RIM and RIM-binding protein localize synaptic CaV2 channels to differentially regulate transmission in neuronal circuits.

At chemical synapses, voltage-gated Ca2+-channels (VGCCs) translate electrical signals into a trigger for synaptic vesicle (SV) fusion. VGCCs and the Ca2+ microdomains they elicit must be located precisely to primed SVs, to evoke rapid transmitter release. Localization is mediated by Rab3 interacting molecule (RIM) and RIM-binding proteins (RIM-BPs), which interact and bind to the C-terminus of the CaV2 VGCC α-subunit. We studied this machinery at the mixed cholinergic/GABAergic neuromuscular junction (NMJ) of Caenorhabditis elegans hermaphrodites. rimb-1 mutants had mild synaptic defects, through loosening the anchoring of UNC-2/CaV2 and delaying the onset of SV fusion. UNC-10/RIM deletion much more severely affected transmission. Even though postsynaptic depolarization was reduced, rimb-1 mutants had increased cholinergic (but reduced GABAergic) transmission, to compensate for the delayed release. This did not occur when the excitation-inhibition balance was altered by removing GABA transmission. Further analyses of GABA defective mutants and GABAA or GABAB receptor deletions, as well as cholinergic rescue of RIMB-1, emphasized that GABA neurons may be more affected than cholinergic neurons. Thus RIMB-1 function differentially affects excitation/inhibition balance in the different motor neurons, and RIMB-1 thus may differentially regulate transmission in mixed circuits. Untethering the UNC-2/CaV2 channel by removing its C-terminal PDZ ligand exacerbated the rimb-1 defects, and similar phenotypes resulted from acute degradation of the CaV2 ß-subunit CCB-1. Therefore, untethering of the CaV2 complex is as severe as its elimination, yet does not abolish transmission, likely due to compensation by CaV1. Thus, robustness and flexibility of synaptic transmission emerges from VGCC regulation.Significance statement The machinery for chemical synaptic transmission is organized in a precise spatial arrangement in order to enable efficient and temporally accurate coupling of action potentials with the rise of the Ca2+ concentration through CaV2 P/Q-type voltage gated Ca2+ channels. This triggers the fusion of synaptic vesicles with the plasma membrane and the release of transmitters. Here, we analyzed the molecular and functional interplay of proteins of the active zone scaffold, RIM and RIM-binding protein (RIMB-1), with the CaV2 channel in the C. elegans neuromuscular junction, a tripartite synapse with cholinergic and GABAergic neuronal input. Our work shows a differential requirement of RIMB-1 in cholinergic vs. GABAergic neurons, that affects the regulation of excitation-inhibition balance at circuit, cellular and ultrastructural levels.

Synapsin Is Required for Dense Core Vesicle Capture and cAMP-Dependent Neuropeptide Release.

Release of neuropeptides from dense core vesicles (DCVs) is essential for neuromodulation. Compared with the release of small neurotransmitters, much less is known about the mechanisms and proteins contributing to neuropeptide release. By optogenetics, behavioral analysis, electrophysiology, electron microscopy, and live imaging, we show that synapsin SNN-1 is required for cAMP-dependent neuropeptide release in Caenorhabditis elegans hermaphrodite cholinergic motor neurons. In synapsin mutants, behaviors induced by the photoactivated adenylyl cyclase bPAC, which we previously showed to depend on ACh and neuropeptides (Steuer Costa et al., 2017), are altered as in animals with reduced cAMP. Synapsin mutants have slight alterations in synaptic vesicle (SV) distribution; however, a defect in SV mobilization was apparent after channelrhodopsin-based photostimulation. DCVs were largely affected in snn-1 mutants: DCVs were ∼30% reduced in synaptic terminals, and their contents not released following bPAC stimulation. Imaging axonal DCV trafficking, also in genome-engineered mutants in the serine-9 protein kinase A phosphorylation site, showed that synapsin captures DCVs at synapses, making them available for release. SNN-1 colocalized with immobile, captured DCVs. In synapsin deletion mutants, DCVs were more mobile and less likely to be caught at release sites, and in nonphosphorylatable SNN-1B(S9A) mutants, DCVs traffic less and accumulate, likely by enhanced SNN-1 dependent tethering. Our work establishes synapsin as a key mediator of neuropeptide release.SIGNIFICANCE STATEMENT Little is known about mechanisms that regulate how neuropeptide-containing dense core vesicles (DCVs) traffic along the axon, how neuropeptide release is orchestrated, and where it occurs. We found that one of the longest known synaptic proteins, required for the regulation of synaptic vesicles and their storage in nerve terminals, synapsin, is also essential for neuropeptide release. By electrophysiology, imaging, and electron microscopy in Caenorhabditis elegans, we show that synapsin regulates this process by tethering the DCVs to the cytoskeleton in axonal regions where neuropeptides are to be released. Without synapsin, DCVs cannot be captured at the release sites and, consequently, cannot fuse with the membrane, and neuropeptides are not released. We suggest that synapsin fulfills this role also in vertebrates, including humans.

