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

Abstract

The nerve-cell cytoskeleton is essential for the regulation of intrinsic neuronal activity. For example, neuronal migration defects are associated with microtubule regulators, such as LIS1 and dynein, as well as with actin regulators, including Rac GTPases and integrins, and have been thought to underlie epileptic seizures in patients with cortical malformations. However, it is plausible that post-developmental functions of specific cytoskeletal regulators contribute to the more transient nature of aberrant neuronal activity and could be masked by developmental anomalies. Accordingly, our previous results have illuminated functional roles, distinct from developmental contributions, for Caenorhabditis elegans orthologs of LIS1 and dynein in GABAergic synaptic vesicle transport. Here, we report that C. elegans with function-altering mutations in canonical Rac GTPase-signaling-pathway members demonstrated a robust behavioral response to a GABA A receptor antagonist, pentylenetetrazole. Rac mutants also exhibited hypersensitivity to an acetylcholinesterase inhibitor, aldicarb, uncovering deficiencies in inhibitory neurotransmission. RNA interference targeting Rac hypomorphs revealed synergistic interactions between the dynein motor complex and some, but not all, members of Rac-signaling pathways. These genetic interactions are consistent with putative Rac-dependent regulation of actin and microtubule networks and suggest that some cytoskeletal regulators cooperate to uniquely govern neuronal synchrony through dynein-mediated GABAergic vesicle transport in C. elegans.

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

“…These data support the idea that APC mutants have reduced GABA signaling. Because PTZ-induced convulsions are primarily mediated by the GABAergic RME neurons in the head of the animal (Locke et al, 2009), while the activity of the D-type motor neurons (DD and VD neurons) contributes to the body wall muscle activity measured in the aldicarb assay, the presence of both aldicarb-hypersensitivity and PTZ-induced convulsions in the APC mutants suggests that the APC may play a broad role in regulating GABA transmission in C. elegans .…”

GABAergic Synaptic Plasticity during a Developmentally Regulated Sleep-Like State inC. elegans

“…Pentylenetetrazole (PTZ, Sigma; # P6500) was included in the NGM at a concentration of 5 mg/mL as described (Locke et al, 2009). These plates were seeded with the bacteria DA837.…”

“…Mid L4, lethargus, and adult worms were plated on the PTZ plates pre-warmed to room temperature and then observed at 15 min, 30 min, and 60 min. At each of these time points, worm behavior was categorized as (1) normal crawling behavior, (2) posterior paralysis, (3) complete paralysis, and (4) convulsions (Also referred to as “head bobs”) (Williams et al, 2004; Locke et al, 2009) . In this assay, paralyzed worms were neither prodded nor removed from the plate.…”

Epilepsy research methods update: Understanding the causes of epileptic seizures and identifying new treatments using non-mammalian model organisms

“…32 It is likely that cytoskeletal transport processes are important in this regard, as both microtubule-and actin-based mechanisms have been implicated due to the effect of dynein and Rac mutations, respectively. 31,33 An alternative C. elegans epilepsy model emerged from studies of neuronal acetylcholine receptors. A gain-of-function mutation in the acr-2 gene, which encodes a subunit of the acetylcholine receptor, was found to cause spontaneous 'shrinking' convulsions in the absence of seizure-inducing agents.…”