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Pesticide exposure during in utero and early postnatal development can cause a wide range of neurological defects. However, relatively few insecticides have been recognized as developmental neurotoxicants, so far. Recently, discovery of the insecticide, fipronil, in chicken eggs has raised public concern. The status of fipronil as a potential developmental neurotoxicant is still under debate. Whereas several in vivo and in vitro studies suggest specific toxicity, other in vitro studies could not confirm this concern. Here, we tested fipronil and its main metabolic product, fipronil sulfone both at concentrations between 1.98 and 62.5 µM, alongside with the established developmental neurotoxicant, rotenone (0.004–10 µM) in vitro on the human neuronal precursor cell line NT2. We found that rotenone impaired all three tested DNT endpoints, neurite outgrowth, neuronal differentiation, and precursor cell migration in a dose-dependent manner and clearly separable from general cytotoxicity in the nanomolar range. Fipronil and fipronil sulfone specifically inhibited cell migration and neuronal differentiation, but not neurite outgrowth in the micromolar range. The rho-kinase inhibitor Y-27632 counteracted inhibition of migration for all three compounds (EC50 between 12 and 50 µM). The antioxidant, n-acetyl cysteine, could ameliorate the inhibitory effects of fipronil on all three tested endpoints (EC 50 between 84 and 164 µM), indicating the involvement of oxidative stress. Fipronil sulfone had a stronger effect than fipronil, confirming the importance to test metabolic products alongside original pesticides. We conclude that in vitro fipronil and fipronil sulfone display specific developmental neurotoxicity on developing human model neurons. Supplementary Information The online version contains supplementary material available at 10.1007/s12640-021-00364-8.

Developmental neurotoxicity (DNT) of chemical compounds disrupts the formation of a normal brain. There is impressive progress in the development of alternative testing methods for DNT potential in chemicals, some of which also incorporate invertebrate animals. This review briefly touches upon studies on the genetically tractable model organisms of Caenorhabditis elegans and Drosophila melanogaster about the action of specific developmental neurotoxicants. The formation of a functional nervous system requires precisely timed axonal pathfinding to the correct cellular targets. To address this complex key event, our lab developed an alternative assay using a serum-free culture of intact locust embryos. The first neural pathways in the leg of embryonic locusts are established by a pair of afferent pioneer neurons which use guidance cues from membrane-bound and diffusible semaphorin proteins. In a systematic approach according to recommendations for alternative testing, the embryo assay quantifies defects in pioneer navigation after exposure to a panel of recognized test compounds for DNT. The outcome indicates a high predictability for test-compound classification. Since the pyramidal neurons of the mammalian cortex also use a semaphorin gradient for neurite guidance, the assay is based on evolutionary conserved cellular mechanisms, supporting its relevance for cortical development.

Exposure to environmental chemicals during in utero and early postnatal development can cause a wide range of neurological defects. Since current guidelines for identifying developmental neurotoxic chemicals depend on the use of large numbers of rodents in animal experiments, it has been proposed to design rapid and cost-efficient in vitro screening test batteries that are mainly based on mixed neuronal/glial cultures. However, cell culture tests do not assay correct wiring of neuronal circuits. The establishment of precise anatomical connectivity is a key event in the development of a functional brain. Here, we expose intact embryos of the locust (Locusta migratoria) in serum-free culture to test chemicals and visualize correct navigation of identified pioneer axons by fluorescence microscopy. We define separate toxicological endpoints for axonal elongation and navigation along a stereotyped pathway. To distinguish developmental neurotoxicity (DNT) from general toxicity, we quantify defects in axonal elongation and navigation in concentration-response curves and compare it to the biochemically determined viability of the embryo. The investigation of a panel of recognized DNT-positive and -negative test compounds supports a rather high predictability of this invertebrate embryo assay. Similar to the semaphorin-mediated guidance of neurites in mammalian cortex, correct axonal navigation of the locust pioneer axons relies on steering cues from members of this family of cell recognition molecules. Due to the evolutionary conserved mechanisms of neurite guidance, we suggest that our pioneer axon paradigm might provide mechanistically relevant information on the DNT potential of chemical agents on the processes of axon elongation, navigation, and fasciculation.

Developmental neurotoxicity (DNT) of chemicals poses a serious threat to human health worldwide. Current in vivo test methods for assessing DNT require the use of high numbers of laboratory animals. Most alternative in vitro testing methods monitor rather simple toxicological endpoints, whereas the formation of a functional brain requires precisely timed navigation of axons within a complex tissue environment. We address this complexity by monitoring defects in axonal navigation of pioneer axons of intact locust embryos after exposure to chemicals. Embryos develop in serum-free culture with test chemicals, followed by immunolabeling of pioneer neurons. Defects in axon elongation of pioneer axons are quantified in concentration-response curves and compared to the general viability of the embryo, as measured by a resazurin assay. We show that selected chemical compounds interfering with calcium signaling, the cytoskeletal organization, and the reference developmental neurotoxicant rotenone, can be classified as DNT positive. The pesticide rotenone inhibits pioneer neuron elongation with a lower IC50 than the viability assay. The rho kinase inhibitor Y27632 can partially rescue outgrowth inhibition, supporting the classification of rotenone as a specific DNT positive compound. Since mechanisms of axonal guidance, such as growth cone navigation along molecular semaphorin gradients are conserved between locust and mammalian nervous systems, we will further explore the potential of this invertebrate preparation as an assay for testing the DNT potential of chemicals in humans.

The ventral nerve cord of Tetraconata contains a comparably low number of serotonin-immunoreactive neurons, facilitating individual identification of cells and their characteristic neurite morphology. This offers the rather unique possibility of establishing homologies at the single cell level. Because phylogenetic relationships within Tetraconata are still discussed controversially, comparisons of individually identifiable neurons can help to unravel these issues. Serotonin immunoreactivity has been investigated in numerous tetraconate taxa, leading to reconstructions of hypothetical ground patterns for major lineages. However, detailed descriptions of basal insects are still missing, but are crucial for meaningful evolutionary considerations. We investigated the morphology of individually identifiable serotonin-immunoreactive neurons in the ventral nerve cord of Zygentoma (Thermobia domestica, Lepisma saccharina, Atelura formicaria) and Archaeognatha (Machilis germanica, Dilta hibernica). To improve immunocytochemical resolution, we also performed preincubation experiments with 5-hydroxy-L-tryptophan and serotonin. Additionally, we checked for immunolabeling of tryptophan hydroxylase, an enzyme associated with the synthesis of serotonin. Besides the generally identified groups of anterolateral, medial, and posterolateral neurons within each ganglion of the ventral nerve cord, we identified several other immunoreactive cells, which seem to have no correspondence in other tetraconates. Furthermore, we show that not all immunoreactive neurons produce serotonin, but have the capability for serotonin uptake. Comparisons with the patterns of serotonin-containing neurons in major tetraconate taxa suggest a close phylogenetic relationship of Remipedia, Cephalocarida, and Hexapoda, supporting the Miracrustacea hypothesis. J. Comp. Neurol., 2016. © 2016 Wiley Periodicals, Inc. J. Comp. Neurol. 525:79-115, 2017. © 2016 Wiley Periodicals, Inc.

