Subtypes of dorsal root ganglion neurons based on different inward currents as measured by whole-cell voltage clamp

1989 ◽  
Vol 80 (1-2) ◽  
Author(s):  
MichaelJ. McLean ◽  
PaulB. Bennett ◽  
RonaldM. Thomas
Keyword(s):  
Dorsal Root Ganglion ◽  
Voltage Clamp ◽  
Dorsal Root ◽  
Cell Voltage ◽  
Dorsal Root Ganglion Neurons ◽  
Whole Cell ◽  
Whole Cell Voltage Clamp ◽  
Inward Currents ◽  
Ganglion Neurons

GABA-Induced Cl− Current in Cultured Embryonic Human Dorsal Root Ganglion Neurons

1999 ◽  
Vol 82 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Alexander Y. Valeyev ◽  
John C. Hackman ◽  
Alice M. Holohean ◽  
Patrick M. Wood ◽  
Jennifer L. Katz ◽  
...  
Keyword(s):  
Dorsal Root Ganglion ◽  
Dorsal Root ◽  
Single Channel ◽  
Dorsal Root Ganglion Neurons ◽  
Hill Coefficient ◽  
Equilibrium Potential ◽  
Single Channels ◽  
Drg Neurons ◽  
Whole Cell ◽  
Ganglion Neurons

γ-Aminobutyric acid (GABA)-activated channels in embryonic (5–8 wk old) human dorsal root ganglion (DRG) neurons in dissociated culture were characterized by whole cell and single-channel techniques. All DRG neurons when held at negative holding membrane potentials displayed inward current to micromolar concentrations of GABA applied by pressure pulses from closely positioned micropipettes. The current was directly proportional to the concentration of GABA (EC50, 111 μM; Hill coefficient, 1.7). DRG neurons also responded to micromolar concentrations of pentobarbital and alphaxalone but not to cis-4-aminocrotonic acid (CACA), glycine, or taurine. Baclofen (100 μM) affected neither the holding currents nor K+ conductance (when patch pipettes were filled with 130 mM KCl) caused by depolarizing pulses. Whole cell GABA-currents were blocked by bicuculline, picrotoxin, and t-butylbicyclophosphorothionate (TBPS; all at 100 μM). The reversal potential of whole cell GABA-currents was close to the theoretical Cl− equilibrium potential, shifting with changes in intracellular Cl− concentration in a manner expected for Cl−-selective channels. The whole cell I-V curve for GABA-induced currents demonstrated slight outward rectification with nearly symmetrical outside and inside Cl− concentrations. Spectral analysis of GABA-induced membrane current fluctuations showed that the kinetic components were best fitted by a triple Lorentzian function. The apparent elementary conductance for GABA-activated Cl− channels determined from the power spectra was 22.6 pS. Single-channel recordings from cell-attached patches with pipettes containing 10 μM GABA indicated that GABA-activated channels have a main and a subconductance level with values of 30 and 19 pS, respectively. Mean open and closed times of the channel were characterized by two or three exponential decay functions, suggesting two or three open channel states and two closed states. Single channels showed a lack of rectification. The actions of GABA on cultured human embryonic DRG neurons are mediated through the activation of GABAA receptors with properties corresponding to those found in the CNS of human and other mammalian species but differing from those of cultured human adult DRG neurons.

Electrophysiological Analysis of Dorsal Root Ganglion Neurons Pre- and Post-Coexpression of Green Fluorescent Protein and Functional 5-HT3Receptor

1997 ◽  
Vol 77 (6) ◽  
pp. 3115-3121 ◽  
Author(s):  
George M. Smith ◽  
Richard L. Berry ◽  
Jay Yang ◽  
Darrell Tanelian
Keyword(s):  
Green Fluorescent Protein ◽  
Dorsal Root Ganglion ◽  
Dorsal Root ◽  
Fluorescent Protein ◽  
Dorsal Root Ganglion Neurons ◽  
Drg Neurons ◽  
Whole Cell ◽  
Electrophysiological Analysis ◽  
Green Fluorescent ◽  
Ganglion Neurons

Smith, George M., Richard L. Berry, Jay Yang, and Darrell Tanelian. Electrophysiological analysis of dorsal root ganglion neurons pre- and post-coexpression of green fluorescent protein and functional 5-HT3receptor. J. Neurophysiol. 77: 3115–3121, 1997. Aequorea green fluorescent protein (GFP) is an excellent marker to examine genetically altered live cells in whole animals or culture. Its potential use in identifying genetically modified neurons, however, has not been investigated extensively. To examine the usefulness, toxicity, and potential electrophyiological effects of GFP expression in neurons, we generated adenovirus containing the mGFP4 cDNA. One week after virus transfection of dorsal root ganglion neurons (DRG), 10% of postnatal DRG neurons appeared brightly fluorescent, labelling the soma and neurites. Temporal examination of these neurons demonstrated no toxicity to DRG neurons even after several weeks in culture with repeated daily epifluorescent exposure. Electrophysiological analysis and comparison of control and viral exposed (GFP− and GFP+) DRG neurons did not demonstrate any differences in whole cell resistance, resting potential, action potential (AP) threshold, AP duration, AP amplitude, or whole cell capacitance. To investigate the usefulness of GFP as a marker for identifying neurons genetically altered to express a novel neurotransmitter receptor, a second adenovirus construct was generated containing both GFP and serotonin type 3 (5-HT3) receptor cDNAs. Transfection of DRG neurons with this virus produced an inward current in the presence of serotonin only in DRG neurons that were GFP-positive. It is concluded that adenoviral transfection of neurons with GFP, for cellular labeling, and coexpression of GFP-neurotransmitter constructs are safe, nontoxic, methods for electrophysiologically investigating neurons over several weeks. The uniqueness of the vector used in these experiments is that it was constructed to express GFP in a second cassette so that it would label the transduced cells, but have no potential for interfering with the function of the foreign 5-HT3receptor.

