Sabine Kreissl

Stimulatory effect of octopamine on juvenile hormone biosynthesis in honey bees (Apis mellifera) : Physiological and immunocytochemical evidence

Abstract The effect of octopamine on the activity of corpora allata of adult worker honey bees has been examined in vitro and correlated to the local distribution of this biogenic amine in brain and retrocerebral complex as studied immunocytochemically by means of an highly specific antiserum. Octopamine causes a dose-dependent increase in juvenile hormone release from corpora allata. Maximum increase is obtained with concentrations of 10−6 M in nurse and foraging bees by 45.3 or 32.3%, respectively. Octopamine-like immunoreactivity occurs in about 45 somata of the median neurosecretory cells in the pars intercerebralis of the bee brain. They project via immunopositive nervus corporis cardiaci I into the corpora cardiaca, where interspersed varicose structures and 8–10 cell bodies in the ventral part of this gland are stained. A network of immunoreactive fine varicose nerve fibres surrounds each gland cell of the corpora allata. Immunoreactivity in these neuronal structures is detectable if bees were starved over night, a condition in which corpora allata elicit the highest juvenile hormone production ever observed in bees. Both, the stimulatory effect of octopamine and the presence of immunoreactive nerve fibers in the corpora allata, strongly indicate a physiological role of this biogenic amine in the regulation of juvenile hormone biosynthesis in adult honey bees.

Dissociated neurons of the pupal honeybee brain in cell culture

SummaryPrimary cell cultures were prepared from specific regions of the pupal honeybee brain which are involved in proboscis extension learning. Defined areas could be dissociated purely by mechanical treatment. We show that cultured neurons regenerate new neurites and remain viable for up to three weeks in a serum-free, chemically-defined medium. Several labelling techniques were employed to identify subpopulations of cultured neurons. For example, acetylcholinesterase staining; fluorescent beads to distinguish identified cell populations of co-cultured brain areas; various markers for surface antigens such as a monoclonal antibody to olfactory projection neurons of the antennoglomerular tracts and monopolar cells of the optic lobes, as well as anti-HRP immunoreactivity and α-bungarotoxin binding; and various antisera for detecting transmitter phenotype. The appearance of transmitter-immunoreactive cells agreed closely with that expected from their known distributionin situ. Our results suggest that cultured cells retain surface properties and transmitter phenotype of theirin vivo counterparts, despite differences in basic morphology. Thus our culture system provides the important initial step for futurein vitro investigations of the cellular and electrophysiological properties of neurons mediating proboscis extension learning.

Allatostatin immunoreactivity in the honeybee brain

Information transmission and processing in the brain is achieved through a small family of chemical neurotransmitters and neuromodulators and a very large family of neuropeptides. In order to understand neural networks in the brain it will be necessary, therefore, to understand the connectivity, morphology, and distribution of peptidergic neurons, and to elucidate their function in the brain. In this study we characterize the distribution of substances related to Dip‐allatostatin I in the honeybee brain, which belongs to the allatostatin‐A (AST) peptide family sharing the conserved c‐terminal sequence ‐YXFGL‐NH2. We found about 500 AST‐immunoreactive (ASTir) neurons in the brain, scattered in 18 groups that varied in their precise location across individuals. Almost all areas of the brain were innervated by ASTir fibers. Most ASTir neurites formed networks within functionally distinct areas, e.g., the antennal lobes, the mushroom bodies, or the optic lobes, indicating local functions of the peptide. A small number of very large neurons had widespread arborizations and neurites were found in the corpora cardiaca and in the cervical connectives, suggesting that AST also has global functions. We double‐stained AST and GABA and found that a subset of ASTir neurons were GABA‐immunoreactive (GABAir). Double staining AST with backfills of olfactory receptor neurons or mass fills of neurons in the antennal lobes and in the mushroom bodies allowed a more fine‐grained description of ASTir networks. Together, this first comprehensive description of AST in the bee brain suggests a diverse functional role of AST, including local and global computational tasks. J. Comp. Neurol. 518:1391–1417, 2010.