Serotonergic neuron ADF modulates avoidance behaviors by inhibiting sensory neurons in C. elegans.

Serotonin plays an essential role in both the invertebrate and vertebrate nervous systems. ADF, an amphid neuron with dual ciliated sensory endings, is considered to be the only serotonergic sensory neuron in the hermaphroditic Caenorhabditis elegans. This neuron is known to be involved in a range of behaviors including pharyngeal pumping, dauer formation, sensory transduction, and memory. However, whether ADF neuron is directly activated by environmental cues and how it processes these information remains unknown. In this study, we found that ADF neuron responds reliably to noxious stimuli such as repulsive odors, copper, sodium dodecyl sulfonate (SDS), and mechanical perturbation. This response is mediated by cell-autonomous and non-cell autonomous mechanisms. Furthermore, we show that ADF can modulate avoidance behaviors by inhibiting ASH, an amphid neuron with single ciliated ending. This work greatly furthers our understanding of 5-HT's contributions to sensory information perception, processing, and the resulting behavioral responses.

Melatonin receptor type 1 signals to extracellular signal-regulated kinase 1 and 2 via Gi and Gs dually coupled pathways in HEK-293 cells.

The pineal gland hormone melatonin exerts its regulatory roles in a variety of physiological and pathological responses through two G protein-coupled receptors, melatonin receptor type 1 (MT1) and melatonin receptor type 2 (MT2), which have been recognized as promising targets in the treatment of a number of human diseases and disorders. The MT1 receptor was identified nearly 20 years ago; however, the molecular mechanisms by which MT1-mediated signaling affects physiology remain to be further elucidated. In this study, using HEK293 cells stably expressing the human MT1 receptor, melatonin induced a concentration-dependent activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2). The melatonin-mediated phosphorylation of ERK1/2 at later time points (≥5 min) was strongly suppressed by pretreatment with pertussis toxin, but only a slight, if any, inhibition of ERK1/2 activation at early time points (≤2 min) was detected. Further experiments demonstrated that the Gßγ subunit, phosphoinositide 3-kinase, and calcium-insensitive protein kinase C were involved in the MT1-mediated activation of ERK1/2 at later time points (≥5 min). Moreover, results derived from cAMP assays combined with a MT1 mutant indicated that the human MT1 receptor could also couple to Gs protein, stimulating intracellular cAMP formation, and that the MT1-induced activation of ERK1/2 at early time points (≤2 min) was mediated by the Gs/cAMP/PKA cascade. Our findings may provide new insights into the pharmacological effects and physiological functions modulated by the MT1-mediated activation of ERK1/2.

Activation of BNGR-A24 by direct interaction with tachykinin-related peptides from the silkworm Bombyx mori leads to the Gq- and Gs-coupled signaling cascades.

