Patterns of appearance of serotonin and proctolin immunoreactivities in the developing nervous system of the American lobster

Serotonin (5-HT) and proctolin, neurohormones widely distributed in the lobster nervous system, have been implicated in a variety of behaviors and also are known to coexist in large pairs of identified neurons in the fifth thoracic (T5) and first abdominal ganglia (A1) of adults (Siwicki, Beltz, and Kravitz, 1987). Earlier studies also have shown that these paired neurons already contain 5-HT in embryos approximately halfway through development, whereas proctolin immunoreactivity does not appear in these cells until near the time of hatching (Beltz and Kravitz, 1987a). In the current studies, the brain and ventral nerve cord have been screened for the appearance of serotonin and proctolin immunoreactivities using immunocytochemical and biochemical methods, in order to determine whether the late appearance of proctolin in the paired T5 and A1 cells is a general feature of development in other neurons as well. In embryos approximately halfway through development, the adult complement of 5-HT-staining cells is already present. In several cases, embryonic serotonin cells are proportionally very large and prominent, suggesting possible developmental roles. In contrast to serotonin, fewer than 10% of the proctolin-staining neurons of juvenile animals are seen in embryos halfway through development. The number of immunoreactive cells gradually increases, but even by the sixth larval stage only half the number of cells that will eventually stain for proctolin are observed. Therefore, the developmental appearance of proctolin in lobster neurons, assayed using immunocytochemical methods, is relatively late and protracted compared to the appearance of serotonin. Quantitative measurements for 5-HT in lobster larvae were performed using high pressure liquid chromatography (HPLC) with dual electrochemical detection and for proctolin using radioimmunoassay. A gradual, probably growth-related increase in the amounts of serotonin and proctolin were seen during larval development. The implications of the biochemical data, in light of the immunocytochemical studies, are discussed.     

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     

Glomerular organization in developing olfactory and accessory lobes of American lobsters: Stabilization of numbers and increase in size after metamorphosis

Olfactory glomeruli are columnar and radially arranged at the periphery of the primary chemosensory areas, the olfactory lobes (OLs), in the American lobster Homarus americanus. The number of olfactory glomeruli reaches nearly 100/lobe in midembryonic life, increases rapidly during larval life, and stabilizes at about 200 in juvenile and adult lobsters. The accessory lobes (ALs), higher order integration areas, are composed of cortical columns and of spherical glomeruli. Two populations of spherical glomeruli are defined, the cortical glomeruli located at the bases of the columns, and the medullary glomeruli scattered throughout the ALs. Both cortical columns and spherical glomeruli are seen for the first time in the second larval stage. There are about 1000 cortical columns and 1700 glomeruli/AL in the postlarva and these numbers remain constant during the life of the lobster. In both OLs and ALs, it is the size of the interglomerular spaces and of the glomeruli themselves that increases. Therefore, the data suggest that in both OLs and ALs the glomeruli were already generated when the lobster metamorphoses (stage III to IV) and switches from a planktonic to a benthic existence, and that the new sensory neurons that are formed at each molt in the antennulae grow into existing olfactory glomeruli. Stability of the glomerular population in the primary olfactory centers, once the full complement of glomeruli is acquired, has also been reported in insects, fish, and mammals. © 1996 John Wiley & Sons, Inc.     

Effects of serotonin depletion on local interneurons in the developing olfactory pathway of lobsters

During embryonic life, the growth of the olfactory and accessory lobes of the lobster brain is retarded by serotonin depletion using 5,7-dihydroxytryptamine (5,7-DHT) (Benton et al., 1997). The local and projection interneurons that synapse with chemosensory cells in the olfactory lobes are potential targets of this depletion. This study documents proliferation and survival in the local interneuron cell clusters, and examines the differentiation of a prominent local interneuron, the serotonergic dorsal giant neuron (DGN), following serotonin depletion. An increase in dye coupling between the DGN and nearby cells is seen after serotonin depletion. However, morphometric analyses of individual DGNs in normal, sham-injected, and 5,7-DHT-treated embryos show that the general morphology and size of the DGNs are not significantly altered by serotonin depletion. Thus, the DGN axonal arbor occupies a greater proportion of the reduced olfactory lobes in the 5,7-DHT-treated embryos than in normal and sham-injected groups. The paired olfactory globular tract neutrophils (OGTNs), where olfactory interneurons synapse onto the DGNs, are 75% smaller in volume than the comparable region in either sham-injected or normal embryos. In vivo experiments using bromodeoxyuridine (BrdU) show that proliferation in the local interneuron soma clusters is reduced by 5,7-DHT treatment and that survival of newly proliferated local interneurons is also compromised. Our data suggest that alterations in the growth of the DGNs do not contribute to the dramatic reduction in size of the olfactory neutrophils following serotonin depletion, but that cell proliferation and survival among the local interneurons are regulated by serotonin during development. Reduced numbers of local interneurons are therefore one likely reason for the growth reduction observed after serotonin depletion.     