Traumatic injury or the pathogenesis of some neurological disorders is accompanied by inflammatory cellular mechanisms, mainly resulting from the activation of central nervous system (CNS) resident microglia. Under inflammatory conditions, microglia up-regulate the inducible isoform of NOS (iNOS), leading to the production of high concentrations of the radical molecule nitric oxide (NO). At the onset of inflammation, high levels of microglial-derived NO may serve as a cellular defense mechanism helping to clear the damaged tissue and combat infection of the CNS by invading pathogens. However, the excessive overproduction of NO by activated microglia has been suggested to govern the inflammation-mediated neuronal loss causing eventually complete neurodegeneration. Here, we investigated how NO influences phagocytosis of neuronal debris by BV-2 microglia, and how neurite outgrowth of human NT2 model neurons is affected by microglial-derived NO. The presence of NO greatly increased microglial phagocytic capacity in a model of acute inflammation comprising lipopolysaccharide (LPS)-activated microglia and apoptotic neurons. Chemical manipulations suggested that NO up-regulates phagocytosis independently of the sGC/cGMP pathway. Using a transwell system, we showed that reactive microglia inhibit neurite outgrowth of human neurons via the generation of large amounts of NO over effective distances in the millimeter range. Application of a NOS blocker prevented the LPS-induced NO production, totally reversed the inhibitory effect of microglia on neurite outgrowth, but reduced the engulfment of neuronal debris. Our results indicate that a rather simple notion of treating excessive inflammation in the CNS by NO synthesis blocking agents has to consider functionally antagonistic microglial cell responses during pharmaceutic therapy.

Clearance of infected and apoptotic neuronal corpses during inflammatory conditions is a fundamental process to create a favorable environment for neuronal recovery. Microglia are the resident immune cells and the predominant phagocytic cells of the CNS, showing a multitude of cellular responses upon activation. Here, we investigated in functional assays how the CO generating enzyme heme oxygenase 1 (HO-1) influences BV-2 microglial migration, clearance of debris, and neurite outgrowth of human NT2 neurons. Stimulation of HO-1 activity attenuated microglial migration in a scratch wound assay, and phagocytosis in a cell culture model of acute inflammation comprising lipopolysaccharide (LPS)-activated microglia and apoptosis-induced neurons. Application of a CO donor prevented the production of NO during LPS stimulation, and reduced microglial migration and engulfment of neuronal debris. LPS-activated microglia inhibited neurite elongation of human neurons without requiring direct cell–cell surface contact. The inhibition of neurite outgrowth was totally reversed by application of exogenous CO or increased internal CO production through supply of the substrate hemin to HO. Our results point towards a vital cytoprotective role of HO-1/CO signaling after microglial activation. In addition, they support a therapeutic potential of CO releasing chemical agents in the treatment of excessive inflammatory conditions in the CNS. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 854–876, 2015

The olfactory pathway of the locust Locusta migratoria is characterized by a multiglomerular innervation of the antennal lobe (AL) by olfactory receptor neurons (ORNs). After crushing the antenna and thereby severing ORN axons, changes in the AL were monitored. First, volume changes were measured at different times post-crush with scanning laser optical tomography in 5th instar nymphs. AL volume decreased significantly to a minimum volume at 4 days post-crush, followed by an increase. Second, anterograde labeling was used to visualize details in the AL and antennal nerve (AN) during de- and regeneration. Within 24 h post-crush (hpc) the ORN fragments distal to the lesion degenerated. After 48 hpc, regenerating fibers grew through the crush site. In the AL, labeled ORN projections disappeared completely and reappeared after a few days. A weak topographic match between ORN origin on the antenna and the position of innervated glomeruli that was present in untreated controls did not reappear after regeneration. Third, the cell surface marker fasciclin I that is expressed in ORNs was used for quantifying purposes. Immunofluorescence was measured in the AL during de- and regeneration in adults and 5th instar nymphs: after a rapid but transient, decrease, it reappeared. Both processes happen faster in 5th instar nymphs than in adults.

Inflammation within the central nervous system (CNS) is a major component of many neurodegenerative diseases. The underlying mechanisms of neuronal loss are not fully understood, but the activation of CNS resident phagocytic microglia seems to be a significant element contributing to neurodegeneration. At the onset of inflammation, high levels of microglial phagocytosis may serve as an essential prerequisite for creating a favorable environment for neuronal regeneration. However, the excessive and long-lasting activation of microglia and the augmented engulfment of neurons have been suggested to eventually govern widespread neurodegeneration. Here, we investigated in a functional assay of acute inflammation how the small GTPase RhoA and its main target the Rho kinase (ROCK) influence microglial phagocytosis of neuronal debris. Using BV-2 microglia and human NT2 model neurons, we demonstrate that the pain reliever Ibuprofen decreases RhoA activation and microglial phagocytosis of neuronal cell fragments. Inhibition of the downstream effector ROCK with the small-molecule agents Y-27632 and Fasudil reduces the engulfment of neuronal debris and attenuates the production of the inflammatory mediator nitric oxide during stimulation with lipopolysaccharide. Our results support a therapeutic potential for RhoA/ROCK-inhibiting agents as an effective treatment of excessive inflammation and the resulting progression of microglia-mediated neurodegeneration in the CNS.

Botulinum neurotoxins (BoNTs) internalize into nerve terminals and block the release of neurotransmitters into the synapse. BoNTs are widely used as a therapeutic agent for treatment of movement disorders and recently gained more attention as a biological weapon. Consequently, there is strong interest to develop a cell-based assay platform to screen the toxicity and bioactivity of the BoNTs. In this study, we present an in vitro screening assay for BoNT/A based on differentiated human embryonal carcinoma stem (NT2) cells. The human NT2 cells fully differentiated into mature neurons that display immunoreactivity to cytoskeletal markers (βIII-tubulin and MAP2) and presynaptic proteins (synapsin and synaptotagmin I). We showed that the human NT2 cells undergo a process of exo-endocytotic synaptic vesicle recycling upon depolarization with high K(+) buffer. By employing an antibody directed against light chain of BoNT/A, we detected internalized toxin as a punctate staining along the neurites of the NT2 neurons. Using well-established methods of synaptic vesicle exocytosis assay (luminal synaptotagmin I and FM1-43 imaging) we show that pre-incubation with BoNT/A resulted in a blockade of vesicle release from human NT2 neurons in a dose-dependent manner. Moreover, this blocking effect of BoNT/A was abolished by pre-adsorbing the toxin with neutralizing antibody. In a proof of principle, we demonstrate that our cell culture assay for vesicle release is sensitive to BoNT/A and the activity of BoNT/A can be blocked by specific neutralizing antibodies. Overall our data suggest that human NT2 neurons are suitable for large scale screening of botulinum bioactivity.

