ABSTRACT
Growth differentiation factor 15 (GDF-15) is a member of the transforming growth factor-β superfamily. It is widely distributed in the central and peripheral nervous systems. Whether and how GDF-15 modulates nociceptive signaling remains unclear. Behaviorally, we found that peripheral GDF-15 significantly elevated nociceptive response thresholds to mechanical and thermal stimuli in naïve and arthritic rats. Electrophysiologically, we demonstrated that GDF-15 decreased the excitability of small-diameter dorsal root ganglia (DRG) neurons. Furthermore, GDF-15 concentration-dependently suppressed tetrodotoxin-resistant sodium channel Nav1.8 currents, and shifted the steady-state inactivation curves of Nav1.8 in a hyperpolarizing direction. GDF-15 also reduced window currents and slowed down the recovery rate of Nav1.8 channels, suggesting that GDF-15 accelerated inactivation and slowed recovery of the channel. Immunohistochemistry results showed that activin receptor-like kinase-2 (ALK2) was widely expressed in DRG medium- and small-diameter neurons, and some of them were Nav1.8-positive. Blockade of ALK2 prevented the GDF-15-induced inhibition of Nav1.8 currents and nociceptive behaviors. Inhibition of PKA and ERK, but not PKC, blocked the inhibitory effect of GDF-15 on Nav1.8 currents. These results suggest a functional link between GDF-15 and Nav1.8 in DRG neurons via ALK2 receptors and PKA associated with MEK/ERK, which mediate the peripheral analgesia of GDF-15.
ABSTRACT
Growth differentiation factor 15 (GDF-15) is a member of the transforming growth factor-β superfamily. It is widely distributed in the central and peripheral nervous systems. Whether and how GDF-15 modulates nociceptive signaling remains unclear. Behaviorally, we found that peripheral GDF-15 significantly elevated nociceptive response thresholds to mechanical and thermal stimuli in naïve and arthritic rats. Electrophysiologically, we demonstrated that GDF-15 decreased the excitability of small-diameter dorsal root ganglia (DRG) neurons. Furthermore, GDF-15 concentration-dependently suppressed tetrodotoxin-resistant sodium channel Nav1.8 currents, and shifted the steady-state inactivation curves of Nav1.8 in a hyperpolarizing direction. GDF-15 also reduced window currents and slowed down the recovery rate of Nav1.8 channels, suggesting that GDF-15 accelerated inactivation and slowed recovery of the channel. Immunohistochemistry results showed that activin receptor-like kinase-2 (ALK2) was widely expressed in DRG medium- and small-diameter neurons, and some of them were Nav1.8-positive. Blockade of ALK2 prevented the GDF-15-induced inhibition of Nav1.8 currents and nociceptive behaviors. Inhibition of PKA and ERK, but not PKC, blocked the inhibitory effect of GDF-15 on Nav1.8 currents. These results suggest a functional link between GDF-15 and Nav1.8 in DRG neurons via ALK2 receptors and PKA associated with MEK/ERK, which mediate the peripheral analgesia of GDF-15.
