Daniel L. Chase

Mechanism of extrasynaptic dopamine signaling in Caenorhabditis elegans

D1-like and D2-like dopamine receptors have synergistic and antagonistic effects on behavior. To understand the mechanisms underlying these effects, we studied dopamine signaling genetically in Caenorhabditis elegans. Knocking out a D2-like receptor, DOP-3, caused locomotion defects similar to those observed in animals lacking dopamine. Knocking out a D1-like receptor, DOP-1, reversed the defects of the DOP-3 knockout. DOP-3 and DOP-1 have their antagonistic effects on locomotion by acting in the same motor neurons, which coexpress the receptors and which are not postsynaptic to dopaminergic neurons. In a screen for mutants unable to respond to dopamine, we identified four genes that encode components of the antagonistic Gαo and Gαq signaling pathways, including Gαo itself and two subunits of the regulator of G protein signaling (RGS) complex that inhibits Gαq. Our results indicate that extrasynaptic dopamine regulates C. elegans locomotion through D1- and D2-like receptors that activate the antagonistic Gαq and Gαo signaling pathways, respectively.

Two RGS proteins that inhibit Gαo and Gαq signaling in C. elegans neurons require a Gβ5-like subunit for function

Abstract Background: Gβ proteins have traditionally been thought to complex with Gγ proteins to function as subunits of G protein heterotrimers. The divergent Gβ 5 protein, however, can bind either Gγ proteins or r egulator of G protein s ignaling (RGS) proteins that contain a G g amma– l ike (GGL) domain. RGS proteins inhibit G protein signaling by acting as Gα GTPase activators. While Gβ 5 appears to bind RGS proteins in vivo, its association with Gγ proteins in vivo has not been clearly demonstrated. It is unclear how Gβ 5 might influence RGS activity. In C. elegans there are exactly two GGL-containing RGS proteins, EGL-10 and EAT-16, and they inhibit Gα o and Gα q signaling, respectively. Results: We knocked out the gene encoding the C. elegans Gβ 5 ortholog, GPB-2, to determine its physiological roles in G protein signaling. The gpb-2 mutation reduces the functions of EGL-10 and EAT-16 to levels comparable to those found in egl-10 and eat-16 null mutants. gpb-2 knockout animals are viable, and exhibit no obvious defects beyond those that can be attributed to a reduction of EGL-10 or EAT-16 function. GPB-2 protein is nearly absent in eat-16; egl-10 double mutants, and EGL-10 protein is severely diminished in gpb-2 mutants. Conclusions: Gβ 5 functions in vivo complexed with GGL-containing RGS proteins. In the absence of Gβ 5 , these RGS proteins have little or no function. The formation of RGS–Gβ 5 complexes is required for the expression or stability of both the RGS and Gβ 5 proteins. Appropriate RGS–Gβ 5 complexes regulate both Gα o and Gα q proteins in vivo.

A Specific Subset of Transient Receptor Potential Vanilloid-Type Channel Subunits in Caenorhabditis elegans Endocrine Cells Function as Mixed Heteromers to Promote Neurotransmitter Release

Transient receptor potential (TRP) channel subunits form homotetramers that function in sensory transduction. Heteromeric channels also form, but their physiological subunit compositions and functions are largely unknown. We found a dominant-negative mutant of the C. elegans TRPV (vanilloid-type) subunit OCR-2 that apparently incorporates into and inactivates OCR-2 homomers as well as heteromers with the TRPV subunits OCR-1 and -4, resulting in a premature egg-laying defect. This defect is reproduced by knocking out all three OCR genes, but not by any single knockout. Thus a mixture of redundant heteromeric channels prevents premature egg laying. These channels, as well as the G-protein Gαo, function in neuroendocrine cells to promote release of neurotransmitters that block egg laying until eggs filling the uterus deform the neuroendocrine cells. The TRPV channel OSM-9, previously suggested to be an obligate heteromeric partner of OCR-2 in sensory neurons, is expressed in the neuroendocrine cells but has no detectable role in egg laying. Our results identify a specific set of heteromeric TRPV channels that redundantly regulate neuroendocrine function and show that a subunit combination that functions in sensory neurons is also present in neuroendocrine cells but has no detectable function in these cells.

