Background

Dopamine is reduced in the brain of rats treated with fipronil, a broad-spectrum insecticide. VGF (no acronym) is a neurotrophin-inducible protein expressed as the 75 kDa form (precursor or pro-VGF) or its truncated peptides. VGF immunostaining has been revealed using an antibody against the C-terminal nonapeptide of the rat pro-VGF in the nerve terminals of the rat substantia nigra, where it was reduced after 6-hydroxydopamine treatment. It is unknown whether pro-VGF and/or its shortened peptides are present in these neurons. Therefore, the aim of this study was first to determine which types of VGF are expressed in the normal substantia nigra (and striatum) and then to determine VGF modulations and whether they occur in parallel with locomotor changes after fipronil injection.

Methods

Rats were divided into two groups that received a unilateral intranigral infusion of either fipronil (25 µg) diluted in dimethyl sulfoxide (DMSO) or DMSO alone, and then were tested for locomotor activity. An untreated group of rats (n=4) was used for identification of the VGF fragments using high performance liquid chromatography-mass spectrometry and western blot, while changes in treated groups (fipronil vs DMSO, each n=6) were investigated by immunohistochemistry using an antibody against the rat pro-VGF C-terminal nonapeptide in parallel with the anti-tyrosine hydroxylase antibody.

Results

In untreated rats, the VGF C-terminal antibody identified mostly a 75 kDa band in the substantia nigra and striatum, supporting the finding of high-resolution mass spectrometry, which revealed fragments covering the majority of the pro-VGF sequence. Furthermore, several shortened VGF C-terminal forms (varying from 10 to 55 kDa) were also found by western blot, while high-resolution mass spectrometry revealed a C-terminal peptide overlapping the immunogen used to create the VGF antibody in both substantia nigra and striatum. In the substantia nigra of fipronil-treated rats, immunostaining for tyrosine hydroxylase and VGF was reduced compared to DMSO-treated rat group, and this was related with significant changes in locomotor activity.

Conclusion

Fipronil has the ability to modulate the production of pro-VGF and/or its C-terminal truncated peptides in the nigrostriatal system indicating its intimate interaction with the dopaminergic neurotransmission and implying a potential function in modulating locomotor activity.

中文翻译：

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VGF 修饰与成年雄性大鼠农药氟虫腈诱导的黑质纹状体多巴胺能神经变性相关

 背景

用氟虫腈（一种广谱杀虫剂）处理的大鼠大脑中的多巴胺减少。 VGF（无缩写词）是一种神经营养素诱导蛋白，以 75kDa 形式（前体或 VGF 前体）或其截短肽表达。使用针对大鼠黑质神经末梢中大鼠原 VGF 的 C 端九肽的抗体揭示了 VGF 免疫染色，该抗体在 6-羟基多巴胺治疗后减少。目前尚不清楚这些神经元中是否存在 VGF 前体和/或其缩短的肽。因此，本研究的目的首先是确定哪些类型的 VGF 在正常黑质（和纹状体）中表达，然后确定 VGF 调节以及它们是否与氟虫腈注射后运动变化同时发生。

 方法

大鼠被分为两组，接受单侧黑质内输注二甲亚砜（DMSO）稀释的氟虫腈（25 µg）或单独的DMSO，然后测试运动活性。使用高效液相色谱-质谱法和蛋白质印迹法，使用未治疗组的大鼠（n = 4）来鉴定 VGF 片段，而使用免疫组织化学方法研究治疗组（氟虫腈与 DMSO，每个 n = 6）的变化与抗酪氨酸羟化酶抗体平行的抗大鼠 VGF 原 C 端九肽的抗体。

 结果

在未经治疗的大鼠中，VGF C 端抗体在黑质和纹状体中识别出大部分 75 kDa 的条带，支持高分辨率质谱的发现，该结果揭示了覆盖大部分 pro-VGF 序列的片段。此外，通过蛋白质印迹还发现了几种缩短的 VGF C 末端形式（从 10 到 55kDa），而高分辨率质谱分析显示 C 末端肽与用于在黑质和黑质中产生 VGF 抗体的免疫原重叠。纹状体。在氟虫腈处理的大鼠黑质中，与DMSO处理的大鼠组相比，酪氨酸羟化酶和VGF的免疫染色减少，这与运动活性的显着变化有关。

 结论

氟虫腈能够调节黑质纹状体系统中 VGF 前体和/或其 C 端截短肽的产生，表明其与多巴胺能神经传递密切相互作用，并暗示其在调节运动活性方面具有潜在功能。