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Articles by Damian J. Williams in JoVE
JoVE General
Intranucleares microinjeção de DNA em Dissociated neurônios de mamíferos adultos
Intranuclear injeção direta de cDNA é uma técnica eficaz para transfecção células pós-mitóticas. Este método oferece altos níveis de expressão da proteína heteróloga a partir de uma ou várias construções de cDNA e permite que a função da proteína a ser estudada em um ambiente fisiologicamente relevantes com uma variedade de ensaios única célula.
Other articles by Damian J. Williams on PubMed
The Sources and Sequestration of Ca(2+) Contributing to Neuroeffector Ca(2+) Transients in the Mouse Vas Deferens
The detection of focal Ca(2+) transients (called neuroeffector Ca(2+) transients, or NCTs) in smooth muscle of the mouse isolated vas deferens has been used to detect the packeted release of ATP from nerve terminal varicosities acting at postjunctional P2X receptors. The present study investigates the sources and sequestration of Ca(2+) in NCTs. Smooth muscle cells in whole mouse deferens were loaded with the Ca(2+) indicator Oregon Green 488 BAPTA-1 AM and viewed with a confocal microscope. Ryanodine (10 microM) decreased the amplitude of NCTs by 45 +/- 6 %. Cyclopiazonic acid slowed the recovery of NCTs (from a time course of 200 +/- 10 ms to 800 +/- 100 ms). Caffeine (3 mM) induced spontaneous focal smooth muscle Ca(2+) transients (sparks). Neither of the T-type Ca(2+) channel blockers NiCl2 (50 microM) or mibefradil dihydrochloride (10 microM) affected the amplitude of excitatory junction potentials (2 +/- 5 % and -3 +/- 10 %) or NCTs (-20 +/- 36 % and 3 +/- 13 %). In about 20 % of cells, NCTs were associated with a local, subcellular twitch that remained in the presence of the alpha1-adrenoceptor antagonist prazosin (100 nM), showing that NCTs can initiate local contractions. Slow (5.8 +/- 0.4 microm s(-1)), spontaneous smooth muscle Ca(2+) waves were occasionally observed. Thus, Ca(2+) stores initially amplify and then sequester the Ca(2+) that enters through P2X receptors and there is no amplification by local voltage-gated Ca(2+) channels.
Inhibition of N-type Calcium Channels by Activation of GPR35, an Orphan Receptor, Heterologously Expressed in Rat Sympathetic Neurons
GPR35 is a G protein-coupled receptor recently "de-orphanized" using high-throughput intracellular calcium measurements in clonal cell lines expressing a chimeric G-protein alpha-subunit. From these screens, kynurenic acid, an endogenous metabolite of tryptophan, and zaprinast, a synthetic inhibitor of cyclic guanosine monophosphate-specific phosphodiesterase, emerged as potential agonists for GPR35. To investigate the coupling of GPR35 to natively expressed neuronal signaling pathways and effectors, we heterologously expressed GPR35 in rat sympathetic neurons and examined the modulation of N-type (Ca(V)2.2) calcium channels. In neurons expressing GPR35, calcium channels were inhibited in the absence of overt agonists, indicating a tonic receptor activity. Application of kynurenic acid or zaprinast resulted in robust voltage-dependent calcium current inhibition characteristic of Gbetagamma-mediated modulation. Both agonist-independent and -dependent effects of GPR35 were blocked by Bordetella pertussis toxin pretreatment indicating the involvement of G(i/o) proteins. In neurons expressing GPR35a, a short splice variant of GPR35, zaprinast was more potent (EC(50) = 1 microM) than kynurenic acid (58 microM) but had a similar efficacy (approximately 60% maximal calcium current inhibition). Expression of GPR35b, which has an additional 31 residues at the N terminus, produced similar results but with much greater variability. Both GPR35a and GPR35b appeared to have similar expression patterns when fused to fluorescent proteins. These results suggest a potential role for GPR35 in regulating neuronal excitability and synaptic release.
N-arachidonoyl L-serine, a Putative Endocannabinoid, Alters the Activation of N-type Ca2+ Channels in Sympathetic Neurons
The effect of N-arachidonoyl l-serine (ARA-S), a recently discovered lipoamino acid found in the CNS, on N-type Ca2+ channels of rat sympathetic ganglion neurons was determined using whole cell patch clamp. Application of ARA-S produced a rapid and reversible augmentation of Ca2+ current that was voltage dependent and resulted from a hyperpolarizing shift in the activation curve. ARA-S did not influence G protein modulation of Ca2+ channels and appeared to act independently of G-protein-coupled receptors. These findings provide a foundation for investigating possible roles for ARA-S in nervous system function.
Rapid Modification of Proteins Using a Rapamycin-inducible Tobacco Etch Virus Protease System
The ability to disrupt the function of a specific protein on a rapid time scale provides a powerful tool for biomedical research. Specific proteases provide a potential method to selectively cleave a chosen protein, but rapid control of protease activity is difficult.
