Парадигмы для Фармакологические Характеристика C. Элеганс Synaptic Мутанты передачи
Summary
В этом видеоролике показано, как использовать два нервных стимуляторов, альдикарб и pentylenetetrazole (PTZ), в дополнительных способов изучения синаптической функции в нематода C. Элеганс. Этот дополнительный подход может также использоваться, чтобы пролить свет на эволюционно консервативных механизмов для модуляции нейронной синхронии и имеет значение для лечения эпилепсии и эпилептические припадки.
Abstract
The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the anatomy and connectivity of its nervous system has been determined from electron micrographs and refined by pharmacological assays. In this video, we describe how two complementary neural stimulants, an acetylcholinesterase inhibitor, called aldicarb, and a gamma-aminobutyric acid (GABA) receptor antagonist, called pentylenetetrazole (PTZ), may be employed to specifically characterize signaling at C. elegans neuromuscular junctions (NMJs) and facilitate our understanding of antagonistic neural circuits.
Of 302 C. elegans neurons, nineteen GABAergic D-type motor neurons innervate body wall muscles (BWMs), while four GABAergic neurons, called RMEs, innervate head muscles. Conversely, thirty-nine motor neurons express the excitatory neurotransmitter, acetylcholine (ACh), and antagonize GABA transmission at BWMs to coordinate locomotion. The antagonistic nature of GABAergic and cholinergic motor neurons at body wall NMJs was initially determined by laser ablation and later buttressed by aldicarb exposure. Acute aldicarb exposure results in a time-course or dose-responsive paralysis in wild-type worms. Yet, loss of excitatory ACh transmission confers resistance to aldicarb, as less ACh accumulates at worm NMJs, leading to less stimulation of BWMs. Resistance to aldicarb may be observed with ACh-specific or general synaptic function mutants. Consistent with antagonistic GABA and ACh transmission, loss of GABA transmission, or a failure to negatively regulate ACh release, confers hypersensitivity to aldicarb. Although aldicarb exposure has led to the isolation of numerous worm homologs of neurotransmission genes, aldicarb exposure alone cannot efficiently determine prevailing roles for genes and pathways in specific C. elegans motor neurons. For this purpose, we have introduced a complementary experimental approach, which uses PTZ.
Neurotransmission mutants display clear phenotypes, distinct from aldicarb-induced paralysis, in response to PTZ. Wild-type worms, as well as mutants with specific inabilities to release or receive ACh, do not show apparent sensitivity to PTZ. However, GABA mutants, as well as general synaptic function mutants, display anterior convulsions in a time-course or dose-responsive manner. Mutants that cannot negatively regulate general neurotransmitter release and, thus, secrete excessive amounts of ACh onto BWMs, become paralyzed on PTZ. The PTZ-induced phenotypes of discrete mutant classes indicate that a complementary approach with aldicarb and PTZ exposure paradigms in C. elegans may accelerate our understanding of neurotransmission. Moreover, videos demonstrating how we perform pharmacological assays should establish consistent methods for C. elegans research.
Protocol
Discussion
Действующие протоколы по альдикарб экспозиции с C. Элеганс не позволяют экспериментаторам различать мутанты с конкретными дефицита ацетилхолина передачи и мутанты с обобщенными дефицит в синаптической передаче, так как оба класса мутантов выставку сопротивление альдикарб. Кроме того, алди…
Acknowledgements
Мы хотим отметить дух сотрудничества всех членов Колдуэлл Lab. Василий О'Коннор ученый награду от марте Dimes и КАРЬЕРА награду от Национального научного фонда в GAC, а также Бакалавриат Научные исследования грантовой программы из Медицинского института Говарда Хьюза в университете штата Алабама, финансировали нейронную возбудимость и эпилепсии исследований в Лаборатории Колдуэлл.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Aldicarb | Reagent | Supelco, Inc. | PS734 | Purchasable from Sigma |
| Pentylenetetrazole | Reagent | Sigma | P6500 |
References
- Mahoney, T. R., Luo, S., Nonet, M. L. Analysis of synaptic transmission in Caenorhabditis elegans using an aldicarb-sensitivity assay. Nat. Protoc. 1, 1772-1777 (2006).
- Williams, S. N., Locke, C. J., Braden, A. L., Caldwell, K. A., Caldwell, G. A. Epileptic-like convulsions associated with LIS-1 in the cytoskeletal control of neurotransmitter signaling in Caenorhabditis elegans. Hum. Mol. Genet.. 13, 2043-2059 (2004).