The insect olfactory system consists of thousands of sensory neurons on each antenna, which project into the primary olfactory center, the glomerular antennnal lobe. There, they form synapses with local interneurons and projection neurons, which relay olfactory information to the second-order olfactory center, the mushroom body. Olfactory afferents of adult locusts (Locusta migratoria) were axotomized by crushing the base of the antenna. We studied the resulting degeneration and regeneration in the antennal lobe by size measurements, anterograde dye labeling through the antennal nerve, and immunofluorescence staining of cell surface markers. Within 3 days postcrush, the antennal lobe size was reduced by 30% and from then onward regained size back to normal by 2 weeks postinjury. Concomitantly, anterograde labeling revealed regenerating afferents reaching the antennal lobe by day 4 postcrush, and reinnervating the olfactory neuropil almost back to normal within 2 weeks. Regenerated fibers were directed precisely into the antennal lobe, where they reinnervated glomeruli. As a remarkable exception, a few regenerating fibers projected erroneously into the mushroom body on a pathway that is normally chosen by second-order projection neurons. Regenerating afferents expressed the cell surface proteins lachesin and fasciclin I. The antennal lobe neuropil expressed the cell surface marker semaphorin 1a. In conclusion, axonal regeneration in the locust olfactory system appears to be possible, precise, and fast, opening the possibility of future functional and mechanistic studies.

Summary Purpose: The selection of a minimal active sequence of erythropoietin allowed the design of peptide mimetics that exert beneficial effects in the central nervous system but lack an erythropoietic effect. Erythropoietin has been suggested as a promising therapeutic and prophylactic for epilepsies based on its neuroprotective, neuroregenerative, and antiinflammatory potency. Therefore, it is of particular interest to evaluate whether the nonerythropoietic erythropoietin-derived peptide pHBSP can affect epileptogenesis. Methods: In a post–status epilepticus model in rats, we determined the effects of pHBSP and of recombinant human erythropoietin with short-term administration following status epilepticus. Key Findings: Both pHBSP and erythropoietin further enhanced the status epilepticus–associated increase in hippocampal cell proliferation. Thereby, pHBSP seemed to promote neuronal differentiation and survival resulting in a significant increase in neurogenesis. Neither pHBSP nor erythropoietin affected the number of animals exhibiting spontaneous recurrent seizures as well as the seizure frequency in the chronic phase. In the Morris water maze, pHBSP attenuated cognitive deficits in epileptic animals. Significance: In conclusion, the helix B–derived erythropoietin peptide pHBSP can modulate the cellular and cognitive consequences of a status epilepticus. The impact of pHBSP on spatial learning might indicate that the peptide allows beneficial effects on epileptogenesis-associated cognitive deficits. However, it needs to be considered that learning deficits were not abolished by pHBSP and that the effects were not observed consistently until the end of the study. Therefore, adjustment of timing, duration, and dose of peptide administration might be necessary to further evaluate the efficacy of pHBSP.

An Expert Meeting on The Current Scientific and Legal Status of Alternative Methods to the LD50 Test for Botulinum Neurotoxin (BoNT) Potency Testing was organised by the Centre for Documentation and Evaluation of Alternatives to Animal Experiments (ZEBET) at the Federal Institute for Risk Assessment (BfR), and took place on 27-28 April 2009, in Berlin, Germany. The goal of ZEBET is to promote primarily the replacement of legally required animal experiments with valid alternative test methods, to reduce the numbers of test animals used to the absolutely necessary level, and to alleviate their pain and suffering. ZEBET's tasks are the documentation, assessment and promotion of alternative methods to animal experiments on a national and international level, and the initiation of research and development, including the funding of smaller research projects.

Postmitotic neurons were generated from the human NT2 teratocarcinoma cell line in a novel cell aggregate differentiation procedure. The NT2 model neurons express punctate immunoreactivity for synapsin and for cell markers related to GABAergic and glutamatergic neurotransmission. Using the outside-out patch-clamp configuration, we characterized the kinetics of currents elicited by a rapid application of the amino acid neurotransmitters. Moreover, we detected spontaneous postsynaptic currents in glia free cell cultures that may result from the firing activity of glutamatergic and GABAergic NT2 neurons. These cultured spontaneously active networks may be a useful tool to analyze factors that modulate the formation and efficacy of synapses between human neurons.

The human Ntera2 (NT2) teratocarcinoma cell line can be induced to differentiate into post-mitotic neurons. Here, we report that the human NT2 neurons generated by a spherical aggregate cell culture method express increasing levels of typical pre-synaptic proteins (synapsin and synaptotagmin I) along the neurite depending on the length of in vitro culture. By employing an antibody directed against the luminal domain of synaptotagmin I and the fluorescent dye N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide, we show that depolarized NT2 neurons display calcium-dependent exo-endocytotic synaptic vesicle recycling. NT2 neurons express the neuronal isoform of neuronal nitric oxide synthase and soluble guanylyl cyclase (sGC), the major receptor for nitric oxide (NO). We tested whether NO signal transduction modulates synaptic vesicle turnover in human NT2 neurons. NO donors and cylic guanosine-monophosphate analogs enhanced synaptic vesicle recycling while a sGC inhibitor blocked the effect of NO donors. Two NO donors, sodium nitroprusside, and and N-Ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino) ethanamine evoked vesicle exocytosis which was partially blocked by the sGC inhibitor. The activator of adenylyl cyclase, forskolin, and a cAMP analog induced synaptic vesicle recycling and exocytosis via a parallel acting protein kinase A pathway. Our data from NT2 neurons suggest that NO/cyclic nucleotide signaling pathways may facilitate neurotransmitter release in human brain cells.