Coexpressed D1- and D2-Like Dopamine Receptors Antagonistically Modulate Acetylcholine Release in Caenorhabditis elegans

Dopamine acts through two classes of G protein-coupled receptor (D1-like and D2-like) to modulate neuron activity in the brain. While subtypes of D1- and D2-like receptors are coexpressed in many neurons of the mammalian brain, it is unclear how signaling by these coexpressed receptors interacts to modulate the activity of the neuron in which they are expressed. D1- and D2-like dopamine receptors are also coexpressed in the cholinergic ventral-cord motor neurons of Caenorhabditis elegans. To begin to understand how coexpressed dopamine receptors interact to modulate neuron activity, we performed a genetic screen in C. elegans and isolated mutants defective in dopamine response. These mutants were also defective in behaviors mediated by endogenous dopamine signaling, including basal slowing and swimming-induced paralysis. We used transgene rescue experiments to show that defects in these dopamine-specific behaviors were caused by abnormal signaling in the cholinergic motor neurons. To investigate the interaction between the D1- and D2-like receptors specifically in these cholinergic motor neurons, we measured the sensitivity of dopamine-signaling mutants and transgenic animals to the acetylcholinesterase inhibitor aldicarb. We found that D2 signaling inhibited acetylcholine release from the cholinergic motor neurons while D1 signaling stimulated release from these same cells. Thus, coexpressed D1- and D2-like dopamine receptors act antagonistically in vivo to modulate acetylcholine release from the cholinergic motor neurons of C. elegans.

Genetic analysis of RGS protein function in Caenorhabditis elegans.

Caenorhabditis elegans has close homologs or orthologs of most mammalian (RGS) and G proteins, and mutants for all the RGS and G-protein genes of C. elegans have been generated. C. elegans RGS proteins can be matched to the specific Galpha proteins they regulate in vivo by comparing the defects in animals lacking or transgenically overexpressing an RGS protein with defects in a specific Galpha mutant. Transgenic expression of mutated RGS proteins or subdomains in C. elegans has also been used to carry out structure/function studies of RGS proteins. We propose that similar strategies can be used to understand the function of RGS proteins from other organisms by expressing them in C. elegans. This article describes general considerations regarding such experiments and provides detailed protocols for quantitatively measuring G-protein signaling phenotypes in C. elegans.

D1 Dopamine Receptor Signaling Is Modulated by the R7 RGS Protein EAT-16 and the R7 Binding Protein RSBP-1 in Caenoerhabditis elegans Motor Neurons

Dopamine signaling modulates voluntary movement and reward-driven behaviors by acting through G protein-coupled receptors in striatal neurons, and defects in dopamine signaling underlie Parkinsons disease and drug addiction. Despite the importance of understanding how dopamine modifies the activity of striatal neurons to control basal ganglia output, the molecular mechanisms that control dopamine signaling remain largely unclear. Dopamine signaling also controls locomotion behavior in Caenorhabditis elegans. To better understand how dopamine acts in the brain we performed a large-scale dsRNA interference screen in C. elegans for genes required for endogenous dopamine signaling and identified six genes (eat-16, rsbp-1, unc-43, flp-1, grk-1, and cat-1) required for dopamine-mediated behavior. We then used a combination of mutant analysis and cell-specific transgenic rescue experiments to investigate the functional interaction between the proteins encoded by two of these genes, eat-16 and rsbp-1, within single cell types and to examine their role in the modulation of dopamine receptor signaling. We found that EAT-16 and RSBP-1 act together to modulate dopamine signaling and that while they are coexpressed with both D1-like and D2-like dopamine receptors, they do not modulate D2 receptor signaling. Instead, EAT-16 and RSBP-1 act together to selectively inhibit D1 dopamine receptor signaling in cholinergic motor neurons to modulate locomotion behavior.

A Novel Strategy for Cell-Autonomous Gene Knockdown in Caenorhabditis elegans Defines a Cell-Specific Function for the G-Protein Subunit GOA-1

We developed a novel knockdown strategy to examine cell-specific gene function in Caenorhabditis elegans. In this strategy a null mutation in any gene is replaced with a genetically stable transgene that contains a wild-type copy of the gene fused to a 3′ tag that targets the mRNA transcript for degradation by the host nonsense-mediated decay (NMD) machinery. In NMD-defective animals, tagged transgene mRNA is expressed at levels similar to the endogenous gene it replaced and is translated into wild-type protein that fully rescues gene function. Cell-specific activation of NMD cell autonomously knocks down transgene expression in specific cell types without affecting its expression or function in other cells of the organism. To demonstrate the utility of this system, we replaced the goa-1 gene, encoding the pan-neuronally expressed G-protein subunit GOA-1, with a degradation-tagged transgene. We then knocked down expression of the transgene from only two neurons, the hermaphrodite-specific neurons (HSNs), and showed that GOA-1 acts cell autonomously in the HSNs to inhibit egg-laying behavior.

Large-scale gene knockdown in C. elegans using dsRNA feeding libraries to generate robust loss-of-function phenotypes.

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.