A Simple, Highly Efficient Method for Heterologous Expression in Mammalian Primary Neurons Using Cationic Lipid-mediated MRNA Transfection
Expression of heterologous proteins in adult mammalian neurons is a valuable technique for the study of neuronal function. The post-mitotic nature of mature neurons prevents effective DNA transfection using simple, cationic lipid-based methods. Adequate heterologous protein expression is often only achievable using complex techniques that, in many cases, are associated with substantial toxicity. Here, a simple method for high efficiency transfection of mammalian primary neurons using in vitro transcribed mRNA and the cationic lipid transfection reagent Lipofectamine™ 2000 is described. 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 Ca(2+) 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. Functional expression of a cannabinoid receptor (CB1R), a G protein inwardly rectifying K(+) channel (GIRK4) and a dominant-negative G protein α-subunit mutant (G(oA) G203T) indicate that the levels of heterologous protein expression attainable using mRNA transfection are suitable for most functional protein studies. This study demonstrates that mRNA transfection is a straightforward and effective method for heterologous expression in neurons and is likely to have many applications in neuroscience research.
A Functionally Characterized Test Set of Human Induced Pluripotent Stem Cells
Human induced pluripotent stem cells (iPSCs) present exciting opportunities for studying development and for in vitro disease modeling. However, reported variability in the behavior of iPSCs has called their utility into question. We established a test set of 16 iPSC lines from seven individuals of varying age, sex and health status, and extensively characterized the lines with respect to pluripotency and the ability to terminally differentiate. Under standardized procedures in two independent laboratories, 13 of the iPSC lines gave rise to functional motor neurons with a range of efficiencies similar to that of human embryonic stem cells (ESCs). Although three iPSC lines were resistant to neural differentiation, early neuralization rescued their performance. Therefore, all 16 iPSC lines passed a stringent test of differentiation capacity despite variations in karyotype and in the expression of early pluripotency markers and transgenes. This iPSC and ESC test set is a robust resource for those interested in the basic biology of stem cells and their applications.
Modulation of Neurite Outgrowth by Activation of Calcium-permeable Kainate Receptors Expressed by Rat Nociceptive-like Dorsal Root Ganglion Neurons
Neurite outgrowth is a fundamental step in establishing proper neuronal connections in the developing central nervous system. Dynamic control of outgrowth has been attributed to changes in growth cone Ca2+ levels in response to extracellular cues. Here we have investigated a possible role for Ca2+ permeable kainate (KA) receptors in regulating neurite outgrowth of nociceptive-like dorsal root ganglion (DRG) neurons. To identify KA receptor subunits likely to be involved, we used quantitative RT-PCR on acutely dissociated DRG and dorsal horn neurons. DRG neurons expressed more GluK1, particularly the GluK1b spice variant, than dorsal horn neurons. Conversely, dorsal horn neurons expressed more GluK2, particularly GluK2a, than DRG neurons. Further, an RNA editing assay indicated that the majority of GluK1 and GluK2 mRNA transcripts in DRG were unedited. Imaging Ca2+ transients following application of a KA receptor agonist to DRG and dorsal horn co-cultures revealed increases in intracellular Ca2+ in the growth cones of DRG neurons. In the majority of cases, this increase in Ca2+ was partly or completely blocked by Joro spider toxin (JSTX), an antagonist for Ca2+-permeable AMPA and KA receptors. Treatment of DRG/dorsal horn co-cultures with KA for 18 hours suppressed neurite outgrowth while application of the rapidly desensitizing KA receptor agonist SYM 2081, the competitive AMPA/KA receptor antagonist, CNQX, and JSTX or philanthotoxin enhanced neurite outgrowth and prevented KA effects on neurite outgrowth. Thus, Ca2+ entry through KA receptors at the growth cone of DRG neurons may be an important regulator of neurite outgrowth.
Mechanisms Involved in Nicotinic Acetylcholine Receptor-induced Neurotransmitter Release from Sympathetic Nerve Terminals in the Mouse Vas Deferens
Prejunctional nicotinic acetylcholine receptors (nAChRs) amplify postganglionic sympathetic neurotransmission, and there are indications that intraterminal Ca(2+) stores might be involved. However, the mechanisms by which nAChR activation stimulates neurotransmitter release at such junctions is unknown. Rapid local delivery (picospritzing) of the nAChR agonist epibatidine was combined with intracellular sharp microelectrode recording to monitor spontaneous and field-stimulation-evoked neurotransmitter release from sympathetic nerve terminals in the mouse isolated vas deferens. Locally applied epibatidine (1 µM) produced 'epibatidine-induced depolarisations' (EIDs) that were similar in shape to spontaneous excitatory junction potentials (SEJPs) and were abolished by nonselective nAChR antagonists and the purinergic desensitizing agonist α,β-methylene ATP. The amplitude distribution of EIDs was only slightly shifted towards lower amplitudes by the selective α7 nAChR antagonists α-bungarotoxin and methyllcaconitine, the voltage-gated Na(+) channel blocker tetrodotoxin or by blocking voltage-gated Ca(2+) channels with Cd(2+). Lowering the extracellular Ca(2+) concentration reduced the frequency of EIDs by 69%, but more surprisingly, the Ca(2+)-induced Ca(2+) release blocker ryanodine greatly decreased the amplitude (by 41%) and the frequency of EIDs by 36%. Ryanodine had no effect on electrically-evoked neurotransmitter release, paired-pulse facilitation, SEJP frequency, SEJP amplitude or SEJP amplitude distribution. These results show that activation of non-α7 nAChRs on sympathetic postganglionic nerve terminals induces high-amplitude junctional potentials that are argued to represent multipacketed neurotransmitter release synchronized by intraterminal Ca(2+)-induced Ca(2+) release, triggered by Ca(2+) influx directly through the nAChR. This nAChR-induced neurotransmitter release can be targeted pharmacologically without affecting spontaneous or electrically-evoked neurotransmitter release.
