Neurosecretory fibres in the median eyes of the scorpion,Androctonus australis L.

Summary The retina of the median eyes of the North African scorpion, Androctonus australis L., is supplied with numerous neurosecretory nerve fibres which establish synaptoid contacts on the retinula cells. The number of fibres or profiles of varicosities of fibre terminals associated with a retinular unit (five retinula cells with a fused rhabdom) varies between 10 and 20. Electron-opaque vesicles with a diameter of 80–100 nm are abundant within the axonal profiles. The synaptoid junctions are characterized by postsynaptic electron-dense material on the inner leaflet of the retinula cell membrane and, frequently, presynaptic submembranous dense material. Because of these ultrastructural features, the junctions observed here resemble typical interneuronal synaptic contacts. Hence this kind of neurosecretory junction appears to be unique among arthropods.It is suggested that the neurosecretory fibres within the retina represent the efferent pathways for the control of the circadian pigment movements within the retinula cells.Supported by the Deutsche Forschungsgemeinschaft (F1 77/7)     

The lateral eyes of the scorpion,Androctonus australis

Summary The dioptric apparatus of the lateral eyes of the scorpion, Androctonus austrails, consists of a cuticular lens, but lacks a vitreous body. The retina is formed by (1) retinula cells displaying a contiguous network of rhabdoms; (2) arhabdomeric cells bearing a distal dendrite that contacts retinula cells via numerous projections and ends before the rhabdomere of the retinula cells; (3) pigment cells that ensheath retinula and arhabdomeric cells with the exception of the contact regions; and (4) neurosecretory fibres possibly originating in the supraesophageal ganglion. The ratio of the number of retinula to arhabdomeric cells is determined to be close to 2 1 in the three larger anterolateral eyes, in contrast to the median eyes where the ratio is 5 1.The construction of the dioptric apparatus as well as the anatomy of the retina imply that in the lateral eyes of Androctonus australis visual acuity is reduced. A certain degree of spatial discrimination, however, may be retained by the presence of a relatively high number of arhabdomeric cells. It is suggested that the lateral eyes of A. australis mainly function as light detectors, e.g., for Zeitgeber stimuli.Supported by grant no. FL 77/8-10 from the Deutsche Forschungsgemeinschaft     

The Androctonus australis garzoni scorpion venom contains toxins that selectively affect voltage-dependent K+-channels in cerebellum granular cells

A purified peptide from Androctonus australis Garzoni venom (AaG) affects selectively a K⁺-current recorded from cerebellum granular cells. This current is characterized by fast activating and inactivating kinetics similar to an I_A-type current. Addition of 2 μm peptide Aa1 (from Androctonus australis, toxin 1) to the external side of the channel suppressed completely and in a selective manner the I_A-type current, with an IC50 value of 130 nm, whereas in the same conditions, the other potassium current, identified as delayed rectifier (I_d), was not affected. Additionally, we show that another partially purified peptide (III-12) from the same venom was able to block reversibly both K⁺-currents. Received: 10 February 1997 / Accepted: 7 August 1997     

Neuronal components of the circadian clock in the scorpion,Androctonus australis: Central origin of the efferent neurosecretory elements projecting to the median eyes

Summary The central origin of efferent neurosecretory fibers (ENSF) supplying the median eyes of the scorpion, Androctonus australis, with circadian information was investigated at the light- and electron-microscopic levels by the use of several tracer techniques. The results obtained convincingly show that the central neurons stained via the optic nerve are the source of the ENSF in the retina. Almost all of these neurons give rise to two different axon collaterals, one of which projects into the ipsilateral, the other into the contralateral median-eye nerve. Three protocerebral contact zones of the ENSF system are described, and their functional role within the circadian clock system is discussed.This investigation represents a portion of the Ph. D. thesis of one of the authors (S.H.) and was supported by grants to G.F. from the Deutsche Forschungsgemeinschaft (SFB 45).     

Diabody mixture providing full protection against experimental scorpion envenoming with crude Androctonus australis venom

Androctonus australis is primarily involved in envenomations in North Africa, notably in Tunisia and Algeria, and constitutes a significant public health problem in this region. The toxicity of the venom is mainly due to various neurotoxins that belong to two distinct structural and immunological groups, group I (the AahI and AahIII toxins) and group II (AahII). Here, we report the use of a diabody mixture in which the molar ratio matches the characteristics of toxins and polymorphism of the venom. The mixture consists of the Db9C2 diabody (anti-group I) and the Db4C1op diabody (anti-AahII), the latter being modified to facilitate in vitro production and purification. The effectiveness of the antivenom was tested in vivo under conditions simulating scorpion envenomation. The intraperitoneal injection of 30 μg of the diabody mixture protected almost all the mice exposed to 3 LD(50) s.c. of venom. We also show that the presence of both diabodies is necessary for the animals to survive. Our results are the first demonstration of the strong protective power of small quantities of antivenom used in the context of severe envenomation with crude venom.     

