Published: Vol 8, Iss 17, Sep 5, 2018 DOI: 10.21769/BioProtoc.2989 Views: 5311
Reviewed by: Alessandro DidonnaYusuke TominaDURAI SELLEGOUNDERAnonymous reviewer(s)
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Abstract
Pentylenetetrazole (PTZ) is a GABAA receptor antagonist and is used to monitor presynaptic defects in the release of the inhibitory neurotransmitter GABA. PTZ is a competitive inhibitor of GABA, and prevents binding of GABA on the GABAA receptors present on the surface of muscle. In the absence of GABA binding, the excitatory to inhibitory signal ratio increases resulting in a convulsive phenotype. This assay provides a fast and reliable method to detect presynaptic defects in GABAergic synaptic transmission. The assay is based on correlating the extent of convulsions with the degree of presynaptic GABA release defects.
Keywords: PTZBackground
Synapses are the asymmetric intercellular junctions that mediate synaptic transmission. They constitute the fundamental units of brain circuitry that enable execution of complex behaviors. The arrival of an action potential causes calcium influx via voltage-gated calcium channels resulting in depolarization of the plasma membrane. Upon this pre-synaptic depolarization, synaptic vesicles fuse with the plasma membrane releasing their contents into the synaptic cleft via a highly controlled process (Sudhof, 1995; Sudhof, 2004).
Locomotion is a prominent behavioral output in Caenorhabditis elegans. Neural circuits that generate coordinated dorso-ventral sinusoidal bends allow for normal locomotion in C. elegans. Locomotory behavior is orchestrated at multiple levels and involves the integration of diverse sensory cues by multiple sensory neurons that are processed by the interneurons and ultimately direct changes at the neuromuscular junctions (NMJ) (de Bono and Maricq, 2005; Bargmann, 2012). The C. elegans NMJ has three main components; the excitatory cholinergic motor neurons, the inhibitory GABAergic motor neurons and the postsynaptic muscle cells (White et al., 1976; White et al., 1986). Cholinergic signaling mediates muscle contraction while GABAergic signaling fine-tunes C. elegans locomotion by exerting a regulatory control on excitatory signaling. Any defect in GABAergic signaling results in an increased excitatory signal at the NMJ resulting in more muscle contraction.
GABA released from the presynaptic GABAergic motor neuron functions via GABAA receptors, which encode GABA-gated chloride channels (Schofield et al., 1987). GABAA receptors contain a neurosteroid or PTZ-binding site, right next to the subunit containing the GABA-binding site. In the event of reduced presynaptic GABA release and in the presence of PTZ, PTZ binds the neurosteroid-binding site hampering the binding of GABA at the GABA- binding site. A reduction in GABA binding causes a decrease in the inhibitory signaling, resulting in a convulsive phenotype called ‘head bobs’ or anterior convulsions (Williams et al., 2004; Locke et al., 2008) and depicted in Figure 1. The convulsive phenotype occurs because of an altered excitatory to inhibitory input ratio to the body-wall muscles. The PTZ assay is often used as a secondary assay in conjunction with the Aldicarb assay (Oh and Kim, 2017), to investigate the role of genes involved in GABA synaptic transmission. The results from this assay can be further supported by additional experimentation including electrophysiological recordings.
Figure 1. Mechanism of PTZ-induced anterior head bobs. PTZ is a competitive antagonist of GABA. If the presynaptic GABA release is lower than that seen in WT animals, PTZ binds at the neurosteroid binding site, thus hampering the binding of GABA at GABA-binding site. In the absence of GABA signaling, excitatory cholinergic signaling increases resulting in anterior convulsions called ‘head-bobs’. Hence, mutants showing anterior convulsions on PTZ plates suggest defective presynaptic GABA release.
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Acknowledgments
This protocol has been adapted from Thapliyal et al. (2018). ST thanks the Council of Scientific and Industrial Research (CSIR) for a graduate fellowship. KB is an Intermediate Fellow of the DBT-Wellcome Trust India Alliance and thanks the Alliance for funding support (grant number IA/12/500516).
Competing interests
Authors declare no conflict of interest or competing interest.
References
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Copyright
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Thapliyal, S. and Babu, K. (2018). Pentylenetetrazole (PTZ)-induced Convulsion Assay to Determine GABAergic Defects in Caenorhabditis elegans. Bio-protocol 8(17): e2989. DOI: 10.21769/BioProtoc.2989.
Category
Neuroscience > Cellular mechanisms > Intracellular signalling
Neuroscience > Behavioral neuroscience > Animal model
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