scholarly journals Properties of Wild-Type and Fluorescent Protein-Tagged Mouse Tetrodotoxin-Resistant Sodium Channel (NaV1.8) Heterologously Expressed in Rat Sympathetic Neurons

2008 ◽  
Vol 99 (4) ◽  
pp. 1917-1927 ◽  
Author(s):  
Geoffrey G. Schofield ◽  
Henry L. Puhl ◽  
Stephen R. Ikeda
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Sympathetic Neurons ◽  
Functional Expression ◽  
Nodose Ganglion Neurons ◽  
Similar Expression Pattern ◽  
Cervical Ganglion ◽  
Clonal Cell Lines ◽  
Mouse Dorsal Root Ganglion ◽  
Ganglion Neurons

The tetrodotoxin (TTX)-resistant Na+ current arising from NaV1.8-containing channels participates in nociceptive pathways but is difficult to functionally express in traditional heterologous systems. Here, we show that injection of cDNA encoding mouse NaV1.8 into the nuclei of rat superior cervical ganglion (SCG) neurons results in TTX-resistant Na+ currents with amplitudes equal to or exceeding the currents arising from natively expressing channels of mouse dorsal root ganglion (DRG) neurons. The activation and inactivation properties of the heterologously expressed NaV1.8 Na+ channels were similar but not identical to native TTX-resistant channels. Most notably, the half-activation potential of the heterologously expressed NaV1.8 channels was shifted about 10 mV toward more depolarized potentials. Fusion of fluorescent proteins to the N- or C-termini of NaV1.8 did not substantially affect functional expression in SCG neurons. Unexpectedly, fluorescence was not concentrated at the plasma membrane but found throughout the interior of the neuron in a granular pattern. A similar expression pattern was observed in nodose ganglion neurons expressing the tagged channels. In contrast, expression of tagged NaV1.8 in HeLa cells revealed a fluorescence pattern consistent with sequestration in the endoplasmic reticulum, thus providing a basis for poor functional expression in clonal cell lines. Our results establish SCG neurons as a favorable surrogate for the expression and study of molecularly defined NaV1.8-containing channels. The data also indicate that unidentified factors may be required for the efficient functional expression of NaV1.8 with a biophysical phenotype identical to that found in sensory neurons.

scholarly journals Role of PI 3-kinase, Akt and Bcl-2–related proteins in sustaining the survival of neurotrophic factor–independent adult sympathetic neurons

2001 ◽  
Vol 154 (5) ◽  
pp. 995-1006 ◽  
Author(s):  
Nina Orike ◽  
Gayle Middleton ◽  
Emma Borthwick ◽  
Vladimir Buchman ◽  
Timothy Cowen ◽  
...  
Keyword(s):  
Neurotrophic Factors ◽  
Molecular Mechanisms ◽  
Sympathetic Neurons ◽  
Dominant Negative ◽  
Downstream Effector ◽  
Cervical Ganglion ◽  
Negative Class ◽  
Natural Inhibitor ◽  
Ganglion Neurons ◽  
Related Proteins

By adulthood, sympathetic neurons have lost dependence on NGF and NT-3 and are able to survive in culture without added neurotrophic factors. To understand the molecular mechanisms that sustain adult neurons, we established low density, glial cell-free cultures of 12-wk rat superior cervical ganglion neurons and manipulated the function and/or expression of key proteins implicated in regulating cell survival. Pharmacological inhibition of PI 3-kinase with LY294002 or Wortmannin killed these neurons, as did dominant-negative Class IA PI 3-kinase, overexpression of Rukl (a natural inhibitor of Class IA PI 3-kinase), and dominant-negative Akt/PKB (a downstream effector of PI 3-kinase). Phospho-Akt was detectable in adult sympathetic neurons grown without neurotrophic factors and this was lost upon PI 3-kinase inhibition. The neurons died by a caspase-dependent mechanism after inhibition of PI 3-kinase, and were also killed by antisense Bcl-xL and antisense Bcl-2 or by overexpression of Bcl-xS, Bad, and Bax. These results demonstrate that PI 3-kinase/Akt signaling and the expression of antiapoptotic members of the Bcl-2 family are required to sustain the survival of adult sympathetic neurons.