Serotonin immunoreactive neurons in the central nervous system of an insect (Periplaneta americana)

Serotonin-like immunoreactivity was mapped in the central nervous system (CNS) of the cockroach, Periplaneta americana. Immunoreactive staining occurred in every ganglion of the CNS. The largest numbers of immunoreactive somata were detected in the optic lobes and the brain, and lowest numbers in the first and second thoracic ganglia. Dense stained fibers, ramifications, and varicosities were found in all ganglia, and numerous axon like processes occurred in all interganglionic connectives. Immunoreactive processes were not, however, detected in most of the peripherally projecting nerve roots. Processes were found only in roots of the suboesophageal ganglion and the tritocerebral lobes of the brain. A comparison of the map for serotonin immunoreactivity with one generated for the pentapeptide transmitter proctolin suggests that the two systems overlap only in the suboesophageal ganglion and the tritocerebrum. The amine and peptide may co-occur in neurons in these regions. The serotonin immunoreactive system appeared significantly different from the octopaminergic system of the ventral nerve cord. Seventy-two potentially identifiable immunoreactive cells were located in the cockroach CNS. Some of these may be suitable for physiological study of the functional role of serotonin.     

Development of the nasal chemosensory organs in two terrestrial anurans: the directly developing frog, Eleutherodactylus coqui (Anura: Leptodactylidae), and the metamorphosing toad, Bufo americanus (Anura: Bufonidae)

Nearly all vertebrates possess an olfactory organ but the vomeronasal organ is a synapomorphy for tetrapods. Nevertheless, it has been lost in several groups of tetrapods, including aquatic and marine animals. The present study examines the development of the olfactory and vomeronasal organs in two terrestrial anurans that exhibit different developmental modes. This study compares the development of the olfactory and vomeronasal organs in metamorphic anurans that exhibit an aquatic larva (Bufo americanus) and directly developing anurans that have eliminated the tadpole (Eleutherodactylus coqui). The olfactory epithelium in larval B. americanus is divided into dorsal and ventral branches in the rostral and mid-nasal regions. The larval olfactory pattern in E. coqui has been eliminated. Ontogeny of the olfactory system in E. coqui embryos starts to vary substantially from the larval pattern around the time of operculum development, the temporal period when the larval stage is hypothesized to have been eliminated. The nasal anatomy of the two frogs does not appear morphologically similar until the late stages of embryogenesis in E. coqui and the terminal portion of metamorphosis in B. americanus. Both species and their respective developing offspring, aquatic tadpoles and terrestrial egg/embryos, possess a vomeronasal organ. The vomeronasal organ develops at mid-embryogenesis in E. coqui and during the middle of the larval period in B. americanus, which is relatively late for neobatrachians. Development of the vomeronasal organ in both frogs is linked to the developmental pattern of the olfactory system. This study supports the hypothesis that the most recent common ancestor of tetrapods possessed a vomeronasal organ and was aquatic, and that the vomeronasal organ was retained in the Amphibia, but lost in some other groups of tetrapods, including aquatic and marine animals.     

Organization of the Circadian System in Insects

The circadian systems of different insect groups are summarized and compared. Emphasis is placed on the anatomical identification and characterization of circadian pacemakers, as well as on their entrainment, coupling, and output pathways. Cockroaches, crickets, beetles, and flies possess bilaterally organized pacemakers in the optic lobes that appear to be located in the accessory medulla, a small neuropil between the medulla and the lobula. Neurons that are immunoreactive for the peptide pigment-dispersing hormone (PDH) arborize in the accessory medulla and appear to be important components of the optic lobe pacemakers. The neuronal architecture of the accessory medulla with associated PDH-immunoreactive neurons is best characterized in cockroaches, while the molecular machinery of rhythm generation is best understood in fruit flies. One essential component of the circadian clock is the period protein (PER), which colocalizes with PDH in about half of the fruit fly's presumptive pacemaker neurons. PER is also found in the presumptive pacemaker neurons of beetles and moths, but appears to have different functions in these insects. In moths, the pacemakers are situated in the central brain and are closely associated with neuroendocrine functions. In the other insects, neurons associated with neuroendocrine functions also appear to be closely coupled to the optic lobe pacemakers. Some crickets and flies seem to possess central brain pacemakers in addition to their optic lobe pacemakers. With respect to neuronal organization, the circadian systems of insects show striking similarities to the vertebrate circadian system. (Chronobiology International, 15(6), 567-594, 1998)     

