the antennal lobe (AL) of moths are convergence sites for a large number of olfactory receptor neurons (ORNs). The ORNs target single glomeruli. In the male-specific cluster of glomeruli, the macroglomerular complex (MGC), the input is chemotypic in that each glomerulus of the MGC receives information about a specific component of the conspecific female sex pheromone. Little is known about how neurons that detect other odorants arborize in and amongst glomeruli. The present study focuses on how sex pheromones and biologically relevant semiochemicals are represented in the ALs of both sexes of the moth Spodoptera littoralis. To assess this, we optically measured odour-evoked changes of calcium concentration in the ALs. Foci of calcium increase corresponded in size and shape with anatomical glomeruli. More than one glomerulus was normally activated by a specific non-pheromonal odorant and the same glomerulus was activated by several odorants. All odorants and pheromone components tested evoked unique patterns of glomerular activity that were highly reproducible at repeated stimulations within an individual. Odour-evoked patterns were similar between individuals for a given odorant, implicating a spatial olfactory code. In addition, we demonstrated that activity patterns evoked by host-plant related volatiles are similar between males and females.     

The brighter side of climate change: How local oceanography amplified a lobster boom in the Gulf of Maine

Ocean warming can drive poleward shifts of commercially important species with potentially significant economic impacts. Nowhere are those impacts greater than in the Gulf of Maine where North America's most valuable marine species, the American lobster (Homarus americanus Milne Edwards), has thrived for decades. However, there are growing concerns that regional maritime economies will suffer as monitored shallow water young‐of‐year lobsters decline and landings shift to the northeast. We examine how the interplay of ocean warming, tidal mixing, and larval behavior results in a brighter side of climate change. Since the 1980s lobster stocks have increased fivefold. We suggest that this increase resulted from a complex interplay between lobster larvae settlement behavior, climate change, and local oceanographic conditions. Specifically, postlarval sounding behavior is confined to a thermal envelope above 12°C and below 20°C. Summer thermally stratified surface waters in southwestern regions have historically been well within the settlement thermal envelope. Although surface layers are warming fastest in this region, the steep depth‐wise temperature gradient caused thermally suitable areas for larval settlement to expand only modestly. This contrasts with the northeast where strong tidal mixing prevents thermal stratification and recent ocean warming has made an expansive area of seabed more favorable for larval settlement. Recent declines in lobster settlement densities observed at shallow monitoring sites correlate with the expanded area of thermally suitable habitat associated with warmer summers. This leads us to hypothesize that the expanded area of suitable habitat may help explain strong lobster population increases in this region over the last decade and offset potential future declines. It also suggests that the fate of fisheries in a changing climate requires understanding local interaction between life stage‐specific biological thresholds and finer scale oceanographic processes.     

中华蜜蜂嗅叶胚后发育过程中的神经胶质

本研究通过形态解剖、免疫组织化学等技术,对中华蜜蜂Apis cerana cerana Fabricius工蜂嗅叶胚后发育中神经胶质的模式进行了比较研究。结果表明:神经胶质在蜜蜂嗅叶的发育过程中起着重要的作用,它们在嗅叶发育早期就划定了神经纤维网的边界;在嗅觉神经轴突进入嗅叶之前,对神经轴突进行"分选",并引导它们进入嗅叶特定的区域,形成神经纤维球;它们不仅规定了神经纤维网的边界和区域,还为神经纤维网提供内部的分隔。中华蜜蜂神经胶质增殖的高峰期集中在幼虫发育末期和预蛹期。     

Mas-allatotropin in the developing antennal lobe of the sphinx moth Manduca sexta: distribution, time course, developmental regulation, and colocalization with other neuropeptides

