Serotonin signals through postsynaptic Gαq, Trio RhoGEF, and diacylglycerol to promote Caenorhabditis elegans egg-laying circuit activity and behavior.

Activated Gαq signals through phospholipase-Cß and Trio, a Rho GTPase exchange factor (RhoGEF), but how these distinct effector pathways promote cellular responses to neurotransmitters like serotonin remains poorly understood. We used the egg-laying behavior circuit of Caenorhabditis elegans to determine whether phospholipase-Cß and Trio mediate serotonin and Gαq signaling through independent or related biochemical pathways. Our genetic rescue experiments suggest that phospholipase-Cß functions in neurons while Trio Rho GTPase exchange factor functions in both neurons and the postsynaptic vulval muscles. While Gαq, phospholipase-Cß, and Trio Rho GTPase exchange factor mutants fail to lay eggs in response to serotonin, optogenetic stimulation of the serotonin-releasing HSN neurons restores egg laying only in phospholipase-Cß mutants. Phospholipase-Cß mutants showed vulval muscle Ca2+ transients while strong Gαq and Trio Rho GTPase exchange factor mutants had little or no vulval muscle Ca2+ activity. Treatment with phorbol 12-myristate 13-acetate that mimics 1,2-diacylglycerol, a product of PIP2 hydrolysis, rescued egg-laying circuit activity and behavior defects of Gαq signaling mutants, suggesting both phospholipase-C and Rho signaling promote synaptic transmission and egg laying via modulation of 1,2-diacylglycerol levels. 1,2-Diacylglycerol activates effectors including UNC-13; however, we find that phorbol esters, but not serotonin, stimulate egg laying in unc-13 and phospholipase-Cß mutants. These results support a model where serotonin signaling through Gαq, phospholipase-Cß, and UNC-13 promotes neurotransmitter release, and that serotonin also signals through Gαq, Trio Rho GTPase exchange factor, and an unidentified, phorbol 12-myristate 13-acetate-responsive effector to promote postsynaptic muscle excitability. Thus, the same neuromodulator serotonin can signal in distinct cells and effector pathways to coordinate activation of a motor behavior circuit.

Photochemical Barcodes.

A photochemical strategy to encode fluorescence signals in vivo with spatial control was designed around the unique properties of a photoactivatable borondipyrromethene (BODIPY). The photoinduced disconnection of two oxazines, flanking a single BODIPY, in two consecutive steps produces a mixture of three emissive molecules with resolved fluorescence inside polymer beads. The relative amounts and emission intensities of the three fluorophores can be regulated precisely in each bead by adjusting the dose of activating photons to mark individual particles with distinct codes of fluorescence signals. The visible wavelengths and mild illumination sufficient to induce these transformations permit the photochemical barcoding of beads also in living nematodes. Different regions of the same animal can be labeled with distinct barcodes to allow the monitoring of their dynamics for long times with no toxic effects. Thus, our photochemical strategy for the generation of fluorescence barcodes can produce multiple and distinguishable labels in the same biological sample to enable the spatiotemporal tracking of, otherwise indistinguishable, targets.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans.

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified. Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 µm and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Bioimaging with Macromolecular Probes Incorporating Multiple BODIPY Fluorophores.

Seven macromolecular constructs incorporating multiple borondipyrromethene (BODIPY) fluorophores along a common poly(methacrylate) backbone with decyl and oligo(ethylene glycol) side chains were synthesized. The hydrophilic oligo(ethylene glycol) components impose solubility in aqueous environment on the overall assembly. The hydrophobic decyl chains effectively insulate the fluorophores from each other to prevent detrimental interchromophoric interactions and preserve their photophysical properties. As a result, the brightness of these multicomponent assemblies is approximately three times greater than that of a model BODIPY monomer. Such a high brightness level is maintained even after injection of the macromolecular probes in living nematodes, allowing their visualization with a significant improvement in signal-to-noise ratio, relative to the model monomer, and no cytotoxic or behavioral effects. The covalent scaffold of these macromolecular constructs also permits their subsequent conjugation to secondary antibodies. The covalent attachment of polymer and biomolecule does not hinder the targeting ability of the latter and the resulting bioconjugates can be exploited to stain the tubulin structure of model cells to enable their visualization with optimal signal-to-noise ratios. These results demonstrate that this particular structural design for the incorporation of multiple chromophores within the same covalent construct is a viable one to preserve the photophysical properties of the emissive species and enable the assembly of bioimaging probes with enhanced brightness.