The well-characterized human teratocarcinoma line Ntera2 (NT2) can be differentiated into mature neurons. We have significantly shortened the time-consuming process for generating postmitotic neurons to approximately 4 weeks by introducing a differentiation protocol for free-floating cell aggregates and a subsequent purification step. Here, we characterize the neurochemical phenotypes of the neurons derived from this cell aggregate method. During differentiation, the NT2 cells lose immunoreactivity for vimentin and nestin filaments, which are characteristic for the immature state of neuronal precursors. Instead, they acquire typical neuronal markers such as beta-tubulin type III, microtubule-associated protein 2, and phosphorylated tau, but no astrocyte markers such as glial fibrillary acidic protein. They grow neural processes that express punctate immunoreactivity for synapsin and synaptotagmin suggesting the formation of presynaptic structures. Despite their common clonal origin, neurons cultured for 2-4 weeks in vitro comprise a heterogeneous population expressing several neurotransmitter phenotypes. Approximately 40% of the neurons display glutamatergic markers. A minority of neurons is immunoreactive for serotonin, gamma-amino-butyric acid, and its synthesizing enzyme glutamic acid decarboxylase. We have found no evidence for a dopaminergic phenotype. Subgroups of NT2 neurons respond to the application of nitric oxide donors with the synthesis of cGMP. A major subset shows immunoreactivity to the cholinergic markers choline acetyl-transferase, vesicular acetylcholine transporter, and the non-phosphorylated form of neurofilament H, all indicative of motor neurons. The NT2 system may thus be well suited for research related to motor neuron diseases.

Zusammenfassung Stickstoffmonoxid (NO) ist als gasförmiger Botenstoff im Nervensystem bekannt. Es spielt eine Rolle bei synaptischer Plastizität, aber auch bei neuronalen Entwicklungs- und Regenerationsvorgängen. Wir untersuchen die Funktion von NO und seiner Signalkaskade über zyklisches GMP am Heuschreckenembryo. Dessen sich entwickelndes Nervensystem ist sehr gut für pharmakologische Manipulationen in Gewebekultur geeignet. Wir analysieren die zellulären Mechanismen der NO-Wirkung an drei Beispielen: 1. im peripheren Nervensystem beim Auswachsen von Pionierneuronen in der Antenne, 2. im zentralen Nervensystem bei der axonalen Regeneration serotonerger Interneurone nach Axotomie und 3. im enterischen Nervensystem bei der Migration von Neuronen, die den Darm-Nervenplexus bilden. In allen Fällen dient intern freigesetztes Stickstoffmonoxid bzw. die Synthese von zyklischem GMP als ein permissives Signal für den jeweiligen Entwicklungsvorgang. Kohlenstoffmonoxid (CO) moduliert als ein weiterer gasförmiger Botenstoff antagonistisch zu NO die Wanderung der Darmneurone. Experimente mit humanen Modellneuronen in Zellkultur weisen darauf hin, dass NO auch bei der Bildung des menschlichen Nervensystems Aspekte der Zellmigration reguliert.

In higher vertebrates, the central nervous system (CNS) is unable to regenerate after injury, at least partially because of growth-inhibiting factors. Invertebrates lack many of these negative regulators, allowing us to study the positive factors in isolation. One possible molecular player in neuronal regeneration is the nitric oxide (NO)-cyclic guanosine-monophosphate (cGMP) transduction pathway which is known to regulate axonal growth and neural migration. Here, we present an experimental model in which we study the effect of NO on CNS regeneration in flat-fillet locust embryo preparations in culture after crushing the connectives between abdominal ganglia. Using whole-mount immunofluorescence, we examine the morphology of identified serotonergic neurons, which send a total of four axons through these connectives. After injury, these axons grow out again and reach the neighboring ganglion within 4 days in culture. We quantify the number of regenerating axons within this period and test the effect of drugs that interfere with NO action. Application of exogenous NO or cGMP promotes axonal regeneration, whereas scavenging NO or inhibition of soluble guanylyl cyclase delays regeneration, an effect that can be rescued by application of external cGMP. NO-induced cGMP immunostaining confirms the serotonergic neurons as direct targets for NO. Putative sources of NO are resolved using the NADPH-diaphorase technique. We conclude that NO/cGMP promotes outgrowth of regenerating axons in an insect embryo, and that such embryo-culture systems are useful tools for studying CNS regeneration.

Mouse models for colon cancer that harbor a germ line mutation in the tumor suppressor gene Adenomatous polyposis coli (Apc) exhibit a primary genetic defect that predisposes to a high incidence of adenomatous polyps in the small intestine rather than in the colon. Colon cell culture models expressing quantifiable markers for carcinogenic risk may represent an alternative approach to reduce, refine or replace long-term animal experimentation. The newly developed colon epithelial cell lines 1638N COL-Cl(1) (clonal derivative of the parental Apc mutant cell line 1638N COL) and 1638N COL-Pr(1) (tumor derivative of the clone), established from an Apc1638N [+/-] mutant mouse, exhibit aberrant cell cycle progression, downregulated apoptosis, enhanced carcinogenic risk and tumor formation, indicating that aberrantly proliferative preneoplastic1638N COL-Cl(1) cells exhibit a quantifiable risk for carcinogenesis. Treatment of these preneoplastic Apc mutant cells with a combination of celecoxib and 5-fluorouracil at clinically achievable low concentrations produced a 2.1 fold to 5.5 fold higher efficacy for cytostatic growth arrest and a 40.2% to 52.4% higher efficacy for inhibition of carcinogenic risk, relative to that obtained by these agents used individually. Thus, a low dose combination of mechanistically distinct agents resulted in enhanced efficacy. These data validate a novel cell culture model and a rapid mechanism-based approach to prioritize efficacious drug combinations for animal studies and clinical trials on cancer prevention and, thereby, support the 3R concept by refining and/or reducing the use of animals in biomedical research relevant to prevention/therapy of colon cancer.

in vitro models are often used to investigate pathophysiological mechanisms of brain cell injury as they occur for instance during cerebral ischemia. To analyze the efficacy of potential neuroprotective compounds, cell physiological experiments were performed in a recently improved culture system of human model neurons. The postmitotic neurons were generated from the human NT-2 teratocarcinoma cell line, using a cell sphere culture method to facilitate rapid terminal differentiation. We simulated ischemic conditions in cultures of purified NT-2 neurons and found that low doses of the antihypertensive drug diltiazem protected against excitotoxic neuronal damage in vitro. Experiments with primary cortical mouse neuron cultures demonstrated a similar response to simulated ischemia and confirmed the neuroprotective effect of diltiazem. Calcium imaging experiments showed that diltiazem reduced both NMDA- and glutamate-induced calcium influxes in NT-2 neurons suggesting that its neuroprotective effect is based on the inhibition of voltage-gated calcium channels. These results indicate that diltiazem is an effective blocker of glutamate-induced excitotoxicity. Moreover, we suggest that cell cultures of human model neurons can provide an important initial test system for drug development in stroke therapy. (c) 2006 Elsevier B.V. All rights reserved. (Less)

During the formation of the brain, neuronal cell migration and neurite extension are controlled by extracellular guidance cues. Here, I discuss experiments showing that the messenger nitric oxide (NO) is an additional regulator of cell motility. NO is a membrane permeant molecule, which activates soluble guanylyl cyclase (sGC) and leads to the formation of cyclic GMP (cGMP) in target cells. The analysis of specific cells types in invertebrate models such as molluscs, insects and the medicinal leech provides insight how NO and cyclic nucleotides affect the wiring of nervous systems by regulating cell and growth-cone motility. Inhibition of the NOS and sGC enzymes combined with rescue experiments show that NO signalling orchestrates neurite outgrowth and filopodial dynamics, cell migration of enteric neurons, glial migration and axonogenesis of pioneer fibers. Cultured insect embryos are accessible model systems in which cellular mechanisms of NO-induced cytoskeletal reorganizations can be analyzed in natural settings. Finally, I will outline some indications that NO may also regulate cell motility in the developing and regenerating vertebrate nervous system.

