Synaptic Organization of Microglomerular Clusters in the Lateral and Medial Bulbs of the Honeybee Brain.

The honeybee Apis mellifera is an established model for the study of visual orientation. Yet, research on this topic has focused on behavioral aspects and has neglected the investigation of the underlying neural architectures in the bee brain. In other insects, the anterior optic tubercle (AOTU), the lateral (LX) and the central complex (CX) are important brain regions for visuospatial performances. In the central brain of the honeybee, a prominent group of neurons connecting the AOTU with conspicuous microglomerular synaptic structures in the LX has been recently identified, but their neural organization and ultrastructure have not been investigated. Here we characterized these microglomerular structures by means of immunohistochemical and ultrastructural analyses, in order to evaluate neurotransmission and synaptic organization. Three-dimensional reconstructions of the pre-synaptic and post-synaptic microglomerular regions were performed based on confocal microscopy. Each pre-synaptic region appears as a large cup-shaped profile that embraces numerous post-synaptic profiles of GABAergic tangential neurons connecting the LX to the CX. We also identified serotonergic broad field neurons that probably provide modulatory input from the CX to the synaptic microglomeruli in the LX. Two distinct clusters of microglomerular structures were identified in the lateral bulb (LBU) and medial bulb (MBU) of the LX. Although the ultrastructure of both clusters is very similar, we found differences in the number of microglomeruli and in the volume of the pre-synaptic profiles of each cluster. We discuss the possible role of these microglomerular clusters in the visuospatial behavior of honeybees and propose research avenues for studying their neural plasticity and synaptic function.

Morphological characterization of the antennal lobes in the Mediterranean fruit fly Ceratitis capitata.

The medfly Ceratitis capitata is one of the most important pests for horticulture worldwide. The knowledge about anatomy and function of the medfly olfactory system is still limited. The first brain structure to process olfactory information in insects is the antennal lobe (AL), which is composed of its functional and morphological units, the olfactory glomeruli. Here, we present a morphological three-dimensional reconstruction of AL glomeruli in adult brains. We used unilateral antennal backfills of olfactory receptor neurons (ORNs) with neural tracers, revealing the AL structure. We recorded confocal stacks acquired from whole-mount specimens, and analyzed them with the software AMIRA. The ALs in C. capitata are organized in glomeruli which are more tightly packed in the anterior part than the posterior one. Axons of ORNs bilaterally connect the ALs through a commissure between the two ALs. This commissure is formed by several distinct fascicles. Contralateral dye transfer suggests the presence of gap junctions connecting ORNs from both antennae. There was no statistical difference between the average volumes of female ALs (204,166 ± 12,554 µm(3)) and of male ALs (190,287 ± 11,823 µm(3)). In most specimens, we counted 53 glomeruli in each AL, seven of which were sexually dimorphic in size.

Perception of floral volatiles involved in host-plant finding behaviour: comparison of a bee specialist and generalist.

Specialist and generalist bees use olfactory and visual cues to find and recognise flowering plants. Specialised (oligolectic) bees rely on few host plants for pollen collection. These bee species are suggested to use specific volatiles, but it is unknown whether they have dedicated adaptations for these particular compounds compared to bees not specialised on the same plants. In the present study, we investigated the perception of host odorants and its neuronal substrate with regard to host-plant finding behaviour in oligolectic bees. We reconstructed the antennal lobes (AL) in the Salix specialist, Andrena vaga, and counted about 135 glomeruli and thereby less than the approximately 160 in honeybees. Using calcium imaging experiments to measure neural activity in the bee brain, we recorded odorant-evoked activity patterns in the AL of A. vaga and, for comparison, in the generalist honeybee, Apis mellifera. Our physiological experiments demonstrated that A. vaga bees were particularly sensitive to 1,4-dimethoxybenzene, a behaviour-mediating odorant of Salix host flowers. We found more sensitive glomeruli in the specialised bees as compared to generalist honeybees. This neural adaptation might allow oligolectic A. vaga bees to effectively locate host plants from distances.

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.

Myogenesis in the thoracic limbs of the American lobster.

Newly hatched lobster larvae have biramous thoracic limbs composed of an endopodite, which is used for walking in the adult, and an exopodite used for swimming. Several behavioural and physiological aspects of larval locomotion as well the ontogeny of the neuromuscular system have been examined in developing decapod crustaceans. Nevertheless, the cellular basis of embryonic muscle formation in these animals is poorly understood. Therefore, the present report analyses muscle formation in embryos of the American lobster Homarus americanus Milne Edwards, 1837 (Malacostraca, Eucarida, Decapoda, Homarida) using the monoclonal antibody 016C6 that recognizes an isoform of myosin heavy chain. 016C6 labelling at 25% of embryonic development (E25%) revealed that syncytial muscle precursor cells establish the muscles in the endopodites. During subsequent embryogenesis, these muscle precursors subdivide into several distinct units thereby giving rise to pairs of antagonistic primordial muscles in each of the successive podomeres, the layout of which at E45% already resembles the arrangement in the adult thoracopods. The pattern of primordial muscles was also mapped in the exopodites of thoracic limbs three to eight. Immunohistochemistry against acetylated α-tubulin and against presynaptic vesicle-associated phosphoproteins at E45% demonstrated the existence of characteristic neural tracts within the developing limbs as well as putative neuromuscular synapses in both the embryonic exo- and endopodites. The results are compared to muscle development in other Crustacea.

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-NH(2). 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.

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

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 (1 micromol 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.

The neuromuscular junctions of the slow and the fast excitatory axon in the closer of the crab Eriphia spinifrons are endowed with different Ca2+ channel types and allow neuron-specific modulation of transmitter release by two neuropeptides.

Most crustacean muscle fibers receive double excitatory innervation by functionally different motor neurons termed slow and fast. By using specific omega-toxins we show that the terminals of the slow closer excitor (SCE) and the fast closer excitor (FCE) at a crab muscle are endowed with different sets of presynaptic Ca(2+) channel types. omega-Agatoxin, a blocker of vertebrate P/Q-type channels, reduced the amplitude of EPSCs by decreasing the mean quantal content of transmitter release in both neurons by 70-85%, depending on the concentration. We provide the first evidence that omega-conotoxin-sensitive channels also participate in transmission at crustacean neuromuscular terminals and are colocalized with omega-agatoxin-sensitive channels in an axon-type-specific distribution. omega-Conotoxin, a blocker of vertebrate N-type channels, inhibited release by 20-25% only at FCE, not at SCE endings. Low concentrations of Ni(2+), which block vertebrate R-type channels, inhibited release in endings of the SCE by up to 35%, but had little effects in FCE endings. We found that two neuropeptides, the FMRFamide-like DF(2) and proctolin, which occur in many crustaceans, potentiated evoked transmitter release differentially. Proctolin increased release at SCE and FCE endings, and DF(2) increased release only at FCE endings. Selective blocking of Ca(2+) channels by different omega-toxins in the presence of peptides revealed that the target of proctolin-mediated modulation is the omega-agatoxin-sensitive channel (P/Q-like), that of DF(2) the omega-conotoxin-sensitive channel (N-like). The differential effects of these two peptides allows fine tuning of transmitter release at two functionally different motor neurons innervating the same muscle.