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 Ca2+ 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 (GoA 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

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 heterologous expression system for rapid target-directed proteolysis in mammalian cells was developed. 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) has been inserted. Inducible activity was conferred to the TEVp using rapamycin-controlled TEVp fragment complementation. TEVp activity was assayed using a FRET-based reporter construct. TEVp expression was well tolerated by mammalian cells and complete cleavage of the substrate was possible. Cleavage at 37 degrees C proceeded exponentially with a time constant of approximately 150 minutes. Attempts to improve cleavage efficiency were hampered by substantial background activity, which was attributed to inherent affinity between the TEVp fragments. A second TEVp assay, based on changes in inactivation of a modified K(V)3.4 channel, showed that functional properties of a channel can be using altered using an inducible TEVp system. Similar levels of background activity and variability were observed in both electrophysiological and FRET assays.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. The system is likely to be more suitable for experiments in which the cell-to-cell variability is less of an issue; for example, in experiments involving large populations of cells

A 3.7 kb fragment of the mouse Scn10a gene promoter directs neural crest but not placodal lineage EGFP expression in a transgenic animal.

Under physiological conditions, the voltage-gated sodium channel Nav1.8 is expressed almost exclusively in primary sensory neurons. The mechanism restricting Nav1.8 expression is not entirely clear, but we have previously described a 3.7 kb fragment of the Scn10a promoter capable of recapitulating the tissue-specific expression of Nav1.8 in transfected neurons and cell lines (Puhl and Ikeda, 2008). To validate these studies in vivo, a transgenic mouse encoding EGFP under the control of this putative sensory neuron specific promoter was generated and characterized in this study. Approximately 45% of dorsal root ganglion neurons of transgenic mice were EGFP-positive (mean diameter = 26.5 μm). The majority of EGFP-positive neurons bound isolectin B4, although a small percentage (∼10%) colabeled with markers of A-fiber neurons. EGFP expression correlated well with the presence of Nav1.8 transcript (95%), Nav1.8-immunoreactivity (70%), and TTX-R INa (100%), although not all Nav1.8-expressing neurons expressed EGFP. Several cranial sensory ganglia originating from neurogenic placodes, such as the nodose ganglion, failed to express EGFP, suggesting that additional regulatory elements dictate Scn10a expression in placodal-derived sensory neurons. EGFP was also detected in discrete brain regions of transgenic mice. Quantitative PCR and Nav1.8-immunoreactivity confirmed Nav1.8 expression in the amygdala, brainstem, globus pallidus, lateral and paraventricular hypothalamus, and olfactory tubercle. TTX-R INa recorded from EGFP-positive hypothalamic neurons demonstrate the usefulness of this transgenic line to study novel roles of Nav1.8 beyond sensory neurons. Overall, Scn10a-EGFP transgenic mice recapitulate the majority of the Nav1.8 expression pattern in neural crest-derived sensory neurons.This work was supported by the Intramural program at the National Institutes of Health, National Institute on Alcohol Abuse and Alcoholis

Human GPR42 is a transcribed multisite variant that exhibits copy number polymorphism and is functional when heterologously expressed.

FFAR3 (GPR41) is a G-protein coupled receptor for which short-chain fatty acids serve as endogenous ligands. The receptor is found on gut enteroendocrine L-cells, pancreatic β-cells, and sympathetic neurons, and is implicated in obesity, diabetes, allergic airway disease, and altered immune function. In primates, FFAR3 is segmentally duplicated resulting in GPR42, a gene currently classified as a suspected pseudogene. In this study, we sequenced FFAR3 and GPR42 open reading frames from 56 individuals and found an unexpectedly high frequency of polymorphisms contributing to several complex haplotypes. We also identified a frequent (18.8%) structural variation that results in GPR42 copy number polymorphism. Finally, sequencing revealed that 50.6% of GPR42 haplotypes differed from FFAR3 by only a single non-synonymous substitution and that the GPR42 reference sequence matched only 4.4% of the alleles. Sequencing of cDNA from human sympathetic ganglia and colon revealed processed transcripts matching the GPR42 genotype. Expression of several GPR42 haplotypes in rat sympathetic neurons revealed diverse pharmacological phenotypes that differed in potency and efficacy. Our data suggest that GPR42 be reclassified as a functioning gene and that recognition of sequence and copy number polymorphism of the FFAR3/GPR42 complex be considered during genetic and pharmacological investigation of these receptors

Rem2, a member of the RGK family of small GTPases, is enriched in nuclei of the basal ganglia.

