Gerd Bicker

HISTOCHEMISTRY OF CLASSICAL NEUROTRANSMITTERS IN ANTENNAL LOBES AND MUSHROOM BODIES OF THE HONEYBEE

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 α‐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. Microsc. Res. Tech. 45:174–183, 1999.

Serotonin-immunoreactive neurons in the antennal lobes and suboesophageal ganglion of the honeybee

SummaryWe have used immunohistochemical methods to investigate the morphology of identified, presumptive serotonergic neurons in the antennal lobes and suboesophageal ganglion of the worker honeybee. A large interneuron (deutocerebral giant, DCG) is described that interconnects the deutocerebral antennal and dorsal lobes with the suboesophageal ganglion and descends into the ventral nerve chord. This neuron is accompanied by a second serotonin-immunoreactive interneuron with projections into the protocerebrum. Two pairs of bilateral immunoreactive serial homologues were identified in each of the three suboesophageal neuromeres and were also found in the thoracic ganglia. With the exception of the frontal commissure, no immunoreactive processes could be found in the peripheral nerves of the brain and the suboesophageal ganglion. The morphological studies on the serial homologues were extended by intracellular injections of Lucifer Yellow combined with immunofluorescence.

NO news from insect brains

In nerve cells,the short-lived signalling molecule nitric oxide (NO) is generated by Ca2+-calmodulin-stimulated NO synthases. Nitric oxide activates soluble guanylate cyclase in target cells, leading to the formation of cGMP. Biochemical investigations have shown the presence of a Ca2+-calmodulin-regulated NO-cGMP signalling mechanism in the nervous system of insects. Using NADPH-diaphorase staining as a marker for the enzyme NO synthase and an antiserum against cGMP,the cellular organization of NO donor and target cells has so far been resolved in the locust and fruit fly. This paper provides an overview of the cellular organization of NO signalling in the insect nervous system as well as highlighting its functions in olfactory information processing, formation of olfactory memory, vision, and neuronal development. The resolution of discrete donor and NO-responsive target cells in the developing nervous system of Drosophila will facilitate the genetic and pharmacological analysis of NO-cGMP signal transduction.

Sources and targets of nitric oxide signalling in insect nervous systems

Abstract. Nitric oxide (NO) is a membrane permeant signalling molecule which activates soluble guanylyl cyclase and leads 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 review summarizes neurochemical, cell biological, and physiological investigations of NO/cGMP signalling in the nervous system of insects. The anatomical localization of donor and target cells suggests functions in olfaction, vision, and mechanosensation. Behavioural assays have uncovered contributions of NO signalling in oxygen sensing, habituation to chemosensory stimuli, and associative memory formation. During development, NO regulates cell proliferation, axonal outgrowth, and synaptic maturation. The cellular distribution of NO-responsive cells suggests that NO can serve as a retrograde synaptic messenger, as an intracellular messenger, and as a lateral diffusible messenger irrespective of conventional synaptic connectivity.

A tissue-specific marker of Ecdysozoa

Abstract. 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.

The Nitric Oxide/Cyclic GMP Messenger System in Olfactory Pathways of the Locust Brain

Nitric oxide is generated by a Ca2+/calmodulin‐stimulated nitric oxide synthase and activates soluble guanylyl cyclase. Using NADPH diaphorase (NADPHd) staining as a marker for the enzyme nitric oxide synthase and an antiserum against cGMP, we investigated the cellular organization of nitric oxide donor and target cells in olfactory pathways of the brain of the locust (Schistocerca gregaria). A small subset of neuronal and glial cells expressed cGMP immunoreactivity after incubation of tissue in a nitric oxide donor. Nitric oxide‐induced increases in cGMP immunoreactivity were quantified in a tissue preparation of the antennal lobe and in primary mushroom body cell cultures. The mushroom body neuropil is a potential target of a transcellular nitric oxide/ cGMP messenger system since it is innervated by extrinsic NADPHd‐positive neurons. The mushroom body‐intrinsic Kenyon cells do not stain for NADPHd but can be induced to express cGMP immunoreactivity. The colocalization of NADPHd and cGMP immunoreactivity in a cluster of interneurons of the antennal lobe, the principal olfactory neuropil of the insect brain, suggests a role of the nitric oxide/cGMP system in olfactory sensory processing. Colocalization of NADPHd staining and cGMP immunoreactivity was also found in certain glial cells. The cellular organization of the nitric oxide/cGMP system in neurons and glia raises the possibility that nitric oxide acts not only as an intercellular but also as an intracellular messenger molecule in the insect brain.

Nitric oxide and cyclic nucleotides are regulators of neuronal migration in an insect embryo

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.

Developmental expression of nitric oxide/cyclic GMP synthesizing cells in the nervous system of Drosophila melanogaster.

Nitric oxide (NO) is a membrane-permeant signaling molecule which activates soluble guanylyl cyclase and leads to the formation of cyclic GMP (cGMP). The NO/cGMP signaling system is thought to play essential roles during the development of vertebrate and invertebrate animals. Here, we analyzed the cellular expression of this signaling pathway during the development of the Drosophila melanogaster nervous system. Using NADPH diaphorase histochemistry as a marker for NO synthase, we identified several neuronal and glial cell types as potential NO donor cells. To label NO-responsive target cells, we used the detection of cGMP by an immunocytochemical technique. Incubation of tissue in an NO donor induced cGMP immunoreactivity (cGMP-IR) in individual motoneurons, sensory neurons, and groups of interneurons of the brain and ventral nerve cord. A dynamic pattern of the cellular expression of NADPHd staining and cGMP-IR was observed during embryonic, larval, and prepupal phases. The expression of NADPH diaphorase and cGMP-IR in distinct neuronal populations of the larval central nervous system (CNS) indicates a role of NO in transcellular signaling within the CNS and as potential retrograde messenger across the neuromuscular junction. In addition, the presence of NADPH diaphorase-positive imaginal discs containing NO-responsive sensory neurons suggests that a transcellular NO/cGMP messenger system can operate between cells of epithelial and neuronal phenotype. The discrete cellular resolution of donor and NO-responsive target cells in identifiable cell types will facilitate the genetic, pharmacological, and physiological analysis of NO/cGMP signal transduction in the developing nervous system of Drosophila.

BIOGENIC AMINES IN THE BRAIN OF THE HONEYBEE: CELLULAR DISTRIBUTION, DEVELOPMENT, AND BEHAVIORAL FUNCTIONS

This review provides a summary of the cellular distribution of amine‐containing neurons and the organization of aminergic pathways in the brain and suboesophageal ganglion of the honeybee. Neurons synthesizing the biogenic amines serotonin, dopamine, octopamine, and histamine are stained with well‐defined polyclonal antisera. Since some of these aminergic neurons are uniquely identifiable, it is possible to follow their morphogenesis during brain development. Pharmacological studies show that aminergic mechanisms are involved in various behavioral modifications including associative learning. The immunocytochemical approach resolves at a single cell level the neural pathways that mediate adaptive behavioral changes. Microsc. Res. Tech. 44:166–178, 1999.

Turning teratocarcinoma cells into neurons: rapid differentiation of NT-2 cells in floating spheres

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.