The paired antennal lobes (ALs) of the sphinx moth Manduca sexta serve as a well-established model for studying development of the primary integration centers for odor information in the brain. To further reveal the role of neuropeptides during AL development, we have analyzed cellular distribution, developmental time course, and regulation of the neuropeptide M. sexta allatotropin (Mas-AT). On the basis of morphology and appearance during AL formation, seven major types of Mas-AT-immunoreactive (ir) cells could be distinguished. Mas-AT-ir cells are identified as local, projection, and centrifugal neurons, which are either persisting larval or newly added adult-specific neurons. Complementary immunostaining with antisera against two other neuropeptide families (A-type allatostatins, RFamides) revealed colocalization within three of the Mas-AT-ir cell types. On the basis of this neurochemistry, the most prominent type of Mas-AT-ir neurons, the local AT neurons (LATn), could be divided in three subpopulations. The appearance of the Mas-AT-ir cell types occurring during metamorphosis parallels the rising titer of the developmental hormone 20-hydroxyecdysone (20E). Artificially shifting the 20E titer to an earlier developmental time point resulted in the precocious occurrence of Mas-AT immunostaining. This result supports the hypothesis that the pupal rise of 20E is causative for Mas-AT expression during AL development. Comparing localization and developmental time course of Mas-AT and other neuropeptides with the time course of AL formation suggests various functions for these neuropeptides during development, including an involvement in the formation of the olfactory glomeruli.     

Maturation of odor representation in the honeybee antennal lobe

The antennal lobe (AL) is the first center for processing odors in the insect brain, as is the olfactory bulb (OB) in vertebrates. Both the AL and the OB have a characteristic glomerular structure; odors sensed by olfactory receptor neurons are represented by patterns of glomerular activity. Little is known about when and how an odor begins to be perceived in a developing brain. We address this question by using calcium imaging to monitor odor-evoked neural activity in the ALs of bees of different ages. We find that odor-evoked neural activity already occurs in the ALs of bees as young as 1 or 2 days. In young bees, the responses to odors are relatively weak and restricted to a small number of glomeruli. However, different odors already evoke responses in different combinations of glomeruli. In mature bees, the responses are stronger and are evident in more glomeruli, but continue to have distinct odor-dependent signatures. Our findings indicate that the specific glomerular patterns for odors are conserved during the development, and that odor representations are fully developed in the AL during the first 2 weeks following emergence.     

Postembryonic development of the antennal lobes in Periplaneta americana L.

Summary The postembryonic development of the antennal lobes of Periplaneta americana L. was examined with light- and electron-microscopical methods. There is no difference in the number of glomeruli and neurons in the antennal lobes of larval and adult animals. At hatching, the first larva already possesses the adult number of approximately 125 glomeruli and 500 to 560 deutocerebral neurons in the dorsolateral cell group of each antennal lobe. During postembryonic development the volume of the deutocerebral neurons increases three- to fourfold. The glomeruli of the first larva have about 7 % of the volume of the corresponding adult glomeruli. Since number, pattern, and size ratio of glomeruli (with the exception of the macroglomerulus) are constant in all larval stages and adult animals, it is possible to identify individual glomeruli. During the whole postembryonic development the ordinary glomeruli show a continuous volume increase, which parallels the increase in antennal sensory input. The macroglomerulus develops by way of special growth of two to four neuropil units, but not before the last three to four larval stages and only in males. Its growth precedes the formation of antennal pheromone receptors during the final molt; these receptors are known to project into the macroglomerulus. The development of the macroglomerulus in the last larval stages of the male may be caused by a genetically fixed growth program of specific deutocerebral neurons.Supported by the Deutsche Forschungsgemeinschaft (Scha 291/1)     

The organization of the olfactory lobes in Euphausiacea and Mysidacea (Crustacea,Malacostraca)

Summary The structural organization of the olfactory lobes in representatives of euphausiid and mysid crustaceans was investigated and compared, also with these structures described in other crustaceans and in insects. In the investigated euphasiid and mysid species, the olfactory-globular tract and the position of cell clusters associated with the olfactory lobes show a similar arrangement. This arrangement is in agreement with that described in decapod crustaceans. The olfactory lobe neuropil in representatives of both taxa shows glomerular arrangement. These glomeruli are partly enclosed in a glial wrapping, and they represent the only site where synaptic contacts are established within the olfactory lobes. This glomerular arrangement appears similar to that described in the antennal lobe of insects, but differs from the columnar arrangement described in decapod crustaceans. Furthermore, about 15–20 FMRFamide-like immunoreactive globuli cells were labeled and they occupy a similar position in the investigated species. Neurites from these cells terminate only in the olfactory lobe glomeruli, and they are therefore regarded as intrinsic interneurons. The absence of serotonin-like immunoreactivity in the olfactory lobes is a feature only ascribed to the euphausiids and mysids. A specific neuropil area is present in male mysids, and it occupy a position forward of the olfactory lobe. The male-specific neuropil in mysids and the macro-glomerular in insects complex are interpreted as analogous structures.     

