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. A project explored new methods for the high efficiency transfection of post-mitotic cells such as neurons. Although intranuclear microinjection has been a 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. 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 calcium 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. Reference: 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. Front Neurosci 4:181, 2011. A second 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 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-calcium currents. 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. Reference: Won Y-J, Ono F, Ikeda SR. Identification and modulation of voltage-gated Ca2+ currents in zebrafish Rohon-Beard neurons. Journal of Neurophysiology 105:442-53, 2011. A third project, done in collaboration with Dr. David Lovinger (LIN), resulted in the development of an isolated neuron preparation with intact and functional synaptic boutons suitable for both electrophysiological and imaging experiments. The preparation allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies;superior control of the extracellular environment free from the influence of neighboring cells;suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion;and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches. Reference: Jun, SB, Carlson, VC, Ikeda S, Lovinger D. (2011) Vibrodissociation of neurons from rodent brain slices to study synaptic transmission and image presynaptic terminals. JoVE 51, 2011., doi: 10.3791/2752. Finally, Dr. Henry Puhl, a Staff Scientist in STS/LMP, collaborated independently on two projects with other NIAAA/DICBR sections, one within LMP and the other with LIN. The first study was performed in collaboration with Dr. Steven Vogel's group and investigated the role of dysferlin, a protein involved in certain forms of human muscular dystrophy. The study found that genetic suppression of dysferlin in sea urchin eggs interrupted an ATP-dependent signaling system triggered by membrane wounding. These results may shed light on the mechanisms underlying limb girdle muscular dystrophy-2B (LGMD2B) and Miyoshi muscular dystrophy (MMD). Reference: Covian-Nares et al. Membrane wounding triggers ATP release and dysferlin-mediated intercellular calcium signaling. J Cell Sci 123:1884-93, 2010. A second study, performed in collaboration with Dr. Margaret Davis (LIN), characterized eGFP expression driven by the Nr4a1 (Nuclear receptor subfamily 4, group A, member 1) promoter in transgenic mice. Expression occurred primarily in dopamine receptor 1 (Drd1) expressing neurons but the level of basal and stimulated expression varied with compartment, thereby differentiating Drd1 striosome neurons from Drd1 matrix neurons. The study concludes that these mice will be useful for examination of drug- or activity-dependent Nr4a1 induction in the extended striatum associated with learning and plasticity as well as for in situ identification of neurons differentially activated within the striosome and matrix compartments. Reference: Davis, MI and Puhl, HL. Nr4a1-eGFP is a marker of striosome-matrix architecture, development and activity in the extended striatum. PLoS ONE 6:e16619, 2011.

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Lu, Van B; Ikeda, Stephen R (2016) Strategies for Investigating G-Protein Modulation of Voltage-Gated Ca2+ Channels. Cold Spring Harb Protoc 2016:pdb.top087072
Liput, Daniel J; Lu, Van B; Davis, Margaret I et al. (2016) Rem2, a member of the RGK family of small GTPases, is enriched in nuclei of the basal ganglia. Sci Rep 6:25137
Lu, Van B; Ikeda, Stephen R (2016) G-Protein Modulation of Voltage-Gated Ca2+ Channels from Isolated Adult Rat Superior Cervical Ganglion Neurons. Cold Spring Harb Protoc 2016:pdb.prot091223
Nguyen, Tuan A; Sarkar, Pabak; Veetil, Jithesh V et al. (2015) Covert Changes in CaMKII Holoenzyme Structure Identified for Activation and Subsequent Interactions. Biophys J 108:2158-70
Puhl Iii, Henry L; Won, Yu-Jin; Lu, Van B et al. (2015) Human GPR42 is a transcribed multisite variant that exhibits copy number polymorphism and is functional when heterologously expressed. Sci Rep 5:12880
Lu, Van B; Ikeda, Stephen R; Puhl 3rd, Henry L (2015) A 3.7 kb fragment of the mouse Scn10a gene promoter directs neural crest but not placodal lineage EGFP expression in a transgenic animal. J Neurosci 35:8021-34
Iyer, Malliga R; Cinar, Resat; Liu, Jie et al. (2015) Structural Basis of Species-Dependent Differential Affinity of 6-Alkoxy-5-Aryl-3-Pyridinecarboxamide Cannabinoid-1 Receptor Antagonists. Mol Pharmacol 88:238-44
Puhl 3rd, Henry L; Lu, Van B; Won, Yu-Jin et al. (2014) Ancient origins of RGK protein function: modulation of voltage-gated calcium channels preceded the protostome and deuterostome split. PLoS One 9:e100694
Won, Yu-Jin; Lu, Van B; Puhl 3rd, Henry L et al. (2013) ?-Hydroxybutyrate modulates N-type calcium channels in rat sympathetic neurons by acting as an agonist for the G-protein-coupled receptor FFA3. J Neurosci 33:19314-25
Lu, Van B; Puhl 3rd, Henry L; Ikeda, Stephen R (2013) N-Arachidonyl glycine does not activate G protein-coupled receptor 18 signaling via canonical pathways. Mol Pharmacol 83:267-82

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