Rem2 is a member of the RGK subfamily of RAS small GTPases. Rem2 inhibits high voltage activated calcium channels, is involved in synaptogenesis, and regulates dendritic morphology. Rem2 is the primary RGK protein expressed in the nervous system, but to date, the precise expression patterns of this protein are unknown. In this study, we characterized Rem2 expression in the mouse nervous system. In the CNS, Rem2 mRNA was detected in all regions examined, but was enriched in the striatum. An antibody specific for Rem2 was validated using a Rem2 knockout mouse model and used to show abundant expression in striatonigral and striatopallidal medium spiny neurons but not in several interneuron populations. In the PNS, Rem2 was abundant in a subpopulation of neurons in the trigeminal and dorsal root ganglia, but was absent in sympathetic neurons of superior cervical ganglia. Under basal conditions, Rem2 was subject to post-translational phosphorylation, likely at multiple residues. Further, Rem2 mRNA and protein expression peaked at postnatal week two, which corresponds to the period of robust neuronal maturation in rodents. This study will be useful for elucidating the functions of Rem2 in basal ganglia physiology

Nr4a1-eGFP Is a Marker of Striosome-Matrix Architecture, Development and Activity in the Extended Striatum

Transgenic mice expressing eGFP under population specific promoters are widely used in neuroscience to identify specific subsets of neurons in situ and as sensors of neuronal activity in vivo. Mice expressing eGFP from a bacterial artificial chromosome under the Nr4a1 promoter have high expression within the basal ganglia, particularly within the striosome compartments and striatal-like regions of the extended amygdala (bed nucleus of the stria terminalis, striatal fundus, central amygdaloid nucleus and intercalated cells). Grossly, eGFP expression is inverse to the matrix marker calbindin 28K and overlaps with mu-opioid receptor immunoreactivity in the striatum. This pattern of expression is similar to Drd1, but not Drd2, dopamine receptor driven eGFP expression in structures targeted by medium spiny neuron afferents. Striosomal expression is strong developmentally where Nr4a1-eGFP expression overlaps with Drd1, TrkB, tyrosine hydroxylase and phospho-ERK, but not phospho-CREB, immunoreactivity in “dopamine islands”. Exposure of adolescent mice to methylphenidate resulted in an increase in eGFP in both compartments in the dorsolateral striatum but eGFP expression remained brighter in the striosomes. To address the role of activity in Nr4a1-eGFP expression, primary striatal cultures were prepared from neonatal mice and treated with forskolin, BDNF, SKF-83822 or high extracellular potassium and eGFP was measured fluorometrically in lysates. eGFP was induced in both neurons and contaminating glia in response to forskolin but SKF-83822, brain derived neurotrophic factor and depolarization increased eGFP in neuronal-like cells selectively. High levels of eGFP were primarily associated with Drd1+ neurons in vitro detected by immunofluorescence; however ∼15% of the brightly expressing cells contained punctate met-enkephalin immunoreactivity. The Nr4a1-GFP mouse strain will be a useful model for examining the connectivity, physiology, activity and development of the striosome system

The stigma turbine:A theoretical framework for conceptualizing and contextualizing marketplace stigma

Stigmas, or discredited personal attributes, emanate from social perceptions of physical characteristics, aspects of character, and “tribal” associations (e.g., race; Goffman 1963). Extant research emphasizes the perspective of the stigma target, with some scholars exploring how social institutions shape stigma. Yet the ways stakeholders within the socio-commercial sphere create, perpetuate, or resist stigma remain overlooked. We introduce and define marketplace stigma as the labeling, stereotyping, and devaluation by and of commercial stakeholders (consumers, companies and their employees, stockholders, institutions) and their offerings (products, services, experiences). We offer the Stigma Turbine (ST) as a unifying conceptual framework that locates marketplace stigma within the broader sociocultural context, and illuminates its relationship to forces that exacerbate or blunt stigma. In unpacking the ST, we reveal the critical role market stakeholders can play in (de)stigmatization, explore implications for marketing practice and public policy, and offer a research agenda to further our understanding of marketplace stigma and stakeholder welfare