Overview of Research in the Koelle Lab
The Koelle lab is interested in the molecular mechanisms by which neurons respond to neurotransmitters, and also how neurotransmission is used to control the dynamic activities of neural circuits.
The human brain has ~80,000,000,000 neurons; but underlying all this complexity there are simpler units called neural circuits, small sets of neurons which are physically connected at synaptic junctions. These neurons communicate with each other using chemical signals called neurotransmitters and neuropeptides, and induce dynamic patterns of circuit activity. In this way, neural circuits are the functional units of the brain, and the circuit activity induced by neurotransmitter and neuropeptide signaling is what constitutes thoughts and controls behaviors.
Neurotransmitter Signaling: Molecular Mechanisms
Some neurotransmitters act by binding ion channel receptors, but these neurotransmitters also have G protein coupled receptors (GPCRs), and many neurotransmitters and essentially all neuropeptides act exclusively via GPCRs. Our lab uses a combination of C. elegans genetics along with biochemical experiments carried out in both C. elegans and mouse brain lysates to study the proteins that mediate GPCR signaling in neurons. A current focus is to identify the effector proteins that mediate signaling downstream of the major neural G protein, Gαo, to inhibit neural function. These effectors have remained elusive despite 35 years of effort by many labs to find them. Through a combination of biochemical and genetic efforts, we are characterizing a set of proteins through which Gαo signals.
Neurotransmitter Signaling in Neural Circuits
Why are there so many neurotransmitters and neuropeptides, and why does each neurotransmitter have so many different receptors? How is all this signaling used to set up the dynamic pattern of activity within neural circuits to allow these circuits to think thoughts and control behaviors? Currently, there is not a single neural circuit in any organism in which we know how all the signals and receptors are used to control activity of the circuit. We aim to change this by studying a single simple model circuit in C. elegans, the egg-laying circuit. We are also generating a map of what cells express every G protein coupled neurotransmitter and neuropeptide receptor in C. elegans, so that our analysis of the egg-laying circuit can eventually be broadened to analyzing all neural circuits in C. elegans. By understanding first one and then a few neural circuits in great depth, we hope to uncover general principals that apply to all neural circuits in all organisms.
Gαo is by far the most abundant Gα protein in the brain, and every neurotransmitter has receptors that activate this G protein. Gαo 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 Gαo directly opposes Gαq signaling, and ultimately Gαo signaling inactivates neurons, preventing them from carrying out neurotransmitter release.
We have recently carried out rapid, gentle immunopurifications of Gαo 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 Gαo in both mouse and worms, and verified the association of Gαo with these proteins by co-immunoprecipitation/Western blots. Intriguingly, several of these proteins are regulators of Gαq signaling and/or neurotransmitter release, exactly the types of proteins that could plausibly mediate Gαo signaling. We are carrying out further biochemical studies of the association of Gαo with these proteins, analyzing how Gαo may regulate activity of these proteins, and are using C. elegans genetics to analyze the functional significance of these proteins in Gαo signaling.
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.
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.
Biochemistry; Biophysics; Molecular Biology; Neurobiology; Serotonin; Caenorhabditis elegans; Neurotransmitter Agents; RGS Proteins