Acute Isolation, Culture And The Patch Clamp Study Of The Cultured Neurons From Ornithoctonus Huwena

Ornithoctonus huwena (Selenocosmia huwena) is identified recentlyas a new species of spiders in China. It largely distributes in the hillyareas of Yunnan in the southwest of China and Guangxi in the south ofChina, habitually lives in holes underground and is rather aggressive andvenomous. This spider preys mainly on insects and some smallvertebrates using its venom. It is an ideal material for the research ofcobwebs, spider proteins and the development of new medicine.In this article, the dissociation and culture of neurons isolated fromthe Subesophageal ganglion (SUB) of the Ornithoctonus huwena aredescribed. A novel method was created to anatomize the spider ganglionand named as "anatomical method of removing the Sternum". The basicelectrophysiological properties of voltage-gated Na⁺, K⁺ and Ca²⁺channels on the cultured neurons were studied by means of whole-cellpatch-clamp technique. Using this technique, we observed the effects ofthe crude venom of Selenocosmia huwena (S. huwena) on voltage-gatedcalcium channels (VGCCs) and voltage-gated potassium channels(VGPCs) expressed in the SUB neurons. And we also observed theeffects of HWTX-â… and HWTX-â…¤on VGCCs. In addition, we obtainedsome toxin molecules affecting on the VGPCs from the venom of S. huwena.The experimental results demonstrate that the culture media whichwas suitable for the dissociated spider nerve cells contains: NaC1 223mmol/L; KC1 6.8 mmol/L; CaC12 8 mmol/L; MgCl₂ 5.1 mmol/L; Sucrose5 mmol/L; Hepes 10 mmol/L; Glutamine 1 mmol/L; Penicillin 200 IU/ml; Streptomycin 200μg/ml; Bovin Calf Serum 20ï¼…at pH 7.4. The suitableculture temperature was at 27±2℃and it takes about 2-4 h for the process.Most cells were in good condition and above 70ï¼…cells survived in thecell culture dishes. The shape of the soma of the nerve cell was an ellipseand neural cell like a spoon with a single axon. The size of these cellsvaried from 10 to 30μm. Under whole-cell patch-clamp configuration, weobtained high-voltage-activated (HVA) calcium currents and two types ofoutward potassium currents including delayed rectifier potassium currentsand rapid outward potassium currents on spider neuron cells. Sometimes, small voltage-gated sodium currents were also recorded in the experiment.The potassium currents could be inhibited by TEA-Cl and 4-AR Underthe whole-cell patch-clamp mode, we found that HWTX-â… had no evidenteffect on HVA calcium currents expressed in the SUB neurons, but thecrude venom and HWTX-â…¤obviously inhibited these currents and theinhibition was concentration-dependent. Crude venom with concentrationof 0.01mg/mL, 0.1mg/mL and 1mg/mL inhibited 5.92ï¼…, 19.93ï¼…and59.90ï¼…of the calcium currents, respectively. 1μM and 10μM HWTX-â…¤ reduced the amplitude of calcium currents by 20.59ï¼…and 40.50ï¼…, respectively. After the depression of the calcium current amplitude by 10μM HWTX-â…¤, there were no changes of both the threshold of activationand the active voltage of peak inward currents. In addition, we furtherdetected that the crude venom had inhibitory effects on VGPC expressedin the SUB neurons and 1mg/ml crude venom could inhibit 73.69ï¼…ofpotassium currents at most. We obtained 17 peaks from the crude venomby means of reverse phase high-performance liquid-phasechromatography (RP-HPLC), and 11 peaks were identified byMALDI-TOF. Among these 11 peaks, we found that the fourth peakinhibited delayed rectifier potassium currents and rapid outwardpotassium currents expressed in the SUB neurons, and the eighth andtwelfth peak inhibited the rapid outward potassium currents, but the sixthpeak augmented the delayed rectifier potassium currents.All results not only enrich the content of spider's physiology andtoxicology but also establish good foundation for the development ofspider's neurobiology later.