Caste-specific postembryonic development of primary and secondary olfactory centers in the female honeybee brain

Eusocial insects are characterized by division of labor among a sterile worker caste and a reproductive queen. In the honeybee both female castes are determined postembryonically by environmental factors, and queens develop substantially faster than workers. Since olfaction plays a crucial role in organizing honeybee behavior and social interactions, we compared the development of primary and secondary olfactory centers in the brain. Age-synchronized queen and worker pupae were raised in incubators at 34.5 degrees C, and their external morphology was characterized for all pupal stages. The development of olfactory synaptic neuropil was analyzed using anti-synapsin immunocytochemistry, f-actin-phalloidin labeling and confocal microscopy. In the antennal lobes of queens olfactory glomeruli formed approximately 4 days earlier than in workers. The adult number of olfactory glomeruli was in a similar range, but the total glomerular volume was slightly smaller in queens. Olfactory and visual subdivisions (lip, collar) of the mushroom-body calyx formed early, whereas the basal ring separated late. Synaptic microglomeruli in the olfactory lip were established approximately 3-4 days earlier in queens compared to workers. We propose that developmental heterochrony results in fewer synapses in olfactory centers (smaller glomeruli, fewer microglomeruli) in queens, which may result in poorer performance on olfactory learning tasks compared to workers.     

Organization of the Olfactory and Respiratory Skeleton in the Nose of the Gray Short-Tailed Opossum Monodelphis domestica

The internal nasal skeleton in Monodelphis domestica, the gray short-tailed opossum, primarily supports olfactory and respiratory epithelia, the vomeronasal organ, and the nasal gland. This scaffold is built by the median mesethmoid, and the paired vomer and ethmoid bones. The mesethmoid ossifies within the nasal septum cartilage. The bilateral ethmoid segregates respiratory and olfactory regions, and its geometry offers insight into the functional, developmental, and genomic organization of the nose. It forms through partial coalescence of separate elements known as turbinals, which in Monodelphis comprise the maxilloturbinal, nasoturbinal, five endoturbinals, and two ectoturbinals. Geometry of the ethmoid increases respiratory mucosal surface area by a factor of six and olfactory mucosal surface by nearly an order of magnitude. Respiratory epithelium warms and humidifies inspired air, recovers moisture as air is exhaled, and may help mediate brain temperature. In contrast, the olfactory skeleton functions as a series of small funnels that support growth of new olfactory neurons throughout life. Olfactory mucosa lines the mouth of each funnel, forming blind olfactory recesses known as the ethmoid cells, and neuronal axons are funneled from the epithelium through tiny olfactory foramina in the cribriform plate, into close proximity with target glomeruli in the olfactory bulb of the brain where each axon makes its first synapse. The skeleton may thus mediate topological correspondence between odorant receptor areas in the nose with particular glomeruli in the olfactory bulb, enabling growth throughout life of new olfactory neurons and proper targeting by their axons. The geometric arrangement of odorant receptors suggests that a measure of volatility may be a component in the peripheral olfactory code, and that corresponding glomeruli may function in temporal signal processing. Supporting visualizations for this study are available online at www.DigiMorph.org.     

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.     

Distribution of neuropeptides in the antennal lobes of male Spodoptera littoralis

Olfaction is an important sensory modality that regulates a plethora of behavioural expressions in insects. Processing of olfactory information takes place in the primary olfactory centres of the brain, namely the antennal lobes (ALs). Neuropeptides have been shown to be present in the olfactory system of various insect species. In the present study, we analyse the distribution of tachykinin, FMRFamide-related peptides, allatotropin, allatostatin, myoinhibitory peptides and SIFamide in the AL of the male Egyptian cotton leafworm, Spodoptera littoralis. Immunocytochemical analyses revealed that most neuropeptides were expressed in different subpopulations of AL neurons. Their arborisation patterns within the AL suggest a significant role of neuropeptide signalling in the modulation of AL processing. In addition to local interneurons, our analysis also revealed a diversity of extrinsic peptidergic neurons that connected the antennal lobe with other brain centres. Their distributions suggest that extrinsic neurons perform various types of context-related modulation.