The primary focus of the section is to further our understanding of the molecular basis of signaling between G protein coupled receptors and voltage gated ion channels in neurons using electrophysiological, molecular, and imaging techniques. There were four main areas of progress during the current funding period. A project involving development of a reduced model system to study endocannabinoid mobilization, transport, and signaling was completed. Endocannabinoids (eCB) such as 2-arachidonylglycerol (2-AG) are lipid metabolites that are synthesized in a postsynaptic neurons and act upon CB1 cannabinoid receptors (CB1R) in presynaptic nerve terminals. This retrograde transmission underlies several forms of short and long term synaptic plasticity within the central nervous system. In this study, we constructed a model system based on isolated rat sympathetic neurons in which an eCB signaling cascade could be studied in a reduced, spatially compact, and genetically malleable system. We constructed a complete eCB production/mobilization pathway by sequential addition of molecular components. Heterologous expression of four components were required for eCB production and detection: metabotropic glutamate receptor 5a (mGluR5a), Homer 2b, diacylglycerol lipase alpha, and CB1R. In these neurons, application of L-glutamate produced voltage-dependent modulation of N-type calcium channels mediated by activation of CB1R. Using both molecular dissection and pharmacological agents, we provide evidence that activation of mGluR5a results in rapid enzymatic production of 2 AG followed by activation of CB1R. These experiments define the critical elements required to recapitulate retrograde eCB production and signaling in a single peripheral neuron. The system provides a platform for testing candidate molecules underlying facilitation of eCB transport across the plasma membrane. Won, Y-J, Puhl, H.L., and Ikeda, S.R., Journal of Neuroscience, 29:1360313612, 2009. The second project explored a novel methodology to target specific proteins for modification or ablation on a rapid time scale in living mammalian cells. The system consists of an inducible NIa protease from the tobacco etch virus (TEVp) and a chosen protein into which a TEVp substrate recognition sequence (TRS) was inserted. Inducible activity was conferred to the TEVp using rapamycin-controlled TEVp fragment complementation. TEVp activity was assayed using a fluorescence resonance energy transfect (FRET)-based reporter construct. TEVp expression was well tolerated by mammalian cells and complete cleavage of the substrate was possible. Cleavage at 37 C proceeded exponentially with a time constant of approximately 150 minutes. Two strategies were used to improve cleavage efficiency: 1) The relative concentration of the TEVp fragments to each other was increased 2) The relative concentration of TEVp to the substrate was increased. Both these approaches were hampered by substantial background activity that was attributed to inherent affinity between the TEVp fragments, and demonstrated that TEVp activity was surprisingly insensitive to insertion of large protein domains at the split site. The results suggested that an optimum level of TEVp expression leading to sufficient inducible activity (with minimal background activity) exists but the variability in expression levels between cells makes the present system rather impractical for single cell experiments. Hence there is substantial opportunity for improving the system using structure-guided mutagenesis to reduce the propensity for TEVp self-complementation. Williams, D.J., Puhl, H.L., and Ikeda, S.R. PLoS ONE 4:e7474, 2009. A third project explored new methods for transfection post-mitotic cells such as neurons at high efficiency. Although a intranuclear microinjection has been mainstay technique in the laboratory for many years, the process is tedious, time consuming, and results in a limited number of neurons expressing the gene of interest. We have thus experimented with a variety of nonviral methods over a period of several years to increase transfection rates in adult mammalian neurons. The current effort was inspired by work in dendritic cells using mRNA-based gene expression to induce immunological response. A simple method for high efficiency transfection of mammalian primary neurons using in vitro-transcribed mRNA and the cationic lipid transfection reagent Lipofectamine 2000 was developed. Optimal transfection conditions were established in adult mouse dissociated dorsal root ganglion (DRG) neurons using a 96-well based luciferase activity assay. Using these conditions, a transfection efficiency of 25% was achieved in DRG neurons transfected with EGFP mRNA. High transfection efficiencies were also obtained in dissociated rat superior cervical ganglion (SCG) neurons and mouse cortical and hippocampal cultures. Endogenous clacium currents in EGFP mRNA-transfected SCG neurons were not significantly different from untransfected neurons, which suggested that this technique is well suited for heterologous expression in patch clamp recording experiments. This study demonstrated that mRNA transfection is a straightforward and effective method for heterologous expression in neurons and is likely to have many applications in neuroscience research. Williams, D.J., Puhl H.L., Ikeda S.R. A simple, highly efficient method for heterologous expression in mammalian primary neurons using cationic lipid-mediated mRNA transfection. Submitted, 2010. A fourth project, done in collaboration with Dr. Fumihito Ono, resulted in the development of a zebrafish neuronal system suitable for investigating calcium channel modulation and function during a development stage that leverages the genetic malleability and optical transparency of this model organism. Zebrafish (Danio rerio) are an important model vertebrate for studies of neuronal excitability, circuits, and behavior. However, voltage-gated calcium channel properties remain largely unexplored because a suitable preparation for whole-cell voltage-clamp studies is lacking. Rohon-Beard (R-B) primary sensory neurons represent an attractive candidate for this purpose because of their relatively large somata, appearance during early development, and functional homology to mammalian dorsal root ganglia (DRG) neurons. In this study, we used a transgenic zebrafish line (Isl2b:EGFP)ZC7 in which EGFP expression in R-B neurons was driven by the Isl2b promoter to identify dissociated neurons suitable for whole-cell patch clamp experiments. Based on biophysical and pharmacological properties, zebrafish R-B neurons express both high- and low-voltage-activated calcium current (HVA- and LVA-calcium currents, respectively). Nickel-sensitive LVA-calcium channels occur in the minority of R-B neurons (30%) and omega-conotoxin GVIA-sensitive CaV2.2 (N-type) calcium channels underlie the vast majority (90%) of HVA-ICa. To identify G-protein coupled receptors (GPCRs) that modulate calcium channels, a panel of neurotransmitters was screened. Application of GABA/baclofen or 5-HT produced a voltage-dependent inhibition while DAMGO application resulted in a voltage-independent inhibition. Unlike in mammalian neurons, GPCR-mediated voltage-dependent modulation of calcium current appears to be transduced primarily via a cholera toxin-sensitive G-protein alpha subunit. These results provide the basis for using the zebrafish model system to understand calcium channel function, and in turn, how calcium channels contribute to zebrafish mechanosensory function. Won Y-J, Ono F, Ikeda SR (2010) Identification and modulation of voltage-gated Ca2+ currents in zebrafish Rohon-Beard neurons. Submitted, 2010.
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