We provide evidence for axonal regeneration in the central nervous system (CNS) of the locust (Locusta migratoria). We followed the morphology of a small set of proctolin-immunoreactive neurons in the ventral nerve cord before and after crushing one cervical connective in the third instar. The proximal segments started sprouting within 3 days post lesion and grew into the suboesophageal ganglion within 9 days, covering a distance of approximately 2 mm. Within the suboesophageal ganglion, the regenerated neurites formed arborisations in the appropriate region which closely resemble the original shape. These findings will allow us to compare regeneration to the well-described embryonic development of axonal connectivity in this animal.

The dynamic regulation of nitric oxide synthase (NOS) activity and cGMP levels suggests a functional role in the development of nervous systems. We report evidence for a key role of the NO/cGMP signalling cascade on migration of postmitotic neurons in the enteric nervous system of the embryonic grasshopper. During embryonic development, a population of enteric neurons migrates several hundred micrometers on the surface of the midgut. These midgut neurons (MG neurons) exhibit nitric oxide-induced cGMP-immunoreactivity coinciding with the migratory phase. Using a histochemical marker for NOS, we identified potential sources of NO in subsets of the midgut cells below the migrating MG neurons. Pharmacological inhibition of endogenous NOS, soluble guanylyl cyclase (sGC) and protein kinase G (PKG) activity in whole embryo culture significantly blocks MG neuron migration. This pharmacological inhibition can be rescued by supplementing with protoporphyrin IX free acid,an activator of sGC, and membrane-permeant cGMP, indicating that NO/cGMP signalling is essential for MG neuron migration. Conversely, the stimulation of the cAMP/protein kinase A signalling cascade results in an inhibition of cell migration. Activation of either the cGMP or the cAMP cascade influences the cellular distribution of F-actin in neuronal somata in a complementary fashion. The cytochemical stainings and experimental manipulations of cyclic nucleotide levels provide clear evidence that NO/cGMP/PKG signalling is permissive for MG neuron migration, whereas the cAMP/PKA cascade may be a negative regulator. These findings reveal an accessible invertebrate model in which the role of the NO and cyclic nucleotide signalling in neuronal migration can be analyzed in a natural setting.

Cells from the human teratocarcinoma line NTera-2 can be induced to terminally differentiate into postmitotic neurons when treated with retinoic acid. However, this differentiation process is rather time consuming as it takes between 42 and 54 days. Here, we propose a modified differentiation protocol which reduces the time needed for differentiation considerably without compromising the quantity of the neurons obtained. The introduction of a proliferation step as free floating cell spheres cuts the total time needed to obtain high yields of purified NT-2 neurons to about 24-28 days. The cells obtained show neuronal morphology and migrate to form ganglion-like cell conglomerates. Differentiated cells express neuronal polarity markers such as the cytoskeleton associated proteins MAP2 and Tau. Moreover, the generation of neurons in sphere cultures induced immunoreactivity to the ELAV-like neuronal RNA-binding proteins HuC/D, which have been implicated in mechanisms of nerve cell differentiation.

Clearance of infected and apoptotic neuronal corpses during inflammatory conditions is a fundamental process to create a favorable environment for neuronal recovery. Microglia are the resident immune cells and the predominant phagocytic cells of the CNS, showing a multitude of cellular responses upon activation. Here, we investigated in functional assays how the CO generating enzyme heme oxygenase 1 (HO-1) influences BV-2 microglial migration, clearance of debris, and neurite outgrowth of human NT2 neurons. Stimulation of HO-1 activity attenuated microglial migration in a scratch wound assay, and phagocytosis in a cell culture model of acute inflammation comprising lipopolysaccharide (LPS)-activated microglia and apoptosis-induced neurons. Application of a CO donor prevented the production of NO during LPS stimulation, and reduced microglial migration and engulfment of neuronal debris. LPS-activated microglia inhibited neurite elongation of human neurons without requiring direct cell-cell surface contact. The inhibition of neurite outgrowth was totally reversed by application of exogenous CO or increased internal CO production through supply of the substrate hemin to HO. Our results point towards a vital cytoprotective role of HO-1/CO signaling after microglial activation. In addition, they support a therapeutic potential of CO releasing chemical agents in the treatment of excessive inflammatory conditions in the CNS.

Over the last years, a number of therapeutic strategies have emerged to promote axonal regeneration. An attractive strategy is the implantation of biodegradable and nonimmunogenic artificial scaffolds into injured peripheral nerves. In previous studies, transplantation of decellularized veins filled with spider silk for bridging critical size nerve defects resulted in axonal regeneration and remyelination by invading endogenous Schwann cells. Detailed interaction of elongating neurons and the spider silk as guidance material is unknown. To visualize direct cellular interactions between spider silk and neuronsin vitro, we developed anin vitrocrossed silk fiber array. Here, we describe in detail for the first time that human (NT2) model neurons attach to silk scaffolds. Extending neurites can bridge gaps between single silk fibers and elongate afterwards on the neighboring fiber. Culturing human neurons on the silk arrays led to an increasing migration and adhesion of neuronal cell bodies to the spider silk fibers. Within three to four weeks, clustered somata and extending neurites formed ganglion-like cell structures. Microscopic imaging of human neurons on the crossed fiber arraysin vitrowill allow for a more efficient development of methods to maximize cell adhesion and neurite growth on spider silk prior to transplantation studies.

Microglia are the resident immune cells of the brain, which become rapidly activated and migrate to the site of insult in brain infection and disease. Activated microglia generate large amounts of the highly reactive messenger molecule nitric oxide (NO). NO is able to raise cyclic GMP levels via binding to soluble guanylyl cyclase. We investigated potential mechanistic links between inflammation, NO signaling, and microglial migration. To monitor cell migration, we used a scratch wound assay and compared results obtained in the BV-2 microglial line to primary microglia. Incubation with lipopolysaccharide (LPS) as stimulator of acute inflammatory processes enhanced migration of both microglial cell types. LPS activated NO production in BV-2 cells and application of an NO donor increased BV-2 cell migration while an NO scavenger reduced motility. Pharmacological inhibition of soluble guanylyl cyclase and the resulting decrease in motility can be rescued by a membrane permeant analog of cGMP. Despite differences in the threshold towards stimulation with the chemical agents, both BV-2 cells and primary microglia react in a similar way. The important role of NO/cGMP as positive regulator of microglial migration, the downstream targets of the signaling cascade, and resulting cytoskeletal changes can be conveniently investigated in a microglial cell line.