Activation of Ca2+-Dependent Currents in Dorsal Root Ganglion Neurons by Metabotropic Glutamate Receptors and Cyclic ADP-Ribose Precursors

1997 ◽  
Vol 77 (5) ◽  
pp. 2573-2584 ◽  
Author(s):  
Jane H. Crawford ◽  
John F. Wootton ◽  
Guy R. Seabrook ◽  
Roderick H. Scott
Keyword(s):  
Dorsal Root Ganglion ◽  
Dorsal Root ◽  
Metabotropic Glutamate Receptors ◽  
Metabotropic Glutamate Receptor ◽  
Receptor Activation ◽  
Dorsal Root Ganglion Neurons ◽  
Metabotropic Glutamate ◽  
Intracellular Ca ◽  
Inward Currents ◽  
Ganglion Neurons

Crawford, Jane H., John F. Wootton, Guy R. Seabrook, and Roderick H. Scott. Activation of Ca2+-dependent currents in dorsal root ganglion neurons by metabotropic glutamate receptors and cyclic ADP-ribose precursors. J. Neurophysiol. 77: 2573–2584, 1997. Cultured dorsal root ganglion neurons were voltage clamped at −90 mV to study the effects of intracellular application of nicotinamide adenine dinucleotide (βNAD+), intracellular flash photolysis of caged 3′,5′-cyclic guanosine monophosphate (cGMP), and metabotropic glutamate receptor activation. The activation of metabotropic glutamate receptors evoked inward Ca2+-dependent currents in most cells. This was mimicked both by intracellular flash photolysis of the caged axial isomer of cGMP [P-1-(2-nitrophenyl)ethyl cGMP] and intracellular application of βNAD+. Whole cell Ca2+-activated inward currents were used as a physiological index of raised intracellular Ca2+ levels. Extracellular application of 10 μM glutamate evoked the activation of Ca2+-dependent inward currents, thus reflecting a rise in intracellular Ca2+ levels. Similar inward currents were also activated after isolation of metabotropic glutamate receptor activation by application of 10 μM glutamate in the presence of 20 μM 6-cyano-7-nitroquinoxaline-2,3-dione and 20 μM dizocilpine maleate (MK 801), or by extracellular application of 10 μM trans-(1 S,3 R)-1-amino-1,3-cyclopentanedicarboxylic acid. Intracellular photorelease of cGMP, from its caged axial isomer, in the presence of βNAD+ was also able to evoke similar Ca2+-dependent inward currents. Intracellular application of βNAD+ alone produced a concentration-dependent effect on inward current activity. Responses to both metabotropic glutamate receptor activation and cGMP were suppressed by intracellular ryanodine, chelation of intracellular Ca2+ by bis-( o-aminophenoxy)- N,N,N′,N′-tetraacetic acid, and depletion of intracellular Ca2+ stores, but were insensitive to the removal of extracellular Ca2+. Therefore both cGMP, possibly via a mechanism that involves βNAD+ and/or cyclic ADP-ribose, and glutamate can mobilize intracellular Ca2+ from ryanodine-sensitive stores in sensory neurons.

Pharmacologically novel GABA receptor in human dorsal root ganglion neurons

1996 ◽  
Vol 76 (5) ◽  
pp. 3555-3558 ◽  
Author(s):  
A. Y. Valeyev ◽  
J. C. Hackman ◽  
P. M. Wood ◽  
R. A. Davidoff
Keyword(s):  
Dorsal Root Ganglion ◽  
Gaba Receptors ◽  
Dorsal Root ◽  
Glycine Receptor ◽  
Cell Voltage ◽  
Dorsal Root Ganglion Neurons ◽  
Gamma Aminobutyric Acid ◽  
Drg Neurons ◽  
Adult Rat ◽  
Ganglion Neurons