Subject(s)
Animals , Rats , Analgesia , Ganglia, Spinal , Growth Differentiation Factor 15 , Sensory Receptor Cells , Sodium Channels , Tetrodotoxin/pharmacologyABSTRACT
@#AIM: To investigate the morphology and electrophysiological properties of neurons in the inner nuclear layer(INL)of the adult rat retina slices. The retinal slices were prepared by low-melt agarose embedding and then cutted by vibratome. <p>METHODS: Whole cell patch clamp and intracellular staining with Lucifer Yellow were used in this study to study the morphology and electrophysiological properties of the INL neurons in retinal slices.<p>RESULTS: Retinal slices prepared in this method possessed a very smooth surface. The cells on the retinal slices maitained very good vitality, and some of the cells even retained their dendritic connections with other cells on the slice. According to the size and location of the cell bodies, neurons in the INL were easy to differentiate. Luciifer Yellow contained in the intracellular solution revealed the morphology of the recorded cell very well. Bipolar cells possessed elongated cell bodies and their processes mainly extended along the vertical direction. Horizontal cells and amacrine cells possessed much bigger and round cell bodies, resided in the outermost and inner most of the INL, respectively. The rest membrane potential and membrane capacitance of horizontal cell and amacrine cell were much higher than that of bipolar cells. Under a voltage step from -60mV to +40mV, 10mV per step, 41.7% of the cone bipolar cells and 64.7% of the amacrine cells exhibited inward sodium current and outward potassium current. Other cells only possessed outward potassium current. <p>CONCLUSION: The method of preparing retinal slices was very simple, and the viability of the slices were stable. These facilitated the patch-clamp recording of all the neurons in the INL including horizontal cells. Further investigation of the electrophysiological properties of the neurons in the INL was essential in revealing the mechanism of vision.
ABSTRACT
Vision formation is classically based on projections from retinal ganglion cells (RGC) to the lateral geniculate nucleus (LGN) and the primary visual cortex (V1). Neurons in the mouse V1 are tuned to light stimuli. Although the cellular information of the retina and the LGN has been widely studied, the transcriptome profiles of single light-stimulated neuron in V1 remain unknown. In our study, in vivo calcium imaging and whole-cell electrophysiological patch-clamp recording were utilized to identify 53 individual cells from layer 2/3 of V1 as light-sensitive (LS) or non-light-sensitive (NS) by single-cell light-evoked calcium evaluation and action potential spiking. The contents of each cell after functional tests were aspirated in vivo through a patch-clamp pipette for mRNA sequencing. Moreover, the three-dimensional (3-D) morphological characterizations of the neurons were reconstructed in a live mouse after the whole-cell recordings. Our sequencing results indicated that V1 neurons with a high expression of genes related to transmission regulation, such as Rtn4r and Rgs7, and genes involved in membrane transport, such as Na/K ATPase and NMDA-type glutamatergic receptors, preferentially responded to light stimulation. Furthermore, an antagonist that blocks Rtn4r signals could inactivate the neuronal responses to light stimulation in live mice. In conclusion, our findings of the vivo-seq analysis indicate the key role of the strength of synaptic transmission possesses neurons in V1 of light sensory.
ABSTRACT
Vision formation is classically based on projections from retinal ganglion cells (RGC) to the lateral geniculate nucleus (LGN) and the primary visual cortex (V1). Neurons in the mouse V1 are tuned to light stimuli. Although the cellular information of the retina and the LGN has been widely studied, the transcriptome profiles of single light-stimulated neuron in V1 remain unknown. In our study, in vivo calcium imaging and whole-cell electrophysiological patch-clamp recording were utilized to identify 53 individual cells from layer 2/3 of V1 as light-sensitive (LS) or non-light-sensitive (NS) by single-cell light-evoked calcium evaluation and action potential spiking. The contents of each cell after functional tests were aspirated in vivo through a patch-clamp pipette for mRNA sequencing. Moreover, the three-dimensional (3-D) morphological characterizations of the neurons were reconstructed in a live mouse after the whole-cell recordings. Our sequencing results indicated that V1 neurons with a high expression of genes related to transmission regulation, such as Rtn4r and Rgs7, and genes involved in membrane transport, such as Na/K ATPase and NMDA-type glutamatergic receptors, preferentially responded to light stimulation. Furthermore, an antagonist that blocks Rtn4r signals could inactivate the neuronal responses to light stimulation in live mice. In conclusion, our findings of the vivo-seq analysis indicate the key role of the strength of synaptic transmission possesses neurons in V1 of light sensory.
