Studies on α-Latrotoxin Receptors in Rat Brain Synaptosomes: Correlation Between Toxin Binding and Stimulation of Transmitter Release

Abstract: α-Latrotoxin (α-LT), the major component of black widow spider venom, is a high-molecular-weight protein that acts presynaptically by stimulating the release of stored neurotransmitters. The purified toxin was iodinated to high specific radioactivity by the Bolton-Hunter procedure, without appreciable loss of biological activity. By the use of the ¹²⁵I-toxin, specific receptors were revealed in synaptosome fractions isolated from various regions of the rat brain, but not in nonneural tissues. The density of α-LT receptors [which are probably composed of, or include, membrane protein(s)] varies between 0.6 and 0.88 pmol/mg of synaptosome protein, their affinity is very high ( K _A of the order of 10¹⁰ M ⁻¹), their association rate is fast, and their dissociation rate slow. They might belong to a single, homogeneous class. This last conclusion, however, is still uncertain, because results suggesting a possible heterogeneity were obtained by studying the dissociation of the toxin from synaptosomes incubated in high-salt buffer. Experiments in which the binding of α-LT and its dopamine release activity in striatal synaptosomes were investigated in parallel in a variety of experimental conditions support the hypothesis that occupation of the high-affinity receptors is the initial step in the α-LT activation of the presynaptic response.     

The Effect of Moonlight on Scopoli's Shearwater Calonectris diomedea Colony Attendance Patterns and Nocturnal Foraging: A Test of the Foraging Efficiency Hypothesis

Moonlight is known to affect the nocturnal behaviour and activity rhythms of many organisms. For instance, predators active at night may take advantage from increased visibility afforded by the moon, while prey might regulate their activity patterns to become less detectable. Many species of pelagic seabirds attend their colony only at night, in complete darkness, avoiding approaching their nest sites under moonlight. This behaviour has been most often interpreted as an antipredator adaptation (‘predation avoidance’ hypothesis). However, it may also reflect a lower foraging efficiency during moonlit nights (‘foraging efficiency’ hypothesis). Indeed, moonlight may reduce prey availability because preferred seabird prey is known to occur at higher depths in moonlit nights. Using high‐accuracy behavioural information from data loggers, we investigated the effect of moonlight on colony attendance and at‐sea nocturnal foraging in breeding Scopoli's shearwaters Calonectris diomedea. We found that birds departing for self‐feeding trips around the full moon performed longer trips than those departing around the new moon. On nights when the moon was present only partly, nest burrow entrances took place largely in the moonless portion of the night. Moreover, contrary to predictions from the ‘foraging efficiency’ hypothesis, nocturnal foraging activity increased according to moonlight intensity, suggesting that birds increased their foraging activity when prey became more detectable. This study strengthens the idea that colony attendance behaviour is strictly controlled by moonlight in shearwaters, which is possibly related to the perception of a predation risk.     

Overall and allele-specific expression of the SMC1A gene in female Cornelia de Lange syndrome patients and healthy controls

Cornelia de Lange syndrome (CdLS) is a rare multisystem disorder characterized by facial dysmorphisms, limb anomalies, and growth and cognitive deficits. Mutations in genes encoding subunits (SMC1A, SMC3, RAD21) or regulators (NIPBL, HDAC8) of the cohesin complex account for approximately 65% of clinically diagnosed CdLS cases. The SMC1A gene (Xp11.22), responsible for 5% of CdLS cases, partially escapes X chromosome inactivation in humans and the allele on the inactive X chromosome is variably expressed. In this study, we evaluated overall and allele-specific SMC1A expression. Real-time PCR analysis conducted on 17 controls showed that SMC1A expression in females is 50% higher than in males. Immunoblotting experiments confirmed a 44% higher protein level in healthy females than in males, and showed no significant differences in SMC1A protein levels between controls and patients. Pyrosequencing was used to assess the reciprocal level of allelic expression in six female carriers of different SMC1A mutations and 15 controls who were heterozygous at a polymorphic transcribed SMC1A locus. The two alleles were expressed at a 1:1 ratio in the control group and at a 2:1 ratio in favor of the wild type allele in the test group. Since a dominant negative effect is considered the pathogenic mechanism in SMC1A-defective female patients, the level of allelic preferential expression might be one of the factors contributing to the wide phenotypic variability observed in these patients. An extension of this study to a larger cohort containing mild to borderline cases could enhance our understanding of the clinical spectrum of SMC1A-linked CdLS.     