Electrophysiological and Morphological Diversity of Mouse Sympathetic Neurons

1999 ◽  
Vol 82 (5) ◽  
pp. 2747-2764 ◽  
Author(s):  
Phillip Jobling ◽  
Ian L. Gibbins
Keyword(s):  
Tyrosine Hydroxylase ◽  
Electrical Properties ◽  
Superior Cervical Ganglion ◽  
Sympathetic Neurons ◽  
Inactivation Kinetics ◽  
Celiac Ganglion ◽  
Cervical Ganglion ◽  
Firing Properties ◽  
Ganglion Neurons ◽  
Cell Capacitance

We have used multiple-labeling immunohistochemistry, intracellular dye-filling, and intracellular microelectrode recordings to characterize the morphological and electrical properties of sympathetic neurons in the superior cervical, thoracic, and celiac ganglia of mice. Neurochemical and morphological characteristics of neurons varied between ganglia. Thoracic sympathetic ganglia contained three main populations of neurons based on differential patterns of expression of immunoreactivity to tyrosine hydroxylase, neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP). In the celiac ganglion, nearly all neurons contained immunoreactivity to both tyrosine hydroxylase and NPY. Both the overall size of the dendritic tree and the number of primary dendrites were greater in neurons from the thoracic and celiac ganglia compared with those from the superior cervical ganglion. The electrophysiological properties of sympathetic neurons depended more on their ganglion of origin rather than their probable targets. All neurons in the superior cervical ganglion had phasic firing properties and large afterhyperpolarizations (AHPs). In addition, 34% of these neurons displayed an afterdepolarization preceding the AHP. Superior cervical ganglion neurons had prominent I M, I A, and I Hcurrents and a linear current-voltage relationship between −60 and −110 mV. Neurons from the thoracic ganglia had significantly smaller action potentials, AHPs, and apparent cell capacitance compared with superior cervical ganglion neurons, and only 18% showed an afterdepolarization. All neurons in superior cervical ganglia and most neurons in celiac ganglia received at least one strong preganglionic input. Nearly one-half the neurons in the celiac ganglion had tonic firing properties, and another 15% had firing properties intermediate between those of tonic and phasic neurons. Most celiac neurons showed significant inward rectification below −90 mV. They also expressed I A, but with slower inactivation kinetics than that of superior cervical or thoracic neurons. Both phasic and tonic celiac ganglion neurons received synaptic inputs via the celiac nerves in addition to strong inputs via the splanchnic nerves. Multivariate statistical analysis revealed that the properties of the action potential, the AHP, and the apparent cell capacitance together were sufficient to correctly classify 80% of neurons according to their ganglion of origin. These results indicate that there is considerable heterogeneity in the morphological, neurochemical, and electrical properties of sympathetic neurons in mice. Although the morphological and neurochemical characteristics of the neurons are likely to be related to their peripheral projections, the expression of particular electrophysiological traits seems to be more closely related to the ganglia within which the neurons occur.

scholarly journals Differential gene expression in populations of sympathetic neurons purified from a neuropeptide Y reporter mouse

2017 ◽  
Author(s):  
Kristine M. Sikora ◽  
Paul H. M. Kullmann ◽  
John P. Horn
Keyword(s):  
Neuropeptide Y ◽  
Candidate Genes ◽  
Fluorescent Protein ◽  
Sympathetic Neurons ◽  
Quantitative Polymerase Chain Reaction ◽  
Promoter Sequences ◽  
Reporter Mouse ◽  
Cervical Ganglion ◽  
Differential Gene ◽  
Green Fluorescent

AbstractUsing gene chip methodology, we identified candidate genes differentially expressed by vasomotor and non-vasomotor sympathetic neurons in the superior cervical ganglion. Groups of 10 neurons were manually sorted after isolation from a transgenic neuropeptide Y (NPY) reporter mouse that expresses the humanized Renilla green fluorescent protein (GFP) under control of Npy promoter sequences. Anatomical analysis of GFP and NPY co-expression showed that 98% of GFP-positive neurons and 16% of GFP-negative neurons express NPY. The probability of contamination in sorted cell samples was therefore high. To minimize this problem, we screened amplified cDNA samples by the quantitative polymerase chain reaction. This approach identified 101 candidate genes preferentially expressed by GFP-positive neurons and 74 candidate genes preferentially expressed by GFP-negative neurons.

Postnatal rat sympathetic neurons in culture. I. A comparison with embryonic neurons

1979 ◽  
Vol 42 (5) ◽  
pp. 1410-1425 ◽  
Author(s):  
E. Wakshull ◽  
M. I. Johnson ◽  
H. Burton
Keyword(s):  
Acetylcholine Receptors ◽  
Sympathetic Neurons ◽  
Gamma Aminobutyric Acid ◽  
Potential Amplitude ◽  
Cervical Ganglion ◽  
Dissociated Cell Culture ◽  
Ganglion Neurons ◽  
Embryonic Neurons