Over the past few years, molecular studies of phylogeny have challenged the traditional view of evolutionary relationships among protostomian animal phyla. Based on analysis of 18S ribosomal RNA gene sequences, it has been suggested that some traditional groups, like the articulata and the pseudocoelomata, should be completely abandoned and that instead the protostomians should be split into two major clades: the Ecdysozoa and the Lophotrochozoa. However, this new molecular phylogeny still awaits confirmation by independent methods. In this study, we present a cytological feature that supports the new classification. The carbohydrate epitope that is recognised by antisera against the plant glycoprotein horseradish peroxidase (HRP) is known to be selectively expressed by membrane proteins on the surface of neural tissue in insects. We found that the major ecdysozoan phyla show neural expression of HRP immunoreactivity, which is completely absent in the nervous tissue of lophotrochozoans, deuterostomians, and cnidarians. This suggests that the presence of anti-HRP-reactive glycoproteins in neural tissue is an ecdysozoan autapomorphy.

In vertebrate olfactory receptor neurons, NO synthase (NOS) has been detected in embryonic and early postnatal stages. However, expression of the enzyme in the mature epithelium is still controversial. We analyzed the developmental expression pattern of the histochemical NOS-marker NADPH diaphorase (NADPHd) in the olfactory epithelium of young rats. NADPHd was expressed in a small subset of olfactory receptor neurons as early as P0. Between P0 and P24 the number of labeled neurons increased 10-fold, stabilizing thereafter. Whereas NADPHd was generally found in the somata, a transitory dendritic expression was observed between P2 and P5. This dynamic postnatal regulation of the cellular distribution of NADPHd appears to reflect developmental processes within the olfactory epithelium.

Nitric oxide (NO) is a membrane-permeant messenger molecule generated from the amino acid L-arginine. NO can activate soluble guanylyl cyclase leading to the formation of cyclic GMP (cGMP) in target cells. In the nervous system, NO/cGMP signalling is thought to play essential roles in synaptic plasticity during development and also in the mature animal. This paper examines biochemical, cell biological, and physiological investigations of NO/cGMP signalling in the nervous system of the locust, a commonly used neurobiological preparation. Biochemical investigations suggest that an identical enzyme is responsible for both NO synthase (NOS) and NADPH-diaphorase activity after tissue fixation. Immunocytochemical staining of an olfactory center in the locust brain shows that NOS-immunoreactivity colocalizes with NADPH-diaphorase at the cellular level. The cytochemical staining of NO donor and target cells in adult animals suggests functions in olfaction, vision, and sensorimotor integration. During development, NO is implicated in axonal outgrowth and synaptogenesis. The cellular distribution of NO-responsive cells in neural circuits reflects potential functions of NO as a retrograde synaptic messenger, as an intracellular messenger, and as a lateral diffusible messenger independent of conventional synaptic connectivity.

Nitric oxide (NO) diffuses as short-lived messenger through the plasma membrane and serves, among many other functions, as an activator of the cGMP synthesizing enzyme soluble guanylyl cyclase (sGC). In view of recent genetic investigations that postulated a retrograde signal from the larval muscle fibers to the presynaptic terminals, we looked for the presence of an NO/cGMP signaling system at the neuromuscular junction (NMJ) of Drosophila melanogaster larvae. Application of NO donors induced cGMP immunoreactivity in the presynaptic terminals but not the postsynaptic muscle fibers at an identified NMJ. The NO-induced cGMP immunoreactivity was sensitive to a specific inhibitor (ODQ) of the sGC. Since presynaptic terminals which were surgically isolated from the central nervous system are capable of synthesizing cGMP, we suggest that an NO-sensitive guanylyl cyclase is present in the terminal arborizations. Using a fluorescent dye that is known to stain recycling synaptic vesicles, we demonstrate that NO donors and membrane permeant cGMP analogues cause vesicle release at the NMJ. Moreover, the NO-induced release could be blocked by the specific inhibitor of the sGC. A destaining of synaptic terminals after NO exposure in Ca2+-free solution in the presence of cobalt chloride as a channel blocker suggested that NO stimulates Ca2+-independent vesicle release at the NMJ. The combined immunocytochemical and exocytosis imaging experiments imply the involvement of cGMP and NO in the regulation of vesicle release at the NMJ of Drosophila larvae.

This paper summarizes histochemical and immunocytochemical investigations of cholinergic, GABAergic, and glutamatergic pathways in the central brain and suboesophageal ganglion of the honeybee. Acetylcholinesterase histochemistry, immunocytochemical staining for nicotinic acetylcholine receptors, and mapping for alpha-bungarotoxin binding sites indicate cholinergic synaptic interactions in the antennal lobe and a cholinergic pathway via a subset of olfactory projection neurons into the mushroom bodies. Calcium imaging experiments in cell cultures prepared from mushroom bodies demonstrate the expression of nicotinic cholinergic receptors on Kenyon cells. Neurons synthesizing GABA and glutamate are stained with well-defined polyclonal antisera against the amino acids. GABA-immunoreactivity is mainly localized in local interneurons of the antennal lobe and in extrinsic neurons innervating the mushroom bodies. High levels of glutamate-immunoreactivity are found in motoneurons of the suboesophageal ganglion, the dorsal lobe, and also in interneurons. A subgroup of the Kenyon cells shows distinct but weaker levels of glutamate-immunoreactivity. The detailed knowledge about the chemical neuroanatomy of the bee provides a framework for behavioral pharmacological approaches, which implicate the involvement of cholinergic mechanisms in olfactory learning and GABAergic mechanisms in odor discrimination.

Nitric oxide (NO) is a membrane-permeant messenger molecule which activates the cyclic GMP (cGMP)-synthesizing enzyme soluble guanylyl cyclase. Using cytochemical techniques, we recently reported NO-induced cGMP immunoreactivity in the photoreceptor cells of the compound eye of the locust Schistocerca gregaria and also detected NADPH diaphorase staining, a marker of NO synthase, in a subset of the monopolar cells of the lamina. By recording the corneal electroretinogram (ERG), we found that the application of neurochemicals that raise NO/cGMP levels in the optic lobe increased the ERG amplitude, whereas the experimental reduction of NO levels caused a decrease in the response to light. An increase in the light response was also found in intracellular recordings after application of a NO donor, suggesting that the NO-induced changes in the ERG are not caused by changes in the resistive isolation of the retina. Our cytochemical and electrophysiological data are both consistent with the hypothesis that NO synthesized in monopolar cells is a retrograde messenger to the presynaptic photoreceptor neurones.

