Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants

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. Video LinkThe video component of this article can be found at http://www.jove.com/details.php?id=837 On the first day, ensure that at least thirty young adult stage worms of each geno...

The Anaphase-Promoting Complex (APC) ubiquitin ligase regulates GABA transmission at the C. elegans neuromuscular junction

“…unc-4 ( e120 ) mutants are defective in cholinergic signaling due to mutations in the UNC-4 homeodomain protein that is required for cholinergic DA and VA motor neuron development (Esmaeili et al, 2002; Miller et al, 1992). In contrast, 100% of the unc-25 ( e156 ) mutants, which are defective in the GABA synthesis enzyme glutamic acid decarboxylase (GAD), and snb-1 ( md247 ) mutants, which are defective in general synaptic transmission, show robust anterior convulsions after 30 min of PTZ exposure, as has been previously reported (Locke et al, 2006, 2008). Interestingly, we found that nearly 50% of emb-27 ( g48 ) CDC16 mutant animals exhibit anterior convulsions after 30 min of PTZ exposure and more than 90% of emb-27 ( g48 ) CDC16 animals and 40% of emb-30 ( g53 ) APC4 animals were convulsing after 60 min of PTZ exposure (Fig.…”

“…In C. elegans , PTZ induces seizure-like convulsions in animals with defects in GABA neurotransmission. Wild type animals and those with defects only in cholinergic signaling do not exhibit PTZ-induced convulsions (Locke et al, 2006, 2008; Williams et al, 2004). It should be noted, however, that the precise mechanism of action of PTZ in C. elegans is not completely clear and cannot only be explained by PTZ acting as a GABA signaling antagonist because GABA signaling mutants alone do not exhibit spontaneous seizures and the effects of PTZ on convulsions in C. elegans are only apparent when GABA transmission is also reduced.…”

“…PTZ assays were performed as described previously (Locke et al, 2008). Briefly, NGM agar plates containing 10 mg/ml pentylene tetrazole (PTZ, Sigma-Aldrich) assay were made fresh on the day of the experiment, allowed to dry for 1 h, then spotted with 25 μl OP50 E. coli and dried for approximately 2 h. Twenty to twenty-five adult worms with eggs, which had been previously shifted to 25 °C for 20 h beginning at the L4 stage, were transferred onto each PTZ plate to begin the time course.…”

“…(Full body convulsions and full body paralysis, which are seen in response to PTZ with some strains, were not observed.) (Locke et al, 2006, 2008). The percentage of worms of a given strain displaying each phenotype at 30 and 60 min of exposure was calculated for each plate; results were then pooled for each strain and the average percentages of convulsing worms ± s.e.m.…”

Pharmacogenetic Analysis Reveals a Post-Developmental Role for Rac GTPases inCaenorhabditis elegansGABAergic Neurotransmission

“…Behavioral and pharmacological assays: Convulsion assays were performed, as previously described (Williams et al 2004;Locke et al 2008). Concentrations of PTZ (Sigma) employed are indicated in the text.…”