Leptinotoxin-h Action in Synaptosomes, Neurosecretory Cells, and Artificial Membranes: Stimulation of Ion Fluxes

Leptinotoxin-h (LPTx), a neurotoxin (otherwise designated beta-leptinotarsin-h) known to stimulate the release of neurotransmitters from synapses, was purified from the hemolymph of the potato beetle, Leptinotarsa haldemani, by a simplification of the procedure originally developed by Crosland et al. [Biochemistry 23, 734-741, (1984)]. Highly and partially purified preparations of the toxin were applied to guinea pig synaptosomes and neurosecretory (PC12) cells. When applied in a Ca2+-containing Ringer medium, at concentrations in the 10(-11) - 10(-10) M range, the toxin induced: (a) rapid depolarization of the plasma membrane, which was not inhibited by organic blockers of voltage-dependent Na+ and Ca2+ channels (tetrodotoxin or verapamil); (b) large 45Ca influx; and (c) increased free cytosolic Ca2+ concentration. These latter two effects were unaffected by verapamil. In Ca2+-free media the effects of the toxin were different in the two systems investigated. In synaptosomes, depolarization was still observed, even if the toxin concentrations needed were higher (approximately 10X) than those effective in the complete medium. In contrast, in PC12 cells no effect of the toxin on membrane potential was observed. Binding of LPTx to its cellular targets could not be investigated directly because the toxin was inactivated by the procedures used for its labeling. Indirect evidence suggested however that Ca2+ is necessary for toxin binding to PC12 cells. Interaction of LPTx with air/water interfaces, as well as with cholesterol/phospholipid mono- and bilayer membranes was investigated. The results indicate that the toxin has affinity for hydrophobic surfaces, but lacks the capacity to insert across membranes unless transpositive voltage is applied. Our results are inconsistent with the previous conclusion of Crosland et al. (1984), who suggested opening of the Ca2+ channel as the mechanism of action of LPTx. The effects of the toxin resemble those of alpha-latrotoxin (alpha-LTx) of the black widow spider venom, and therefore the two toxins might act by similar mechanisms. However, the sites recognized by the two toxins might be different, because LPTx does not inhibit alpha-LTx binding.     

Leptinotoxin-h Action in Synaptosomes and Neurosecretory Cells: Stimulation of Neurotransmitter Release

Guinea pig brain cortex synaptosomes and neurosecretory PC12 cells were loaded with [3H]3,4-dihydroxyphenylethylamine ([3H]DA, [3H]dopamine) and then exposed to leptinotoxin-h (LPTx) (purified and partially purified preparations, obtained from the hemolymph of Leptinotarsa haldemani). In a Ca2+-containing Ringer medium the toxin induced prompt and massive release of the neurotransmitter. Half-maximal effects were obtained at concentrations estimated of approximately 3 X 10(-11) M for synaptosomes, and 1.5 X 10(-10) M for PC12 cells. Release responses in the two experimental systems investigated were dependent to different extents on the Ca2+ concentration in the medium. In synaptosomes clear, although slow, release of [3H]DA was elicited by the toxin even in Ca2+-free, EGTA-containing medium, provided that high (in the 10(-10) M range) concentrations were used; near-maximal responses were observed at 10(-5)M Ca2+. In contrast, the toxin-induced release from PC12 cells was appreciable only at 3 X 10(-5) M Ca2+, and was maximal at 2 X 10(-4) M and above. In both synaptosomes and PC12 cells Sr2+ and Ba2+ could substitute for Ca2+; Co2+ was inhibitory, whereas Mn2+ failed to modify the release induced by the toxin in Ca2+-containing medium. Organic blockers of the voltage-dependent Ca2+ channel (verapamil and nitrendipine) and calmodulin blocking drugs (trifluoperazine and calmidazolium) failed to inhibit the toxin-induced release of [3H]DA. LPTx induced profound morphological effects. Synaptosomes treated in the Ca2+-containing medium exhibited fusion of synaptic vesicles, formation of numerous infoldings and large cisternae, and alterations of mitochondria. In the Ca2+-free medium the effects were similar, except that their appearance was delayed, and mitochondria were well preserved. Swelling was observed in PC12 cells, accompanied by enlargement of the Golgi area, accumulation of multivesicular bodies, mitochondrial alterations, and decreased number of secretion granules (Ca2+-containing medium). Morphometric analyses revealed a good correlation between the decrease of both synaptic vesicles (synaptosomes) and neurosecretory granules (PC12 cells), and the release of [3H]DA measured biochemically. This is a good indication that the release effect of the toxin is due to stimulation of exocytosis. Taken as a whole, these results confirm the similarity of the effects of LPTx with alpha-latrotoxin of the black widow spider venom, mentioned in the companion article. However, differences in effect and target specificity suggest that the two toxins are specific to separate binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Breathing pattern and growth: comparative aspects

Summary The resting oxygen consumption and breathing pattern of nine newborn and adult species (ranging in body size from mouse to human) have been compared on the basis of data collected from the literature. Minute ventilation is similarly linked to at both ages, the percent of extracted as O₂ about 2.2. Tidal volume/kg is an interspecies constant in newborns and adults, approximately 8 ml/kg. Breathing frequency decreases with the increase in size in a different way at the two ages: large species have newborns breathing at rates 2–3 times above the corresponding adults' values, while in the small species newborns and adults breathe at almost the same rate. Therefore the newborns of the smallest species have both and below the expected values, implying a greater inability to cope with the external demands than newborns of larger species. Several considerations indicate that in the smallest newborns the mechanical properties of the respiratory system could be a constraint to resting ventilations larger than observed. It is therefore possible that their low is the cause, and not the effect, of the relatively small .