Nitric oxide synthesizing neurons of the locust CNS have been identified by NADPH-diaphorase staining. However, the conventional transmitters of these neurons are unknown. Here we use double labelling for NADPH-diaphorase and GABA-immunofluorescence on sections of the brain to investigate a potential coexpression of both markers. The antennal lobe is innervated by a cluster of about 45-50 NADPH-diaphorase positive local interneurons which express GABA-immunofluorescence. The mushroom bodies are a higher order olfactory center which receive an extrinsic innervation from GABA-immunoreactive and NADPH-diaphorase positive fiber systems. Each optic lobe contains about 4500 GABA-immunoreactive cell bodies. In the visual system, identifiable GABA-immunoreactive neurons arborize in the external plexiform layer of the lamina, in several strata of the medulla, and in the lobula complex. A survey of all NADPH-diaphorase positive cell groups detected a colocalization of GABA-immunoreactivity in a small subpopulation of somata along the anterior rim of the medulla. These cytochemical findings suggest that nitric oxide may be a characteristic cotransmitter of GABAergic circuits of the antennal lobe, while in mushroom bodies and the visual system the majority of nitric oxide and GABA releasing neurons are distinct populations.

Using NADPH-diaphorase staining as a marker for nitric oxide (NO) synthase and an antiserum against cyclic GMP, we recently reported the anatomical distribution of nitric oxide donor and target cells in the antennal lobe, the principal olfactory neuropile of the locust. The most striking NADPH-diaphorase activity in the olfactory pathway is concentrated in a cluster of intensely stained local interneurons innervating the glomeruli. After incubation of tissue in a nitric oxide donor and inhibition of phospodiesterase activity, neurons of this cluster expressed cyclic GMP-immunoreactivity in the cell body and neurites. Here we examine the importance of the arrangement of NO donor and target cells for information processing in the glomeruli. The cellular organization of the NO-cyclic GMP system in olfactory interneurons, and the dendritic branching pattern, suggest that nitric oxide may not only act as intercellular, but also as intracellular messenger molecule in the glomerular neuropile of the antennal lobe.

Developmental neurotoxicity (DNT) of environmental chemicals is a serious threat to human health. Current DNT testing guidelines propose investigations in rodents, which require large numbers of animals. With regard to the "3Rs" (reduction, replacement, and refinement) of animal testing, alternative testing strategies are needed in order to refine and reduce animal experiments and allow faster and less expensive screening. The goal of this study was to establish components for a human cell-based test system to assess DNT potential of chemicals at an early stage of brain development. A human neural precursor cell line should be tested for suitability for semi-automated high-throughput DNT screening. We established assays suitable for detecting disturbances in two basic processes of brain development in 96-well scale: neuronal differentiation and migration using the human Ntera2 (NT2) cell line. We assessed the effects of four test compounds with well-established DNT potential in comparison with three compounds without specific DNT potential. We found that human NT2 cell cultures treated with the morphogen, retinoic acid, imitate neuronal differentiation, and migration in vitro. The developmental neurotoxicants methylmercury chloride, sodium arsenite, sodium valproate, and methylazoxymethanol significantly reduced the expression of the neuronal marker β-tubulin type III and decreased the migration distance in developing NT2 cells. Both endpoints, differentiation and migration, can be read out directly in a standard fluorescence plate reader, enabling high-throughput screening. We conclude that NT2 cell tests are likely to become valuable components of a human cell-based modular in vitro DNT test systems.

Background Remipedia were initially seen as a primitive taxon within Pancrustacea based on characters considered ancestral, such as the homonomously segmented trunk. Meanwhile, several morphological and molecular studies proposed a more derived position of Remipedia within Pancrustacea, including a sister group relationship to Hexapoda. Because of these conflicting hypotheses, fresh data are crucial to contribute new insights into euarthropod phylogeny. The architecture of individually identifiable serotonin-immunoreactive neurons has successfully been used for phylogenetic considerations in Euarthropoda. Here, we identified neurons in three species of Remipedia with an antiserum against serotonin and compared our findings to reconstructed ground patterns in other euarthropod taxa. Additionally, we traced neurite connectivity and neuropil outlines using antisera against acetylated α-tubulin and synapsin. Results The ventral nerve cord of Remipedia displays a typical rope-ladder-like arrangement of separate metameric ganglia linked by paired longitudinally projecting connectives. The peripheral projections comprise an intersegmental nerve, consisting of two branches that fuse shortly after exiting the connectives, and the segmental anterior and posterior nerve. The distribution and morphology of serotonin-immunoreactive interneurons in the trunk segments is highly conserved within the remipede species we analyzed, which allows for the reconstruction of a ground pattern: two posterior and one anterior pair of serotonin-immunoreactive neurons that possess a single contralateral projection. Additionally, three pairs of immunoreactive neurons are found in the medial part of each hemiganglion. In one species (Cryptocorynetes haptodiscus), the anterior pair of immunoreactive neurons is missing. Conclusions The anatomy of the remipede ventral nerve cord with its separate metameric ganglia mirrors the external morphology of the animal’s trunk. The rope-ladder-like structure and principal architecture of the segmental ganglia in Remipedia corresponds closely to that of other Euarthropoda. A comparison of the serotonin-immunoreactive cell arrangement of Remipedia to reconstructed ground patterns of major euarthropod taxa supports a homology of the anterior and posterior neurons in Pancrustacea. These neurons in Remipedia possess unbranched projections across the midline, pointing towards similarities to the hexapod pattern. Our findings are in line with a growing number of phylogenetic investigations proposing Remipedia to be a rather derived crustacean lineage that perhaps has close affinities to Hexapoda.

The insect brain offers a unique opportunity for cell biological investigation of calcium signalling in relatively homogeneous neuronal populations, such as the mushroom body neurons. This review summarizes fluorescence imaging experiments with the indicator dye fluo-3 to investigate the calcium responses to transmitter stimulation of honeybee mushroom body neurons in primary culture. Application of acetylcholine (ACh) or nicotine promoted a calcium influx into the cell body and neurites. The increase in intracellular calcium after ACh stimulation was blocked by α-bungarotoxin. These results support previous histochemical studies that suggested the expression of nicotinic cholinergic receptors on Kenyon cells. An increase in cytoplasmic calcium levels leads in specific neurons to the generation of nitric oxide (NO) by a Ca 2+ /calmodulin activated NO synthase. NO is thought to diffuse as a short-lived messenger molecule through the plasma membrane. Using a sensitive photometric assay it could be shown that dissociated cells from the locust brain release NO after stimulation by agents elevating cytoplasmic Ca 2+ levels and by the excitatory neurotransmitter ACh. The experiments in dissociated culture therefore allow the demonstration of a Ca 2+ -dependent release during nerve cell depolarization, which is a basic requirement for identifying NO as a messenger molecule.