Characterization of three "Birtoxin-like" toxins from the Androctonus amoreuxi scorpion venom

The venom of the North African scorpion Androctonus amoreuxi (Aam) was analyzed using a combination of gel filtration, C18 reverse phase HPLC together with mass spectrometry analysis and bioassays. Three novel Birtoxin-like (BTX-L) peptides of 58 amino acid residues comprising three disulfide bridges were isolated and chemically characterized. One peptide, AamBTX-L3, induced serious toxic symptoms in mice and was lethal at nanogram quantities using intracerebroventricular injection. The three BTX-L peptides were tested in competition experiments on rat brain synaptosomes against the ¹²⁵I-labeled “classical” α- and β-toxins of reference, as well as with the ¹²⁵I-KTX, a voltage-gated potassium channel blocker. Only AamBTX-L3 was able to prevent the equilibrium binding of the β-toxin ¹²⁵I-Css IV to its receptor site 4 with a IC₅₀ value of 189 nM. Even if previous electrophysiological data allowed the classification of other BTX-L peptides among the β-type toxins, this report clearly shows that AamBTX-L3 is pharmacologically a β-toxin, which recognizes the voltage-gated Na⁺ (Na_v) channels from central mammalian neurons. In order to uncover the residues functionally essential for interaction between the AamBTX-L3 with the putative receptor site of ¹²⁵I-Css IV on Na_v1.2, molecular models of the three novel Aam BTX-L molecules were made and their surfaces were compared to the already described Css IV biologically interactive surfaces. A hypothesis is given that in BTX-L3, three residues found in the α-helix play a key role during target binding.     

Neurosecretory system of the ventral ganglia in the dragonfly,Orthetrum chrysis (selys) (Odonata: Libellulidae)

Summary The neurosecretory cells of the ventral ganglia in the adult dragonfly, Orthetrum chrysis, are classified into A, B, C1 and C2 cells. The neurosecretory material in the ventral ganglia is composed of PAS-positive material with 1-, 2-glycol groups and some proteins. The proteins rich in cystine or cysteine occur predominantly in the A cells, moderately in C cells and negligibly in B cells. Proteins containing arginine occur in A and B cells only, and those containing basic amino acids occur in C2 cells. The neurosecretory pathways and the neurohemal organs are also described.     

Reduction and reoxidation of the neurotoxin II from the scorpion Androctonus australis Hector

Reoxidation of the totally reduced scorpion neurotoxin II from Androctonus australis Hector (four disulfide bridges) has been investigated. The totally reduced toxin was highly insoluble in neutral and alkaline conditions, which prevented the use of the usual air oxidation process for renaturation. We tested a new method in which the reduced molecules were first solubilized in 10% (v/v) acetic acid and then oxidized by air through dialysis against a series of buffers with a slow pH gradient from 2.2 to 7.0 or 8.0. In this system, up to 95% of the protein was recovered in solution. Addition of reduced and oxidized glutathione accelerated refolding and also permitted a better recovery of fully active peptide as measured by both toxicity to mice and ability to displace 125I radiolabeled toxin II from its binding site on rat brain synaptosomal fractions. The reoxidation reaction could also be monitored directly by high pressure liquid chromatography. A strong effect of guanidine hydrochloride concentration as well as the temperature was observed both on the solubility of the reoxidation intermediates and on the refolding pathway. Finally, the method used, i.e. dialysis reoxidation with a pH gradient from 2.2 to 8.0 in 0.1 M sodium phosphate, 0.1 M sodium chloride, 20 mM guanidine hydrochloride, 1 mM oxidized and reduced glutathione allowed regeneration in high yield (70%) of a reoxidized toxin form indistinguishable from the native toxin. A minor stable and inactive molecular species (about 30%) showing a difference in mobility by electrophoresis was also detected.     

Purification, synthesis and characterization of AaCtx, the first chlorotoxin-like peptide from Androctonus australis scorpion venom

AaCtx is the first chlorotoxin-like peptide isolated from Androctonus australis scorpion venom. Its amino acid sequence shares 70% similarity with chlorotoxin from Leiurus quinquestriatus scorpion venom, from which it differs by twelve amino acids. Due to its very low concentration in venom (0.05%), AaCtx was chemically synthesized. Both native and synthetic AaCtx were active on invasion and migration of human glioma cells. However, their activity was found to be lower than that of chlorotoxin. The molecular model of AaCtx shows that most of amino acids differing between AaCtx and chlorotoxin are localized on the N-terminal loop and the α-helix. Based on known compounds that block chloride channels, we suggest that the absence of negative charged amino acids on AaCtx structure may be responsible for its weak activity on glioma cells migration and invasion. This finding serves as a starting point for structure-function relationship studies leading to design high specific anti-glioma drugs.     

Acetylcholine-evoked uncoupling restricts the passage of Lucifer Yellow between pancreatic acinar cells

Summary Direct cell to cell movement of the fluorescent dye Lucifer Yellow CH (457 daltons) in exocrine acinar tissue is demonstrated by direct observation of living mouse pancreatic segments. Electrical uncoupling of pancreatic acinar cells by local application of a high concentration of acetylcholine significantly restricts cell to cell passage of the fluorescent dye. This result shows that a secretagogue can control direct movement of organic molecules between cells through junctional channels.     

Neurosecretory cell types in the eyestalk of the freshwater prawn Palaemon paucidens

Summary In the medulla terminalis ganglionic X-organ (MTGX) of the eyestalk of the freshwater prawn, Palaemon paucidens, six peptidergic neuro-secretory cell types (A-, B-, C-, D-, E-, and F-cells) are distinguishable on the basis of the different morphology of their elementary granules and rough endoplasmic reticulum (rER). All of these cell types seem to correspond to Type-IIIa cells or dispersing Type-IV cells, that have previously been differentiated at the light microscopic level (Hisano, 1974), as judged from the dimensions of their cell bodies and nuclei. Two other peptidergic neurosecretory cell types that are apparently comparable to the Type-II and Type-IIIb cells (Hisano, 1974), respectively, are recognized in parts of the optic ganglia other than MTGX, and these are now designated as Gand H-cells, respectively. All the remaining cell types, designated as Type-I, cluster-forming Type-IV, Type-V and Type-VI cells in our previous light microscopic study, have small cored-vesicles in their cytoplasm. It remains undecided whether these, possibly aminergic, neurons are neurosecretory or not.The author wishes to express his sincere appreciation to Prof. T. Aoto for his invaluable advice during the course of this study.