1. Whole cell voltage-clamp studies of gamma-aminobutyric acid (GABA) receptors were performed on large (> 80 microns) cultured human dorsal root ganglion (DRG) neurons. 2. GABA and pentobarbital sodium when applied in micromolar concentrations evoked inward Cl- currents in DRG neurons voltage clamped at negative membrane potentials. 3. Diazepam (10 microM) and pentobarbital (10 microM) upmodulated the GABA current by approximately 149 and 168%, respectively. 4. The GABA currents in human DRG cells were unaffected by the classical GABA antagonists picrotoxin and bicuclline (100 microM). In contrast, the GABA responses evoked in adult rat DRG cells cultured in an identical manner were inhibited by both antagonists. The glycine receptor antagonist strychnine (100 microM) did not alter GABA currents in human DRG cells. 5. Human DRG cells did not respond to glycine (10–100 microM) or taurine (10–100 microM). The GABAB agonist baclofen had no effect on the holding current when patch pipettes were filled with 130 mM KCl. The GABAB antagonists saclofen applied either alone or with GABA was without effect. 6. The differences between the GABA receptors described here and GABA receptors in other species may reflect the presence of receptor subunits unique to human DRG cells.

scholarly journals Altered Purinergic Signaling in Colorectal Dorsal Root Ganglion Neurons Contributes to Colorectal Hypersensitivity

2010 ◽  
Vol 104 (6) ◽  
pp. 3113-3123 ◽  
Author(s):  
Masamichi Shinoda ◽  
Jun-Ho La ◽  
Klaus Bielefeldt ◽  
G. F. Gebhart
Keyword(s):  
Dorsal Root Ganglion ◽  
Dorsal Root ◽  
Structural Changes ◽  
Purinergic Signaling ◽  
Dorsal Root Ganglion Neurons ◽  
Kinetic Properties ◽  
Membrane Excitability ◽  
Drg Neurons ◽  
Whole Cell ◽  
Ganglion Neurons

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by pain and hypersensitivity in the relative absence of colon inflammation or structural changes. To assess the role of P2X receptors expressed in colorectal dorsal root ganglion (c-DRG) neurons and colon hypersensitivity, we studied excitability and purinergic signaling of retrogradely labeled mouse thoracolumbar (TL) and lumbosacral (LS) c-DRG neurons after intracolonic treatment with saline or zymosan (which reproduces 2 major features of IBS—persistent colorectal hypersensitivity without inflammation) using patch-clamp, immunohistochemical, and RT-PCR techniques. Although whole cell capacitances did not differ between LS and TL c-DRG neurons and were not changed after zymosan treatment, membrane excitability was increased in LS and TL c-DRG neurons from zymosan-treated mice. Purinergic agonist adenosine-5′-triphosphate (ATP) and α,β-methylene ATP [α,β-meATP] produced inward currents in TL c-DRG neurons were predominantly P2X3-like fast (∼70% of responsive neurons); P2X2/3-like slow currents were more common in LS c-DRG neurons (∼35% of responsive neurons). Transient currents were not produced by either agonist in c-DRG neurons from P2X3−/− mice. Neither total whole cell Kv current density nor the sustained or transient Kv components was changed in c-DRG neurons after zymosan treatment. The number of cells expressing P2X3 protein and its mRNA and the kinetic properties of ATP- and α,β-meATP-evoked currents in c-DRG neurons were not changed by zymosan treatment. However, the EC50 of α,β-meATP for the fast current decreased significantly in TL c-DRG neurons. These findings suggest that colorectal hypersensitivity produced by intracolonic zymosan increases excitability and enhances purinergic signaling in c-DRG neurons.

Effect of Intracellular Dialysis of ATP on the Hyperpolarization-Activated Cation Current in Rat Dorsal Root Ganglion Neurons

2003 ◽  
Vol 90 (4) ◽  
pp. 2115-2122 ◽  
Author(s):  
You Komagiri ◽  
Naoki Kitamura
Keyword(s):  
Dorsal Root Ganglion ◽  
Adenylate Cyclase ◽  
Voltage Clamp ◽  
Dorsal Root ◽  
Voltage Dependence ◽  
Dorsal Root Ganglion Neurons ◽  
Drg Neurons ◽  
Positive Direction ◽  
Cation Current ◽  
Ganglion Neurons

The mechanism of the effect of intracellular ATP on the hyperpolarization-activated non-selective cation current ( Ih) in rat dorsal root ganglion neurons was investigated using a whole cell voltage-clamp technique. Under voltage-clamp conditions, Ih was activated by hyperpolarizing pulses raised to a voltage of between –70 and –130 mV. The activation curve of Ih in rat dorsal root ganglion (DRG) neurons shifted by about 15 mV in the positive direction with an intracellular solution containing 1 mM cAMP. When ATP (2 mM) was applied intracellularly, the half-maximal activation voltage ( Vhalf) of Ih shifted from –97.4 ± 1.9 to –86.8 ± 1.6 mV, resulting in an increase in the current amplitude of Ih by the pulse to between –80 and –90 mV. In the presence of an adenylate cyclase inhibitor, SQ-22536 (100 μM), the intracellular dialysis of ATP also produced a shift in the voltage-dependence of Ih in rat DRG neurons, indicating that the effect of ATP was not caused by cAMP converted by adenylate cyclase. Intracellular dialysis of a nonhydrolysable ATP analog, AMP-PNP or ATP-γ-S, also produced a positive shift in the voltage-dependence of Ih activation, suggesting that the effect of ATP results from its direct action on the channel protein. These results indicate that cytosolic ATP directly regulates the voltage dependence of Ih activation as an intracellular modulating factor.

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