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1Department of Biological Sciences, University of Alabama
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This video demonstrates how to employ two neural stimulants, aldicarb and pentylenetetrazole (PTZ), in complementary ways to study synaptic function in the nematode, C. elegans. This complementary approach may also be used to shed light on evolutionarily conserved mechanisms for modulating neuronal synchrony and has implications for epilepsy and seizures.
Locke, C., Berry, K., Kautu, B., Lee, K., Caldwell, K., Caldwell, G. Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants. J. Vis. Exp. (18), e837, doi:10.3791/837 (2008).
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.
Aldicarb Exposure Paradigm
PTZ Exposure Paradigm
Behavioral Responses of Selected C. elegans Synaptic Transmission Mutants to Aldicarb and PTZ
|Mutant Name||Synaptic Role||Behavior without Drug||Behavioral Response to Aldicarb (compared to wild-type N2)||Behavioral Response to PTZ|
|tom-1(ok188)||inhibits synaptic transmission||uncoordinated||enhanced rate of paralysis||indistinguishable from wild-type|
|unc-43(n498n1186)||complex||uncoordinated||enhanced rate of paralysis||full-body convulsions|
|unc-25(e156)||promotes GABA transmission||uncoordinated||enhanced rate of paralysis||anterior convulsions, full-body paralysis|
|snb-1(md247)||promotes synaptic transmission||uncoordinated||reduced rate of paralysis||anterior convulsions|
|unc-4(e120)||promotes ACh transmission||uncoordinated||reduced rate of paralysis||indistinguishable from wild-type|
Two neural stimulants, an acetylcholinesterase inhibitor, called aldicarb, and a GABA receptor antagonist, called pentylenetetrazole (PTZ), can be used in a complementary manner to characterize C. elegans synaptic transmission mutants. Excess excitatory acetylcholine (ACh) accumulates at worm body wall neuromuscular junctions (NMJs) from deleterious mutations in negative regulators of ACh transmission (e.g. tom-1 and unc-43) or positive regulators of inhibitory GABA transmission (e.g., unc-25). Conversely, excitatory ACh levels at worm NMJs are diminished by deleterious mutations in positive regulators of general synaptic transmission (e.g., snb-1) or ACh-specific transmission genes (e.g. unc-4). When compared to wild-type N2 worms, mutant worms with elevated excitatory ACh transmission at NMJs exhibit enhanced rates of aldicarb-induced paralysis, whereas mutant worms with lowered excitatory ACh transmission demonstrate reduced rates of aldicarb-induced paralysis. Although PTZ disrupts neuronal synchrony at C. elegans body wall muscles, not unlike aldicarb, PTZ also antagonizes inhibitory GABA at C. elegans head muscles. As a result, aldicarb sensitivity cannot accurately predict PTZ sensitivity. Mutant worms with specific defects in negative or positive regulation of ACh transmission are indistinguishable from wild-type N2 worms in the presence of PTZ, whereas mutant worms with defects in positive regulation of general synaptic transmission or specific defects in inhibitory GABA transmission exhibit robust PTZ-induced anterior convulsions. Moreover, unc-43 loss-of-function mutants display full-body convulsions in the presence of PTZ and likely have additional synaptic transmission abnormalities, which contribute to their unique drug responses.
Current protocols for aldicarb exposure with C. elegans do not allow experimenters to distinguish between mutants with specific deficits in ACh transmission and mutants with generalized deficits in synaptic transmission, as both classes of mutants exhibit resistance to aldicarb. Likewise, aldicarb cannot be used to determine if mutants have specific deficits in GABA transmission or generalized failures to negatively regulate ACh transmission, as both classes of mutants exhibit hypersensitivity to aldicarb. Results from our PTZ exposure assays, when combined with results from aldicarb exposure assays, allows researchers to better characterize synaptic transmission mutants.
C. elegans synaptic transmission mutants may be classified in a straightforward manner by complementary aldicarb and PTZ exposure paradigms. Aldicarb resistant mutants with PTZ-induced anterior convulsions are likely deficient in general synaptic function. Conversely, aldicarb resistant mutants without PTZ-induced anterior convulsions are likely specifically deficient in ACh transmission. Mutants with aldicarb hypersensitivity, which do not exhibit PTZ-induced anterior convulsions, likely fail to negatively regulate ACh transmission. Finally, mutants with aldicarb hypersensitivity, which do exhibit PTZ-induced anterior convulsions, are likely GABA deficient.
The utility of aldicarb exposure is also weakened by its subjectivity, as different experimenters often have varying definitions of paralysis. A single experimenter's technique can also fluctuate. Also, aldicarb-exposed worms move differently in response to diverse forces of prodding. The distinction between a paralyzed worm and a responsive worm can be as subtle as a slight head or tail twitch. In addition to complementing aldicarb assays for better characterization of C. elegans synaptic transmission mutants, PTZ may also be used to isolate synaptic transmission mutants, especially those mutants with hypersensitivity to aldicarb. Experimenters, which utilize PTZ exposure, may simply look for anterior convulsions, instead of subtle differences in aldicarb-induced paralysis.>
We wish to acknowledge the cooperative spirit of all Caldwell Lab members. A Basil O’Connor Scholar Award from the March of Dimes and a CAREER Award from the National Science Foundation to GAC, as well as an Undergraduate Research Science Program Grant from the Howard Hughes Medical Institute to The University of Alabama, have funded neuronal excitability and epilepsy research in the Caldwell Lab.
|Aldicarb||Reagent||Supelco, Sigma-Aldrich||PS734||Purchasable from Sigma|
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