1. A morphological and physiological comparison was made between embryonically and postnatally derived superior cervical ganglion neurons (SCGN) grown in dissociated cell culture. It was found that while morphologically distinct, the physiological properties of the postnatal neurons were the same as their embryonic counterparts. 2. Intracellular injection of horseradish peroxidase (HPR) demonstrated that SCGN from any age of animal elaborated two basic types of processes, although the pattern of process ramification was unique for each neuron. The two types of proceses were 1) the large, smooth, rapidly tapering; and 2) the thin, nontapering variety, which often contained varicosities along its length. It is suggested that the former are dendritic in function, while the latter act as axons. 3. A difference was noted in somal size and the number of primary processes extended by the embryonic and postnatal neurons, with the latter more closely resembling the in vivo morphology. 4. Resting potentials and action-potential amplitudes of postnatal SCGN were comparable to those found previously for embryonic SCGN in vitro. 5. Iontophoretic application of putative neurotransmitter substances revealed the presence of acetylcholine receptors (AChR) on both embryonic and postnatal SCGN. Picrotoxin-sensitive depolarizing responses to iontophoresed gamma-aminobutyric acid (GABA) was seen on a few embryonic neurons, but not on the older cells. No responses were detected when norepinephrine (NE), glutamate, cAMP, substance P, or dopamine were applied to the SCGN of either age group. 6. Synatpic interaction between postnatal SCGN were found at an earlier in vitro age (12 days) than was the case for embryonic neurons (20 days). 7. Synaptic transmission was found to be chemical in nature. This was shown by 1) a dependence on external Ca2+ concentrations; 2) steplike fluctuations in synpatic potential amplitude, and 3) a variation in potential amplitude with changes in membrane potential. 8. It is concluded that the postnatal SCGN are able to survive in culture even when taken from animals up to 12.5 wk old. The elaboration of processes is in many ways strikingly similar to sympathetic neurons in the animal, and they are able to form functional synaptic interactions.

Postnatal rat sympathetic neurons in culture. II. Synaptic transmission by postnatal neurons

1979 ◽  
Vol 42 (5) ◽  
pp. 1426-1436 ◽  
Author(s):  
E. Wakshull ◽  
M. I. Johnson ◽  
H. Burton
Keyword(s):  
Skeletal Muscle ◽  
Synaptic Transmission ◽  
Sympathetic Neurons ◽  
Preceding Paper ◽  
Blocking Agent ◽  
Culture Conditions ◽  
Cervical Ganglion ◽  
Dissociated Cell Culture ◽  
Ganglion Neurons ◽  
Neurotransmitter Plasticity

1. It was shown in the preceding paper that postnatally derived rat superior cervical ganglion neurons (SCGN) will grow in dissociated cell culture and form functional synaptic connections with each other. In this report, synaptic transmission by the postnatal SCGN is detailed. 2. Synaptic interactions between SCGN were blocked by the nicotinic cholinergic antagonist hexamathonium (C-6), indicating that acetylcholine was the transmitter substance used by these neurons. This was found to be the case even for neurons taken from 12.5-wk-old animals. 3. In a few cases, the beta-adrenergic blocking agent, propranolol, was found to block synaptic potentials, suggesting that a catecholamine might be involved in the transmission process. The possible mechanisms of this involvement are discussed. 4. SCGN taken from up to 10-wk-old rats were able to form functional synaptic contacts with cocultured skeletal muscle cells. These interactions were sensitive to low external Ca2+ and to 1--2 microM d-tubocurarine (d-TC). 5. It is concluded that even adult SCGN retain a certain amount of neurotransmitter "plasticity" when grown under appropriate culture conditions. From the data on the neuron-neuron and SCGN-skeletal muscle interactions, it is suggested that a matching of presynaptic transmitter with postsynaptic receptor is a sufficient condition for the formation of functional nerve-target interactions.

Primary and adaptive changes of A-type K+ currents in sympathetic neurons from hypertensive rats

1999 ◽  
Vol 276 (6) ◽  
pp. R1758-R1765 ◽  
Author(s):  
Walter P. Robertson ◽  
Geoffrey G. Schofield
Keyword(s):  
Current Density ◽  
Sympathetic Neurons ◽  
Activation Parameters ◽  
Cell Voltage ◽  
Spontaneously Hypertensive ◽  
Wky Rats ◽  
Type K ◽  
Cervical Ganglion ◽  
Wistar Kyoto ◽  
Ganglion Neurons

The A-type K+ current ( I A) of superior cervical ganglion neurons acutely isolated from spontaneously hypertensive (SHR) and age-matched Wistar-Kyoto (WKY) rats was compared under whole cell voltage clamp. Activation parameters were similar in each strain. Steady-state inactivation was shifted approximately −6 mV in SHR, where one-half inactivation occurred at −81 mV vs. −75 mV in WKY rats. The shift was not present in prehypertensive SHR but remained in adult enalapril-treated SHR and, therefore, may represent a primary alteration of I A properties. I A amplitudes evoked from physiological potentials were similar, despite inactivation of a greater fraction of the current in the SHR. Comparing maximal I A densities revealed that current density is elevated in the SHR, which compensates for the inactivation shift. Current density decreased with age in WKY neurons but did not significantly decline in SHR neurons unless hypertension was prevented with enalapril. Thus adult SHR neurons may retain a high I Adensity as an adaptive response to offset potential hyperexcitability resulting from the hyperpolarized I A inactivation.