Postembryonic development of Arg-Phe-amide-like and cholecystokinin-like immunoreactive neurons in the blowfly optic lobe

Summary The adult optic lobes of the blowfly Calliphora erythrocephala were found to be innervated by more than 2000 neurons immunoreactive to antisera raised against the neuropeptides FMRFamide, its fragment RFamide, and gastrin/cholecystokinin (CCK). All of the CCK-like immunoreactive (CCK-IR) neurons also reacted with antisera to RFamide, FMRFamide and pancreatic polypeptide. A few RFamide/FMRFamide-like immunoreactive (RF-IR) neurons did not react with CCK antisera; they reacted instead with antisera to Leu-enkephalin and Met-enkephalin-Arg⁶-Phe⁷. The RF-IR neurons are, thus, heterogeneous with respect to their contents of immunoreactive peptides. Two of the RF-IR neuron types innervating the adult optic lobes could be traced in their entirety only after following their postembryonic development, because of the complexity of the trajectories of the immunoreactive neuronal process in the adult insect. The majority of the cell bodies of the RF-IR and CCK-IR neurons lie within the optic lobes and are derived from imaginal neuroblasts of the inner and outer optic anlagen. Six of the peptidergic neurons are, however, metamorphosing larval neurons with their cell bodies in the central part of the protocerebrum. The full extent of immunoreactivitiy is not attained in some of the neurons until the late pupal or early adult stage. The larval optic center was also found to be innervated by neurons immuno-reactive with both RFamide and CCK antisera. The cell bodies of these RF-IR/CCK-IR neurons are located near the developing lamina (one on each side). In the 24 h pupa, the cell bodies of these neurons are still immunoreactive, but thereafter they cannot be immunolabeled apparently due to cell death or a change in transmitter phenotype.     

Some effects of proctolin on the cardiac ganglion of the Maine Lobster, Homarus americanus (Milne Edwards)

The neuropeptide proctolin has excitatory effects on the isolated lobster cardiac ganglion. Selective application to the anterior cell body region produces a dose-dependent (10(-8)--10(-5) M) prolonged depolarization of large anterior cells as well as marked increases in burst frequency and/or duration. In ganglia which have been silenced with tetrodotoxin, proctolin application to anterior cells elicits long-lasting depolarizing responses which are accompanied by a 10-30% increase of the apparent membrane input resistance. Higher proctolin concentrations produce high-frequency trains of driver potentials. It is proposed that a proctolin like peptide may serve a neurohumoral role in the lobster cardiac ganglion and that the anterior motor neurons exhibit endogenous rhythmicity in its presence.     

Reorganization of peptidergic systems during brain regeneration in Eisenia fetida (Oligochaeta, Annelida)

After the extirpation of the brain reorganization of the peptidergic (FMRFamide, neuropeptide Y, proctolin) systems was studied in the newly forming cerebral ganglion of the annelid Eisenia fetida. During regeneration, all immunoreactive fibres appear on the 1st-2nd postoperative day. At the beginning of regeneration, immunoreactive neurons and fibres form a mixed structure in the wound tissue. On the 3rd postoperative day, FMRFamide positive and neuropeptide Y-immunoreactive, while on the 7th postoperative day proctolin-immunoreactive neurons appear in the loose wound tissue. From the 25th postoperative day a capsule gradually develops around it. The neurons of the preganglion move to the surface of the newly appearing preganglion. The number of these cells gradually increase, and by the 72th-80th postoperative days the localization and number of peptide-immunoreactive neurons is similar to that in the intact one. The neurons of all examined peptidergic systems may originate from the neuroblasts, situated on the inner and outer surface of the intact ganglia (e.g. suboesophageal and ventral cord ganglia). In addition FMRFamide and proctolin immunoreactive neurons may take their derive by mitotic proliferation from the pharyngeal neurons, too.     