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Objective To investigate the effects of BKCa channel on electrophysiology excitatory regulation in MVN neuron following hypoxia and to reveal its molecular mechanism.Methods C57BL/6 mices were performed MVNs hypoxia mice model,and randomly allowed to normal oxgen group and hypoxia group.The hypoxia group, according to the application of NS1 6 1 9 ,was further divided into the no NS1 6 1 9 pretreatment group and NS1 6 1 9 pre-treatment group.Using the patch clamp experiment technology,we recorded the effects of the MVN abnormal neu-ronal firing and the change of the BKCa currents.Using immunohistochemical technique,the changes of BKCa in the hypoxic MVNs detected were.Results Acute hypoxia increased neuronal activities.NS1619 pretreatment de-creased hypoxia-induced firing rate,and increased and postponed the maximum increase by hypoxia(P<0.05),al-so alleviated 10-min-hypoxia-induced depolarization(P<0.05).Perfusion with hypoxic significantly reduced the BKCa positive neurons(P<0.05).Conclusion These findings suggest that acute hypoxia increases neuronal activi-ties.The decreased MVN BKCa channels contribute to hypoxia-induced abnormal neuronal activities.
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OBJECTlVE To investigate the action mechanism of antidepressant fluoxetine on hERG ( ether-a-go-go-related gene ) potassium channel, and the effect of protein kinase C ( PKC ) agonist phorbol-12-myristate-13-acetate ( PMA) on fluoxetine inhibition. METHODS The whole cell patch clamp technique was used to record the change in hERG potassium current ( IKr ) on HEK293 cells that stably expressed hERG potassium channel ( hERG-HEK293 steady-state cells) , which was treated with fluoxe-tine 0.01, 0.1, 1 and 10μmol·L-1 , to study the concentration-and voltage-dependence of the effects on IKr, and to observe the changes in activation, inactivation and recovery dynamics of hERG potassium channel treated with fluoxetine 1μmol·L-1 . On this basis, the effect of PMA of 1μmol·L-1 on inhibition of fluoxetine 1 μmol·L-1 was explored. RESULTS Fluoxetine 0.01, 0.1, 1 and 10 μmol·L-1 inhibited IKr on hERG-HEK293 steady-state cells in a concentration- and voltage-dependent manner. The half maximal inhibitory concentration ( lC50 ) was about 0. 8 mmol·L-1 , and the Hill coefficient was about 1. 1. Fluoxetine 1 μmol·L-1 could reduce the activation, deactivation and recovery currents of IKr and affect the activation and recovery of hERG potassium channel. After fluoxetine inhibition of IKr became stable, PMA 1 μmol·L-1 could inhibit the blocking effect of fluoxetine on hERG potassium channels. CONCLUSlON Fluoxetine has obvious inhibitory effect on IKr of hERG-HEK293 steady-state cells, but the effect could be inhibited by PKC agonist PMA.
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Objective To study the changes of N-methyl-D-aspartate(NMDA)-receptor-channels current in developing hippocampal neurones during hypoxia and effect of adenosine intervention.Methods Whole-cell patch-clamp techniques cultured hippocampal neurons whose cultured day were 6 days and 16 days respectively,the amplitude of the NMDA-receptor-channels currents of hippocampal neuron were determined.And the effect of hypoxia on the NMDA-receptor-channels current,and adenosine regulatory mechanisms in cultured hippocampal neurons were explored.Results During hypoxia,compared with control group,the amplitude of the NMDA-receptorchannels currents of hippocampal neuron whose cultured day was 6 days were significantly increased(P
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Objective To investigate the electrophysiological characteristics of neurons in developing visual cortex and to observe the reciprocal connection and the function of integration between adjacent neurons. Methods Whole cell recordings of the visual cortex from SD rats (4～28 d old) were obtained by using "blind" whole cell patch clamp recording technique. Bipolar stimulating electrodes were placed in the white border under the visual cortex. Postsynaptic currents(PSCs) were evoked by using stimulation of 0 1 Hz. Results There were three types of PSCs recorded in 156 cells in the first developmental month: irresponse, monosynaptic response and polysynaptic response. The incidence of polysynaptic PSCs increased from 12 36% before eye opening to 31 34% after eye opening ( P
