Our fundamental goal is to understand how neurons communicate in circuits to establish an appropriate level of activity that produces a robust, stable behavior.
Our approach is to analyze in detail a model neural circuit that controls egg-laying behavior in the nematode C. elegans. We are taking advantage of the optical clarity and powerful genetics in this experimental system to literally watch the activity of every cell in the circuit in behaving animals using fluorescent calcium reporters, and also to manipulate their activity using optogenetic tools. Using mutations and transgenes to discover and alter molecular signaling events between cells, we are determining how the complex pattern of activity in a circuit creates a coherent, regulated behavior. We expect these studies will reveal general principles of neurotransmitter signaling and neural circuit function with applications to understanding the human nervous system and its dysfunction in disease.
Anatomy and connectivity of the C. elegans egg-laying circuit
Two serotonergic HSN command motoneurons (one each on left and right side of the animal) and six cholinergic VC motoneurons make synapses onto the vm2 vulval muscles (vm2) that drive egg laying. The vm2 vulval muscles also make gap junctions with the uterine muscles (um) which may detect the accumulation of unlaid eggs in the uterus. The vm1 vulval muscles receive cholinergic inputs from the VA/VB motor neurons that contract the body wall muscles (bwm) for locomotion. The VC motor neurons also innervate the body wall muscles may slow locomotion during egg laying. Egg release mechanically activates the uv1 neuroendocrine cells which release tyramine to inhibit the HSN neurons and terminate egg laying.
Activity of the circuit during the active phase
Ratiometric calcium recording in behaving animals shows a dramatic induction of circuit activity during the egg-laying active state. The HSNs induce the active state, VC neuron and vulval muscle activity is coincident with egg laying, while the uv1 cells are activated in response to egg release.
Recent results from the lab
1) There is a significant change in egg-laying circuit activity during its development. The HSNs mature from tonic firing in late larval (L4) animals to burst firing in adults that drives egg-laying behavior. At the same time, the postsynaptic vulval muscles develop coordinated contractile activity that allows eggs to be efficiently laid. Animal sterilization or silencing of the vulval muscles inhibits adult HSN activity, suggesting that signals from the germ line and/or unlaid eggs activate the HSNs to promote egg laying. Thus, we propose a stretch-dependent homeostat promotes the onset and duration of the egg-laying active state. Paper here.
2) Aversive sensory signals act to inhibit egg-laying circuit behavior via activation of the major G protein Gαo (GOA-1) in the presynaptic, serotonin-releasing HSNs. Gαo signaling hyperpolarizes HSN, reducing Ca2+ activity and input into the postsynaptic vulval muscles. Loss of inhibitory Gαo uncouples presynaptic HSN activity from the stretch-dependent homeostat, causing precocious entry into the egg-laying active state. Paper here.
3) Serotonin signals to promote egg laying via activation of the major G protein Gαq (EGL-30) in both the presynaptic neurons and the postsynaptic vulval muscles. Gαq signaling through the PLC-beta effector branch promotes neurotransmitter release, and signaling through the Trio RhoGEF branch promotes vulval muscle excitability. Interestingly, defects in Gαq signaling mutants can all be rescued by DAG-mimetic phorbol esters, suggesting both pathways may converge to modulate DAG levels which then activate downstream effectors. Paper here.
4) Like the uv1 neuroendocrine cells, the cholinergic VC neurons that innervate the vulval muscles are also mechanically activated. Silencing of the VC neurons reduces egg laying in response to serotonin and reduces the probability of egg laying during vulval muscle Ca2+ activity. We propose that the VC neurons respond to muscle contraction and opening of the vulva, releasing acetylcholine to help fully open the vulva for efficient egg release. Paper here.
5) Gonad microinjection induces vulval muscle activity and release of eggs. We hypothesize this injection response is stimulated by an increase in hydrostatic pressure that mimics the change in uterine stretch that promotes egg-laying behavior and that accompanies insemination during mating. Egg release requires muscle myosin but is independent of the HSNs and synaptic input. Paper here
6) Egg-laying circuit activity also regulates mating behavior. There is significant vulval muscle activity during male spicule insertion that activates the uv1 cells, and deposition of sperm and seminal fluid fills the uterus, driving a resumption of vulval muscle activity that allows spicule withdrawal and ejection of male sperm. We will use our collection of genetic and optical tools to determine how changes in signaling between cells in the egg-laying circuit drive two, mutually exclusive, reproductive behaviors.