Comparison of turnover in the olfactory organ of early juvenile stage and adult Caribbean spiny lobsters

Proliferation and turnover of neurons occurs in the olfactory systems of many animals. In this study, we examined developmental changes in turnover in the olfactory organ of the Caribbean spiny lobster Panulirus argus by examining two life-history stages—early juveniles and young adults. Turnover was compared using external morphology of the olfactory organ before and after molting to determine addition and loss of aesthetascs and other chemosensilla, and BrdU labeling to identify newly proliferated cells. The number of olfactory receptor neurons (ORNs) innervating each aesthetasc increased only slightly over development, but there was a net increase of olfactory sensory units (i.e. aesthetascs and their ORNs) at each molt. This increase was similar in early juveniles and young adults when expressed as absolute number of ORNs neurons but greater in early juveniles when expressed as a proportion of existing ORNs. The net increase in olfactory sensory units in early juveniles is due solely to addition, since virtually no aesthetascs are lost. In contrast, the net increase in olfactory sensory units in adults reflects addition of new units accompanied by considerable loss of old units. These developmental changes result in expansive enlargement of the olfactory organ without turnover in early juveniles, and a more modest growth combined with continuous turnover and replenishment of ORNs in adults.     

Development of the Drosophila mushroom bodies: sequential generation of three distinct types of neurons from a neuroblast.

The mushroom bodies (MBs) are prominent structures in the Drosophila brain that are essential for olfactory learning and memory. Characterization of the development and projection patterns of individual MB neurons will be important for elucidating their functions. Using mosaic analysis with a repressible cell marker (Lee, T. and Luo, L. (1999) Neuron 22, 451-461), we have positively marked the axons and dendrites of multicellular and single-cell mushroom body clones at specific developmental stages. Systematic clonal analysis demonstrates that a single mushroom body neuroblast sequentially generates at least three types of morphologically distinct neurons. Neurons projecting into the (gamma) lobe of the adult MB are born first, prior to the mid-3rd instar larval stage. Neurons projecting into the alpha' and beta' lobes are born between the mid-3rd instar larval stage and puparium formation. Finally, neurons projecting into the alpha and beta lobes are born after puparium formation. Visualization of individual MB neurons has also revealed how different neurons acquire their characteristic axon projections. During the larval stage, axons of all MB neurons bifurcate into both the dorsal and medial lobes. Shortly after puparium formation, larval MB neurons are selectively pruned according to birthdays. Degeneration of axon branches makes early-born gamma neurons retain only their main processes in the peduncle, which then project into the adult gamma lobe without bifurcation. In contrast, the basic axon projections of the later-born (alpha'/beta') larval neurons are preserved during metamorphosis. This study illustrates the cellular organization of mushroom bodies and the development of different MB neurons at the single cell level. It allows for future studies on the molecular mechanisms of mushroom body development.     

Calcium Regulation of Cyclic Nucleotide Signaling in Lobster Olfactory Receptor Neurons

An elevated free Ca2+ concentration reduces odor-stimulated production of cyclic AMP (cAMP) in the outer dendritic membranes of lobster olfactory receptor neurons in vitro. This effect can occur within 50 ms of odor stimulation. The effect is concentration-dependent at submicromolar concentrations of free Ca2+. An elevated free Ca2+ concentration also reduces basal and forskolin-stimulated cAMP levels in a concentration-dependent manner, suggesting that Ca2+ is not targeting the activation of the odor receptor/G protein complex. The degradation of synthetic cAMP by phosphodiesterases is not enhanced by an increased free Ca2+ concentration, suggesting that Ca2+ acts by down-regulating the olfactory adenylyl cyclase. Western blot analysis of the lobster olfactory sensilla that contain the outer dendrites reveals a protein in the transduction zone with a molecular mass of approximately 138 kDa that is immunoreactive to an antiserum against adenylyl cyclase type III. Given earlier evidence that Ca2+ potentially enters the receptor cell through odor-activated inositol 1,4,5-trisphosphate-gated channels, our results suggest a possible route for cross talk between the cyclic nucleotide and the inositol phospholipid signaling pathways in lobster olfactory receptor neurons.     

Postembryonic neurogenesis in the central nervous system of the tobacco hornworm,Manduca sexta. III. Spatial and temporal patterns of proliferation