Tachykinins constitute one of the largest peptide families in the animal kingdom and exert their diverse actions via G protein-coupled receptors (GPCRs). In this study, the Bombyx tachykinin-related peptides (TKRPs) were identified as specific endogenous ligands for the Bombyx neuropeptide GPCR A24 (BNGR-A24) and thus designated BNGR-A24 as BmTKRPR. Using both mammalian cell line HEK293 and insect cell line Sf21, further characterization demonstrated that BmTKRPR was activated, thus resulting in intracellular accumulation of cAMP, Ca(2+) mobilization, and ERK1/2 phosphorylation in a Gs and Gq inhibitor-sensitive manner. Moreover, quantitative reverse transcriptase polymerase chain reaction analysis and dsRNA-mediated knockdown experiments suggested a possible role for BmTKRPR in the regulation of feeding and growth. Our findings enhance the understanding of the Bombyx TKRP system in the regulation of fundamental physiological processes.

Recurrent neural network based high-precision position compensation control of magnetic levitation system.

For improving the dynamic quality and steady-state performance, the hybrid controller based on recurrent neural network (RNN) is designed to implement the position control of the magnetic levitation ball system in this study. This hybrid controller consists of a baseline controller, an RNN identifier, and an RNN controller. In the hybrid controller, the baseline controller based on the control law of proportional-integral-derivative is firstly employed to provide the online learning sample and maintain the system stability at the early control phase. Then, the RNN identifier is trained online to learn the accurate inverse model of the controlled object. Next, the RNN controller shared the same structures and parameters with the RNN identifier is applied to add the precise compensation control quantity in real-time. Finally, the effectiveness and advancement of the proposed hybrid control strategy are comprehensively validated by the simulation and experimental tests of tracking step, square, sinusoidal, and trapezoidal signals. The results indicate that the RNN-based hybrid controller can obtain higher precision and faster adjustment than the comparison controllers and has strong anti-interference ability and robustness.

Molecular Strategies for Intensity-Dependent Olfactory Processing in Caenorhabditis elegans.

Various odorants trigger complex animal behaviors across species in both quality- and quantity-dependent manners. However, how the intensity of olfactory input is encoded remains largely unknown. Here we report that isoamyl alcohol (IAA) induces bi-directional currents through a Gα- guanylate cyclase (GC)- cGMP signaling pathway in Caenorhabditis elegans olfactory neuron amphid wing "C" cell (AWC), while two opposite cGMP signaling pathways are responsible for odor-sensing in olfactory neuron amphid wing "B" cell (AWB): (1) a depolarizing Gα (GPA-3)- phosphodiesterase (PDE) - cGMP pathway which can be activated by low concentrations of isoamyl alcohol (IAA), and (2) a hyperpolarizing Gα (ODR-3)- GC- cGMP pathway sensing high concentrations of IAA. Besides, IAA induces Gα (ODR-3)-TRPV(OSM-9)-dependent currents in amphid wing "A" cell (AWA) and amphid neuron "H" cell with single ciliated sensory ending (ASH) neurons with different thresholds. Our results demonstrate that an elaborate combination of multiple signaling machineries encode the intensity of olfactory input, shedding light on understanding the molecular strategies on sensory transduction.

Sensory Glia Detect Repulsive Odorants and Drive Olfactory Adaptation.

Glia are typically considered as supporting cells for neural development and synaptic transmission. Here, we report an active role of a glia in olfactory transduction. As a polymodal sensory neuron in C. elegans, the ASH neuron is previously known to detect multiple aversive odorants. We reveal that the AMsh glia, a sheath for multiple sensory neurons including ASH, cell-autonomously respond to aversive odorants via G-protein-coupled receptors (GPCRs) distinct from those in ASH. Upon activation, the AMsh glia suppress aversive odorant-triggered avoidance and promote olfactory adaptation by inhibiting the ASH neuron via GABA signaling. Thus, we propose a novel two-receptor model where the glia and sensory neuron jointly mediate adaptive olfaction. Our study reveals a non-canonical function of glial cells in olfactory transduction, which may provide new insights into the glia-like supporting cells in mammalian sensory procession.