Allatostatin modulates skeletal muscle performance in crustaceans through pre‐ and postsynaptic effects

Allatostatins, originally identified in insects as peptide inhibitors of juvenile hormone biosynthesis, are regarded as potent inhibitory regulators of intestinal muscles in insects and crustaceans. However, accumulating data indicate that allatostatins might also be involved in modulation of skeletal neuromuscular events. We show that most ganglia of two isopod crustaceans (Idotea baltica and I. emarginata) contain pairs of large, allatostatin‐immunoreactive motor neurons which supply several segmental muscles. Among them are the dorsal extensor muscles, of which some fibres receive immunoreactive, varicose innervation. We demonstrate, on identified muscle fibres, that allatostatin exerts a twofold inhibitory effect: it reduces contractions of single voltage‐clamped fibres, and it decreases the amplitude of evoked excitatory junctional currents recorded from individual release boutons. No change in excitation‐contraction threshold or in passive membrane parameters was observed. As the amplitude of miniature currents generated by spontaneously released single transmitter quanta was not changed, the inhibitory effect of the peptide on junctional currents must be of presynaptic origin. Supportive results were obtained on leg muscles of the crab Eriphia spinifrons, where allatostatin decreased evoked synaptic currents by reducing the mean number of transmitter quanta released by presynaptic depolarization without affecting the amplitudes of currents generated by single quanta. This effect of allatostatin was similar for two functionally different neurons, the slow and the fast closer excitor.

Inhibitory connections in the honeybee antennal lobe are spatially patchy

The olfactory system is a classical model for studying sensory processing. The first olfactory brain center [the olfactory bulb of vertebrates and the antennal lobe (AL) of insects] contains spherical neuropiles called glomeruli. Each glomerulus receives the information from one olfactory receptor type. Interglomerular computation is accomplished by lateral connectivity via interneurons. However, the spatial and functional organization of these lateral connections is not completely understood. Here we studied the spatial logic in the AL of the honeybee. We combined topical application of neurotransmitters, olfactory stimulations, and in vivo calcium imaging to visualize the arrangement of lateral connections. Suppression of activity in a single glomerulus with γ-aminobutyric acid (GABA) while presenting an odor reveals the existence of inhibitory interactions. Stimulating a glomerulus with acetylcholine (ACh) activates inhibitory interglomerular connections that can reduce odor-evoked responses. We show that this lateral network is patchy, in that individual glomeruli inhibit other glomeruli with graded strength, but in a spatially discontinuous manner. These results suggest that processing of olfactory information requires combinatorial activity patterns with complex topologies across the AL.

Muscle development in the marbled crayfish--insights from an emerging model organism (Crustacea, Malacostraca, Decapoda).

The development of the crustacean muscular system is still poorly understood. We present a structural analysis of muscle development in an emerging model organism, the marbled crayfish—a representative of the Cambaridae. The development and differentiation of muscle tissue and its relation to the mesoderm-forming cells are described using fluorescent and non-fluorescent imaging tools. We combined immunohistochemical staining for early isoforms of myosin heavy chain with phallotoxin staining of F-actin, which distinguishes early and more differentiated myocytes. We were thus able to identify single muscle precursor cells that serve as starting points for developing muscular units. Our investigations show a significant developmental advance in head appendage muscles and in the posterior end of the longitudinal trunk muscle strands compared to other forming muscle tissues. These findings are considered evolutionary relics of larval developmental features. Furthermore, we document the development of the muscular heart tissue from myogenic precursors and the formation and differentiation of visceral musculature.

Muscle precursor cells in the developing limbs of two isopods (Crustacea, Peracarida): an immunohistochemical study using a novel monoclonal antibody against myosin heavy chain

In the hot debate on arthropod relationships, Crustaceans and the morphology of their appendages play a pivotal role. To gain new insights into how arthropod appendages evolved, developmental biologists recently have begun to examine the expression and function of Drosophila appendage genes in Crustaceans. However, cellular aspects of Crustacean limb development such as myogenesis are poorly understood in Crustaceans so that the interpretative context in which to analyse gene functions is still fragmentary. The goal of the present project was to analyse muscle development in Crustacean appendages, and to that end, monoclonal antibodies against arthropod muscle proteins were generated. One of these antibodies recognises certain isoforms of myosin heavy chain and strongly binds to muscle precursor cells in malacostracan Crustacea. We used this antibody to study myogenesis in two isopods, Porcellio scaber and Idotea balthica (Crustacea, Malacostraca, Peracarida), by immunohistochemistry. In these animals, muscles in the limbs originate from single muscle precursor cells, which subsequently grow to form multinucleated muscle precursors. The pattern of primordial muscles in the thoracic limbs was mapped, and results compared to muscle development in other Crustaceans and in insects.