Modulation of Ca2+ Channels by Heterologously Expressed Wild-Type and Mutant Human μ-Opioid Receptors (hMORs) Containing the A118G Single-Nucleotide Polymorphism

2007 ◽  
Vol 97 (2) ◽  
pp. 1058-1067 ◽  
Author(s):  
Wojciech Margas ◽  
Ira Zubkoff ◽  
H. Gregg Schuler ◽  
Piotr K. Janicki ◽  
Victor Ruiz-Velasco
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Rank Order ◽  
Sympathetic Neurons ◽  
Signal Transduction Pathway ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Patch Clamp Technique ◽  
Single Nucleotide ◽  
Cervical Ganglion ◽  
Ganglion Neurons

The most common single-nucleotide polymorphism (SNP) of the human μ-opioid receptor (hMOR) gene occurs at position 118 (A118G) and results in substitution of asparagine to aspartate at the N-terminus. The purpose of the present study was to compare the pharmacological profile of several opioid agonists to heterologously expressed hMOR and N-type Ca2+ channels in sympathetic neurons. cDNA constructs coding for wild-type and mutant hMOR were microinjected in rat superior cervical ganglion neurons and N-type Ca2+ channel modulation was investigated using the whole cell variant of the patch-clamp technique. Concentration–response relationships were generated with the following selective MOR agonists: DAMGO, morphine, morphine-6-glucuronide (M-6-G), and endomorphin I. The estimated maximal inhibition for the agonists ranged from 52 to 64% for neurons expressing either hMOR subtype. The rank order of potencies for estimated EC50 values (nM) in cells expressing wild-type hMOR was: DAMGO (31) ≫ morphine (76) ≅ M-6-G (77) ≅ endomorphin I (86). On the other hand, the rank order in mutant-expressing neurons was: DAMGO (14) ≫ morphine (39) ≫ endomorphin I (74) ≅ M-6-G (82), with a twofold leftward shift for both DAMGO and morphine. The DAMGO-mediated Ca2+ current inhibition was abolished by the selective MOR blocker, CTAP, and by pertussis toxin pretreatment of neurons expressing either hMOR subtype. These results suggest that the A118G variant MOR exhibits an altered signal transduction pathway and may help explain the variability of responses to opiates observed with carriers of the mutant allele.

scholarly journals Generation and validation of homozygous fluorescent knock-in cells using CRISPR/Cas9 genome editing

2017 ◽  
Author(s):  
Birgit Koch ◽  
Bianca Nijmeijer ◽  
Moritz Kueblbeck ◽  
Yin Cai ◽  
Nike Walther ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Cell Lines ◽  
Blot Analysis ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Cell Clone ◽  
Dynamic Properties ◽  
Functional Expression ◽  
Fluorescent Markers ◽  
Target Effects

AbstractGene tagging with fluorescent proteins is essential to investigate the dynamic properties of cellular proteins. CRISPR/Cas9 technology is a powerful tool for inserting fluorescent markers into all alleles of the gene of interest (GOI) and permits functionality and physiological expression of the fusion protein. It is essential to evaluate such genome-edited cell lines carefully in order to preclude off-target effects caused by either (i) incorrect insertion of the fluorescent protein, (ii) perturbation of the fusion protein by the fluorescent proteins or (iii) non-specific genomic DNA damage by CRISPR/Cas9. In this protocol1, we provide a step-by-step description of our systematic pipeline to generate and validate homozygous fluorescent knock-in cell lines.We have used the paired Cas9D10A nickase approach to efficiently insert tags into specific genomic loci via homology-directed repair with minimal off-target effects. It is time- and cost-consuming to perform whole genome sequencing of each cell clone. Therefore, we have developed an efficient validation pipeline of the generated cell lines consisting of junction PCR, Southern Blot analysis, Sanger sequencing, microscopy, Western blot analysis and live cell imaging for cell cycle dynamics. This protocol takes between 6-9 weeks. Using this protocol, up to 70% of the targeted genes can be tagged homozygously with fluorescent proteins and result in physiological levels and phenotypically functional expression of the fusion proteins.Editorial SummaryThis protocol provides a detailed workflow describing how to insert fluorescent markers into all alleles of a gene of interest using CRISPR/Cas 9 technology and how to generate and validate homozygous fluorescent knock-in cell lines.

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