A Comparative Electrophysiological Study of the Effects of Paralytic Shellfish Poisons on Nerves, Nerve-Muscle, and Neuronal Cell Types

Neurosciences, Toxicology

Tierney, Linda Jane

University:University of Glasgow

关键词: Neurosciences, Toxicology;   

Others  :  http://theses.gla.ac.uk/75271/1/13815435.pdf

来源: University of Glasgow

【 摘 要 】

This study has used a variety of electrophysiological techniques to investigate the effects of crude shellfish extracts containing paralytic shellfish poisons (PSP) on the sodium (Na+) channel behaviour and on the electrical excitability of nerves, nerve-muscle systems, and cultured and dissociated neuronal cells. In one series of experiments, four novel in vitro tissue preparations were investigated. A crustacean nerve-muscle system, a crustacean mechanoreceptor-nerve preparation, an avian nerve-muscle system and an amphibian sciatic nerve, were exposed to the marine toxins saxitoxin (STX), tetrodotoxin (TTX), and PSP samples prepared under AOAC procedure by MAFF, Tony, Aberdeen. The nerve-muscle preparation from the Norway lobster, Nephrops norvegicus (L), was chosen as an apparently simple yet robust system. Extracellular electrical stimulation of the motor nerve (R3) produced excitatory post-synaptic muscle potentials (epsp's). The preparation was highly sensitive to STX and TTX, with a minimum inhibitory threshold of 10-12 M. The inhibitory effect was not dose-dependent, toxin concentrations of less than 10-8 M resulted in a variable inhibitory effect on the epsp amplitude, whilst values above this concentration resulted in a sudden and complete loss of the epsp. Several concentrations of toxin could be applied to the same preparation. The nerve preparation from the crab, Carcinus maenas, was also chosen for the simplicity of the system. Extracellularly-recorded action potentials induced by natural stimuli were reduced in amplitude by Na+ channel blocking toxins present in the PSP samples. The chick biventor cervicis (CBC) nerve-muscle preparation was highly sensitive to the sodium channel blocking activity of PSP samples. An inhibitory effect on muscle twitch amplitude could be detected at values as low as 0.4 ng/ml, making it more sensitive than the mouse bioassay, and the inhibition increased in a dose-dependent manner from 400 ng/ml. IT50 values (i.e. the time required for the twitch control amplitude to be reduced by 50%) of the PSP samples tested yielded a correlation of r2 = 0.63. The isolated partially desheathed sciatic nerve of Rana temporaria proved to be a highly robust, simple and reliable assay system for PSP samples with a detection sensitivity of 2 ng/ml. Using a Vaseline gap method of stimulation, inhibition of the compound action potential (CAP) was found to be dose-dependent with mean IT50 values for PSP samples yielding a correlation of r2 = 0.696, and for recovery times a correlaion of r2 = 0.918. A single preparation could be used for measurement of several samples, with a complete recovery of the CAP after each toxin exposure. In another series of experiments the electrical properties of three neuroblastoma (tumour) tissue culture lines: Neuro 2a (N2a), NG108-15, S.K.N.SH, and also acutely dissociated primary cells of dorsal root ganglia (DRG) and superior cervical ganglia (SCG), from new born rat pups were examined. Sodium channel properties of the three neuroblastoma lines were investigated at various stages of differentiation. Cell resting membrane potentials became significantly more hyperpolarized after differentiation. Patch clamp techniques revealed an inconsistency in Na+ channel expression before differentiation. After differentiation, the IC50 values of the N2a and NG108-15 cells for STX were 7.8 nM and 15.5 nM respectively, with correlations of r2 = 0.938 and r2 = 0.979. The effect of STX was concentration-dependent. PSP samples tested on these two cell lines also had an inhibitory effect. At least two types of Na+ current can be recorded in the DRG and SCG cells: an STX-sensitive (STXS) current and an STX-resistant (STXR) current. Exposure to STX blocked >92% of the STXS currents, whilst the mean block of STXR currents was 18%. Intracellular and patch clamp methods were used to examine the principle underlying the use of veratridine (VER) and ouabain (OUB) in the neuroblastoma cell bioassay. Both compounds caused a large depolarisation of cell resting membrane potential. In addition, VER reduced the size of the early transient Na+ current and increased a persistent tail component of the Na+ current. STX could inhibit the effect of VER but had little or no effect on the action of OUB. Photographic studies of the N2a cell line revealed morphological changes during a four day period, including an increase in neurite outgrowth, which was dependent on the concentration of serum in the growth medium. Scanning electron micrographs revealed retraction of neurites, cell membrane damage and a decrease in cell survival rate, especially after exposure to VER. A tetrazolium salt 3-(4,5-dimethylthazol-2-y1)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to detect the cytotoxic action by PSP extracts. Samples containing STX eq values almost equivalent to, or less than, the AOAC safety level (80 ?g) caused a greater decrease in cell survival rates than samples with a higher STX value. VER and OUB caused significant reductions (p = < 0.05) in cell survival rates. The data obtained from these investigations are discussed in relation to our existing knowledge of the action of PSP toxins, and to the development of future detection and screening methods.