Dissociated neurons of the pupal honeybee brain in cell culture

Primary 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 agr-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

Histochemistry of acetylcholinesterase and immunocytochemistry of an acetylcholine receptor-like antigen in the brain of the honeybee

A histochemical staining method for acetylcholinesterase (AChE) and an antiserum raised against nicotinic acetylcholine receptors (AChR) of locust nervous tissue were applied in order to reveal certain candidates of cholinergic pathways in the brain of the honeybee. The AChE staining marked layers in the optic lobes, fibers connecting the two brain hemispheres, and fiber tracts as well as soma clusters within the protocerebrum. The calycal input regions of the mushroom bodies were labelled, whereas the intrinsic Kenyon cells showed no staining. Although the antennal afferents projecting into the dorsal lobe showed strong AChE activity, projections into the antennal lobe showed rather weak staining.Application of the antiserum against the AChR showed immunoreactivity in neuropiles, tracts, somata, and the antennal nerve. The immunoreactivity of the optic lobes coincided with the banding pattern of the AChE staining. A particularly striking overlap of AChR immunoreactivity and AChE staining was found in the lip neuropile of the mushroom bodies, which would suggest a cholinergic input into this neuropile via fibers of the median antennoglomerular tract. Because the antiserum against locust AChR binds in neuropiles displaying AChE activity, we conclude that this antiserum also cross-reacts with the bee's receptor. This interpretation is supported by experiments showing α-bungarotoxin (α-BTX) binding sites in some areas of strong immunoreactivity

Calcium imaging reveals nicotinic acetylcholine receptors on cultured mushroom body neurons

1. We used fluorescence imaging with the visible wavelength indicator fluo-3 to investigate the calcium responses to cholinergic ligands of honeybee Kenyon cells in primary culture.2. Application of acetylcholine ( ACh) or nicotine, but not pilocarpine, promoted a calcium influx into the cell body and neurites. The increase in intracellular calcium after ACh stimulation was blocked by tu-bungarotoxin. These results support previous histochemical studies that suggested the expression of nicotinic cholinergic receptors on Kenyon cells.3. After depolarization with high K+ solution fluorescence increasedin the somata and neurites, which indicates the presence ofvoltage-gated Ca *+ channels in Kenyon cell membranes

Inhibitory connections in the honeybee antennal lobe are spatially patchy

We combine calcium imaging with focalized neurotransmitter application to map inhibitory connections in the antennal lobe network. We find that these connections are patchy, and different among individuals, suggesting that they derive from learning experiences

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 (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

Monoclonal antibody labels olfactory and visual pathways in Drosophila and Apis brains

We employed a monoclonal antibody raised against Drosophila brain homogenate for a comparative immunocytochemical analysis of visual and olfactory pathways in brains of two insect species. On Western blots of Drosophila and Apis nervous tissue, antibody fb45 recognized an antigen with an apparent molecular weight higher than 180 kD. Application of the antibody to sections of Drosophila and Apis brain stained certain interneurons which conspicuously fasciculate in common tracts or neuropilar compartments. Both in Drosophila and in Apis, the antigen was also expressed on the perineural sheath and granular cell compartments in the majority of neuronal cell bodies.The antibody stained monopolar cells in the visual system of both species, and in Apis those fibers of the anterior superior optic tract which link the medulla with the mushroom bodies. In Drosophila, bundles of Kenyon cells of the mushroom bodies were stained. In worker bees and drones, the relay neurons of the median and lateral antennoglomerular tracts were labelled.Since the recognition of the antigen does not require fixation, the antibody can be employed to label selectively living neurons in dissociated cell culture. This opens up the possibility for future functional studies on the role of the antigen in vitro

Multidimensional scaling of color similarity in bees

A multiple choice experiment with free flying bees trained to a color signal is described which allows for multidimensional scaling of color similarity. The choice proportions are analysed by metric (Torgerson 1958) and non-metric (Kruskal 1964a, b) multidimensional scaling. The light reflected from the twelve color signals used differed in spectral composition, intensity, and the proportion of white light. Only two scales are necessary to reconstruct the experimental data. The interpretation of the scale values by Helmholtz-coordinates, derived from the chromaticity diagram for bees, shows that the main perceptual parameters are hue and saturation (or blue/greenness and UV/blue-greenness, respectively). Brightness is ignored by the bees in this choice situation. The total color difference is related to the differences on the two perceptual parameters by the city-block metric (Minkowski exponentp=1)

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

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

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

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