Michael Koelle, PhD

Biochemical and Genetic Studies of the Gαo Signaling Mechanism

Gα_o is by far the most abundant Gα protein in the brain, and every neurotransmitter has receptors that activate this G protein. Gα_o was discovered over 35 years ago, yet we still do not know what “effector” proteins this G protein directly binds and regulates to carry out its functions. We do know from C. elegans genetics that Gα_o directly opposes Gα_q signaling, and ultimately Gα_o signaling inactivates neurons, preventing them from carrying out neurotransmitter release.

We have recently carried out rapid, gentle immunopurifications of Gα_o protein complexes from both mouse brain and C. elegans lysates, and identified the proteins in these complexes by mass spectrometry. We found a number of proteins that copurify with Gα_o in both mouse and worms, and verified the association of Gα_o with these proteins by co-immunoprecipitation/Western blots. Intriguingly, several of these proteins are regulators of Gα_q signaling and/or neurotransmitter release, exactly the types of proteins that could plausibly mediate Gα_o signaling. We are carrying out further biochemical studies of the association of Gα_o with these proteins, analyzing how Gα_o may regulate activity of these proteins, and are using C. elegans genetics to analyze the functional significance of these proteins in Gα_o signaling.

Organization of G protein coupled receptors in neural circuits

C. elegans has 27 G protein coupled neurotransmitter receptors and about 150 G protein coupled neuropeptide receptors, which together facilitate signaling among the 302 neurons (of 118 types) found in the worm. Interestingly, humans have about the same number of G protein coupled neurotransmitter and neuropeptide receptors, even though we have ~80-100 billion neurons. In C. elegans, if each receptor is expressed in 20-30 types of neurons (a reasonable estimate based on existing data), that means the average neuron expresses about 6 neurotransmitter and 32 neuropeptide receptors. As one of the first steps to understanding how a neural circuit functions, we need to know which neurons in the circuit express which receptors.

We are undertaking a large-scale project to map which cells in C. elegans express each of its 27 G protein coupled neurotransmitter receptors. For every receptor, we have created fosmid-based GFP reporter transgenes that cause all cells expressing that receptor to fluoresce green. We are currently mapping out which cells express each receptor within the egg-laying circuit, and then we will broaden our analysis to map neurotransmitter receptor expression in the entire nervous system. We have also begun work mapping the cellular expression patterns of the neuropeptide receptors. The neural signaling maps we produce will be essential tools for future work analyzing any neural circuit or behavior in C. elegans.

An egg-laying circuit as a model for understanding neural circuits in general

Much of what is known about neural circuits comes from past studies of very simple circuits in crustaceans, in which their large neurons could be impaled with electrodes to study their activity. Unfortunately, modern genetic methods cannot be applied to crustaceans, limiting what we can learn from them. Conversely, the nematode C. elegans is readily amenable to genetic manipulation via mutations and transgenes and has one of the best-characterized nervous systems of any organism.

The egg-laying circuit of C. elegans consists of 12 neurons of three types and 16 muscle cells of four types. The circuit is silent most of the time, but about every 20 minutes it becomes rhythmically active for 2-3 minutes, in which a few eggs are laid. Activity of the circuit is controlled by two “command neurons” called the HSNs, whose release of a combination of serotonin and a neuropeptide is sufficient to activate the rest of the circuit. This provides us with an opportunity to study signaling by serotonin, a neurotransmitter involved in human mood disorders.

We (and others before us) have studied the egg-laying circuit for decades, and as a result we now have a large collection of C. elegans mutants in which the functions of individual cells within the circuit have been specifically altered. We also have developed methods to express the calcium-sensitive fluorescent protein GCaMP in individual cells of the circuit so that we can optically record activity of these cells within freely-behaving animals. Further, we have expressed ion channels in individual cells of the circuit that can be controlled by light or chemicals so that we can activate or silence these cells at will. By combining these experimental approaches, we now have a virtually unlimited ability to manipulate the egg-laying circuit and measure the consequent effects on circuit activity and egg-laying behavior.

Our goal is to use this approach to understand all the neurotransmitter and neuropeptide signals used within the egg-laying circuit that set up its dynamic pattern of activity. Because the egg-laying circuit has several features common with other circuits that have been studied in the past, we expect that the insights we gain from studying the egg-laying circuit will give us broad insights into the function of neural circuits in general.