We investigated the distribution and anatomical organization of glial cells in the antennal lobes and mushroom bodies of the honeybee. Reconstructions from serial sections, prepared according to the ethyl gallate method, revealed the entire morphology of glial cells in neuropiles, tracts, and the soma rind. The distribution of the glial cell bodies in the neuropiles was derived from the staining of cell nuclei with a fluorescent dye. There are glial cells of different shape in the soma rind which are wrapped around the neuronal cell bodies of the antennal lobes and the Kenyon cells of the mushroom bodies. Glial cells surround neuropilar areas such as the external and lateral sides of the glomeruli of the antennal lobes. Whereas we could not detect glia in the glomerular neuropile, glial cells with long processes are located in the core of the antennal lobe. Extensions of these glial cells also invade tracts containing the olfactory projection neurons. A layer of glial cells separates the mushroom body neuropile from the surrounding protocerebral neuropile. The neuropile of the mushroom bodies is clearly compartmented by glial cells. There is a high density of astrocyte-like glia in a column of the pedunculus which can be followed to the ventral part of the alpha-lobe. A network of mushroom body intrinsic glial cells separates the alpha-lobe from the beta-lobe and the pedunculus. This anatomical description of glial cell types in olfactory information processing pathways of an insect brain provides a framework for further physiological studies of neuroglia in dissociated cell culture.

Using NADPH-diaphorase (NADPH-d) staining as a marker for the enzyme nitric oxide synthase (NOS) we investigated the possible sites of nitric oxide (NO) synthesis in the olfactory pathways of an insect brain. Staining of frozen sections revealed NADPH-d activity in neurones and in glial cells. A cluster of intensely stained interneurones innervates the neuropile of the antennal lobe. NADPH-d expression in the mushroom bodies showed a compartmentalized pattern. The mushroom body intrinsic Kenyon cells did not express NADPH-d. The pedunculus and lobes showed fine granular staining and were invaded by NADPH-d-positive mushroom body extrinsic fibres. The expression of NADPH-d in glial cells enclosing the mushroom bodies suggest that insect glial cells may release NO as a messenger molecule.

The organization of putative octopaminergic pathways in the brain and subesophageal ganglion of the honeybee was investigated with a well-defined polyclonal antiserum against octopamine. Five prominent groups of just over 100 immunoreactive (IR) somata were found in the cerebral ganglion: Neurosecretory cells in the pars intercerebralis innervating the corpora cardiaca via NCC I, one cluster mediodorsal to the antennal lobe, one scattered on both sides of the midline of the protocerebrum, one between the lateral protocerebral lobes and the dorsal lobes, and a single soma on either side of the central body. With the exception of the pedunculi and β-lobes of the mushroom bodies, varicose immunoreactive fibers penetrate all parts of the cerebral ganglion. Strong labelling was found in the central complex and the protocerebral bridge. Fine networks of labelled processes invade the antennal lobes, the calyces and a small part of the α-lobes of the mushroom bodies, the protocerebrum, and all three optic ganglia. In the subesophageal ganglion, one labelled cell body was found in the lateral soma layer of the mandibular segment. Each of the three neuromeres contains a group of six to ten somata in the ventral median parts. Most of the ventral median cells send their neurites dorsally through the midline tracts, whereas the neurites of a few cells follow the ventral cell body neurite tracts. Octopamine-IR was demonstrated in all neuropils that contain pathways for proboscis extension learning in honeybees. Because octopaminergic mechanisms seem to be involved in the behavioral plasticity of the proboscis extension reflex, our study provides anatomical data on the neurochemical organization of an appetitive learning paradigm. © 1994 Wiley-Liss, Inc.

We examined the development of olfactory neuropils in the hemimetabolous insect Locusta migratoria with an emphasis on the mushroom bodies, protocerebral integration centers implicated in memory formation. Using a marker of the cyclic adenosine monophosphate (cAMP) signaling cascade and lipophilic dye labeling, we obtained new insights into mushroom body organization by resolving previously unrecognized accessory lobelets arising from Class III Kenyon cells. We utilized antibodies against axonal guidance cues, such as the cell surface glycoproteins Semaphorin 1a (Sema 1a) and Fasciclin I (Fas I), as embryonic markers to compile a comprehensive atlas of mushroom body development. During embryogenesis, all neuropils of the olfactory pathway transiently expressed Sema 1a. The immunoreactivity was particularly strong in developing mushroom bodies. During late embryonic stages, Sema 1a expression in the mushroom bodies became restricted to a subset of Kenyon cells in the core region of the peduncle. Sema 1a was differentially sorted to the Kenyon cell axons and absent in the dendrites. In contrast to Drosophila, locust mushroom bodies and antennal lobes expressed Fas I, but not Fas II. While Fas I immunoreactivity was widely distributed in the midbrain during embryogenesis, labeling persisted into adulthood only in the mushroom bodies and antennal lobes. Kenyon cells proliferated throughout the larval stages. Their neurites retained the embryonic expression pattern of Sema 1a and Fas I, suggesting a role for these molecules in developmental mushroom body plasticity. Our study serves as an initial step toward functional analyses of Sema 1a and Fas I expression during locust mushroom body formation. J. Comp. Neurol. 520:2021–2040, 2012. © 2011 Wiley Periodicals, Inc.

The selection of a minimal active sequence of erythropoietin allowed the design of peptide mimetics that exert beneficial effects in the central nervous system but lack an erythropoietic effect. Erythropoietin has been suggested as a promising therapeutic and prophylactic for epilepsies based on its neuroprotective, neuroregenerative, and antiinflammatory potency. Therefore, it is of particular interest to evaluate whether the nonerythropoietic erythropoietin-derived peptide pHBSP can affect epileptogenesis.In a post-status epilepticus model in rats, we determined the effects of pHBSP and of recombinant human erythropoietin with short-term administration following status epilepticus.Both pHBSP and erythropoietin further enhanced the status epilepticus-associated increase in hippocampal cell proliferation. Thereby, pHBSP seemed to promote neuronal differentiation and survival resulting in a significant increase in neurogenesis. Neither pHBSP nor erythropoietin affected the number of animals exhibiting spontaneous recurrent seizures as well as the seizure frequency in the chronic phase. In the Morris water maze, pHBSP attenuated cognitive deficits in epileptic animals.In conclusion, the helix B-derived erythropoietin peptide pHBSP can modulate the cellular and cognitive consequences of a status epilepticus. The impact of pHBSP on spatial learning might indicate that the peptide allows beneficial effects on epileptogenesis-associated cognitive deficits. However, it needs to be considered that learning deficits were not abolished by pHBSP and that the effects were not observed consistently until the end of the study. Therefore, adjustment of timing, duration, and dose of peptide administration might be necessary to further evaluate the efficacy of pHBSP.