A single allatostatin-immunoreactive neuron innervates skeletal muscles of several segments in the locust

In the nervous system of embryos and adult Locusta migratoria, somata, neurites within the ganglia, and axons leaving the thoracic ganglia show allatostatin immunoreactivity. The immunoreactive efferent axons divide to follow different nerve branches and form varicose terminals on skeletal muscles. In the adult locust, one pair of motor neurons is particularly prominent among the allatostatin‐immunoreactive neurons. The somata are located symmetrically in a lateral position in the first abdominal neuromere of the fused metathoracic ganglion. Each neuron gives rise to five axon branches projecting into ipsilateral nerves. Three axons project posteriorly and exit through the dorsal nerves of the abdominal neuromeres A1, A2, and A3. One axon extends into the metathoracic neuromere and exits through metathoracic nerve 1 (N1). The fifth axon extends anteriorly through the connective into the mesothoracic ganglion, where it leaves through the mesothoracic N1. The targets of this neuron, among them the mesothoracic and metathoracic muscles M87, M88, M116 and the dorsal longitudinal muscles M81 and M112, are located in five different segments. In addition to supplying skeletal muscles, the neuron forms neurohaemal‐like structures in the sheath of nerve branches. The authors call this neuron the common lateral neuron (CLN). The innervation of several muscles by Diploptera allatostatin 7‐immunoreactive axon branches with a common cellular origin and the anatomy of one of the corresponding motor neurons in adults, the CLN, suggest that allatostatin acts as a modulator of neuromuscular parameters in insects by multisegmental direct innervation of skeletal muscles. J. Comp. Neurol. 413:507–519, 1999.

The neuropeptide proctolin potentiates contractions and reduces cGMP concentration via a PKC-dependent pathway

SUMMARY As in many other arthropods, the neuropeptide proctolin enhances contractures of muscles in the crustacean isopod Idotea emarginata. The enhancement of high K+-induced contractures by proctolin (1μ mol l-1) was mimicked upon application of the protein kinase C (PKC) activator phorbol-12-myristate 1-acetate (PMA) and was inhibited by the PKC inhibitor bisindolylmaleimide (BIM-1). The potentiation was not inhibited by H89, a protein kinase A (PKA) inhibitor. Proctolin did not change the intracellular concentration of 3′,5′-cyclic adenosine monophosphate (cAMP) whereas it significantly reduced the intracellular concentration of 3′,5′-cyclic guanosine monophosphate (cGMP). The reduction of cGMP was not observed in the presence of the PKC inhibitor BIM-1. 8-Bromo-cGMP, a membrane-permeable cGMP analogue, reduced the potentiating effect of proctolin on muscle contracture. We thus conclude that proctolin in the studied crustacean muscle fibres induces an activation of PKC, which leads to a reduction of the cGMP concentration and, consequently, to the potentiation of muscle contracture.

Localization of a FMRFamide-related peptide in efferent neurons and analysis of neuromuscular effects of DRNFLRFamide (DF2) in the crustacean Idotea emarginata.

In the ventral nerve cord of the isopod Idotea emarginata, FMRFamide‐immunoreactive efferent neurons are confined to pereion ganglion 5 where a single pair of these neurons was identified. Each neuron projects an axon into the ipsilateral ventral and dorsal lateral nerves, which run through the entire animal. The immunoreactive axons form numerous varicosities on the ventral flexor and dorsal extensor muscle fibres, and in the pericardial organs. To analyse the neuromuscular effects of a FMRFamide, we used the DRNFLRFamide (DF2). DF2 acted both pre‐ and postsynaptically. On the presynaptic side, DF2 increased transmitter release from neuromuscular endings. Postsynaptically, DF2 depolarized muscle fibres by approximately 10 mV. This effect was not observed in leg muscles of a crab. The depolarization required Ca2+, was blocked by substituting Ca2+ with Co2+, but not affected by nifedipine or amiloride. In Idotea, DF2 also potentiated evoked extensor muscle contractions. The amplitude of high K+ contractures was increased in a dose dependent manner with an EC50 value of 40 nm. In current‐clamped fibres, DF2 strongly potentiated contractions evoked by current pulses exceeding excitation‐contraction threshold. In voltage‐clamped fibres, the inward current through l‐type Ca2+ channels was increased by the peptide. The observed physiological effects together with the localization of FMRFamide‐immunoreactive efferent neurons suggest a role for this type of peptidergic modulation for the neuromuscular performance in Idotea. The pre‐ and postsynaptic effects of DF2 act synergistically and, in vivo, all should increase the efficacy of motor input to muscles resulting in potentiation of contractions.