Postembryonic neurogenesis leads to a dramatic increase in the number of functional neurons within the segmental ganglia of the moth, Manduca sexta. These adult-specific neurons are generated during larval life by segment-specific arrays of individually identifiable stem cells, or neuroblasts (Nbs). By the end of the feeding larval stage, each Nb has generated a discrete nest of progeny, which ranges in size from less than 10 to more than 70 progeny. The sizes of these identifiable nests of progeny vary in a segment-specific manner, with the thoracic nests containing a greater number of progeny compared with their homologues in the simpler abdominal ganglia. In order to describe those factors that influence the size of the postembryonic neuronal lineages, we examined the spatial and temporal pattern of postembryonic neurogenesis in the segmental ganglia of Manduca. The rates at which the identifiable nests accumulated progeny were estimated by counting the number of progeny within the nests, using sectioned material isolated from animals at stages ranging from embryonic hatching until the end of the feeding larval stage. All of the postembryonic Nbs began to generate progeny at around the time of the molt to the third larval instar. Each nest added progeny at a rate that was a characteristic of its identity and segment of origin. Although all of the nests within the thorax continued to accumulate progeny throughout the feeding larval stage, several of the abdominal nests showed little or no growth following the molt to the fifth larval instar. The thymidine analog 5-bromo 2-deoxyuridine (5-BrdU) was used to estimate the mitotic rates of the identifiable Nbs. The number of labeled progeny within a nest 24 h after application of 5-BrdU ranged from a low of 1 to 2 to a high of 11 to 13 labeled cells. In some instances there was a good correlation between the estimated mitotic rate of an identified Nb and the rate of growth of its associated nest of progeny. However, several of the identifiable nests accumulated progeny at a slower rate than predicted based on the estimated mitotic rate of the Nb. Cell death appears to be responsible for slowing the growth of the nests during the feeding larval stage. We estimate that 10% to 70% of the neurons generated during the feeding larval stage degenerate within 24 h of their birth. The level of cell death observed within a nest was dependent on both its identity and its segment of origin. © 1996 John Wiley & Sons, Inc.     

Mating behavior induces changes of expression of Fos protein,plasma testosterone and androgen receptors in the accessory olfactory bulb (AOB) of the male mandarin vole Microtus mandarinus

In order to investigate the neuroendocrine mechanism of the mating behavior in the adult male mandarin voles Microtus mandarinus,the radioimmunoassay(RIA)and immunohistochemistry methods were used to investigate the differences in plasma testosterone(T)concentrations and distribution of T immunoreactive neurons(T-IRs),androgen receptor immunoreactive neurons(AR-IRs)and Fos protein immunoreactive neurons(Fos-IRs)in the accessory olfactory bulb(AOB)and the main olfactory bulb(MOB)following exposure to clean h...     

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)     

Serotonin in the developing stomatogastric system of the lobster,Homarus americanus

We studied the development of the serotonergic modulation of the stomatogastric nervous system of the lobster, Homarus americanus. Although the stomatogastric ganglion (STG) is present early in embryonic development, serotonin immunoreactivity is not visible in the STG until the second larval stage. However, incubation of the STG with exogenous serotonin showed that a serotonin transporter is present in embryonic and early larval stages. Serotonin uptake was blocked by paroxetine and 0% Na(+) saline. The presence of a serotonin transporter in the embryonic STG suggests that hormonally liberated serotonin could be taken up by the STG, and potentially released as a "borrowed transmitter". Consistent with a potential hormonal role, serotonin is found in the pericardial organs, a major neurosecretory structure, by midembryonic development. The rhythmic motor patterns produced by embryonic and larval STGs were decreased in frequency by serotonin. Lateral Pyloric (LP) neuron-evoked excitatory junctional potentials (EJPs) in the embryos and the first larval stage (LI) were larger, slower, and more variable than those in the adult. The amplitude of adult LP neuron-evoked EJPs was increased more than twofold in serotonin, but in embryos and LI preparations this effect was negligible. In embryos and LI preparations, serotonin increased the occurrence of muscle fiber action potentials and altered the EJP wave-form. These data demonstrate that serotonin receptors are present in the stomatogastric nervous system early in development, and suggest that the role of serotonin changes from modulation of muscle fiber excitability early in development to enhancement of neurally evoked EJPs in the adult.     

Distribution of serotonergic neurons in the eyestalk and brain of the crab,Cancer antennarius

1. Serotonin-containing neurons were localized immunocytochemically in crab cerebral ganglia and their extensions in the eyestalk.2. Approximately 155 serotonergic cells were found in identifiable regions of the brain, the largest number being localized in the anterior cell cluster (40 reactive cells) and the bilateral anterior olfactory cell clusters (40 cells each).3. Serotonin immunoreactive cells were found in all three ganglionic divisions of the eyestalk. The medulla terminalis contains up to 15 reactive cells, of which only one occurs in the X-organ (origin of neurosecretory axons in the sinus gland nerve). The m. terminalis also contains three identifiable cells in the mediolateral border adjacent to the sinus gland nerve, of which one is a giant (up to 100 μm diameter), designated MT-1. The axon of MT-1 branches profusely after entering the m. terminalis neuropil.4. No serotonin immunoreactivity was apparent within the sinus gland, the sinus gland nerve or the organ of Bellonci.5. These findings are discussed in relation to the known serotonergic control of peptide hormone secretion by the eyestalk X-organ-sinus gland complex.