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Objective To investigate the electrophysiological properties of neurons in visual cortex of binocular form-deprivated rats during the developmental stages and to explore the synaptic and cellular mechanisms of plasticity in the visual cortex. Methods Whole cell patch clamp technique was applied to the visual cortex slices prepared from 61 Wistar rats of both genders, which were divided into two groups; control group and binocular form-deprivation (BFD) group. Excitatory postsynaptic currents (EPSCs) and long term potentiation (LTP) of the neurons were recorded. Results (1) The percentage of silent response in BFD group was significantly higher than that in the control group(P
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Objective To study the membrane properties of rat retinal ganglion cells during postnatal development, including the passive membrane properties and the action potentials evoked by depolarizing current injections and the relationship between the membrane properties and ages. Methods Whole cell patch clamp recordings of the retinal ganglion cells (RGCs) in retinal slices of postnatal rats (age ranging from postnatal days 7 to 30) were performed. Results (1) A total of 112 RGCs were recorded from postnatal rats. (2) The electrophysiological properties of RGCs changed significantly during development. The excitability of RGCs increased in an age-dependent manner. There was significant difference between before-eye-opening group(P7-13d) and after-eye-opening group(P14-30d) . (3) Three different discharge patterns of RGCs in response to sustained depolarizing current pulses were recorded: single-spike firing, transient firing and sustained firing. During development, retinal ganglion cells of rats exhibited pronounced changes in the discharge patterns(P
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Objective To investigate whether the partially C-terminal deletion of NR2A subunit alters the surface expression and channel function of NMDA receptors in both HEK293 cells and cultured hippocampal neurons of rats. Methods Four plasmids for NR2A mutants with N-terminally GFP-tagged and C-terminal deletion NR2A?C1(?897L-1017S),NR2A?C2(?1024D-1142P),NR2A?C3(?1149D-1347G),and NR2A?C4(?1354S-1464V) were generated,and transfected into HEK293 cells and hippocampal neurons in culture.Surface staining was performed using anti-GFP antibody and Cy3 conjugated secondary antibody.Glutamate evoked currents were also detected using whole-cell recording. Results Positive surface staining was found for all the HEK293 cell co-transfected NR1-1a/NR2A?C1,NR1-1a/NR2A?C2,NR1-1a/NR2A?C3 or NR1-1a/NR2A?C4,and quantitative analysis showed no significant decrease in surface expression level when compared to that from NR1-1a/NR2A transfection.Glutamate-evoked currents were recorded in HEK293 cells co-transfected with NR1-1a/NR2A?C2 or NR1-1a/NR2A?C4.Surface expression level of NMDA receptor clusters on dendrites was significantly decreased in the neurons transfected with NR2A?C1,NR2A?C2 or NR2A?C3 than in those transfected with NR2A.Conclusion C-terminal deletion of NR2A subunit differentially effects surface expression of NMDA receptors in HEK293 cells and in hippocampal neurons in culture.
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To learn the developmental changes in intrinsic electrophysiological properties of the second order taste neurons, whole cell recordings from the developing nucleus of the solitary tract neurons were done in brainstem slices of postnatal rats. Rats aged from postnatal 0 to 21 days (P0-P21) were used, being divided into 3 age groups: postnatal first week (P0-P7 days), second week (P8-P14 days), and third week P15-P21 days). Slices containing gustatory NTS were cut horizontally in the thickness of 300 micrometer. Whole cell recordings were obtained from neurons in response to a series of hyperpolarizing and depolarizing current pulses. The intrinsic electrophysiological properties of the rostral NTS (rNTS) neurons were compared among the age groups. Depolarizing current pulses evoked a train of action potentials in all neurons of all age groups. The resting membrane potential and input resistance of the neurons did not show any significant differences during the ostnatal 3 weeks. The time constant, however, decreased during the development. Duration of action potential measured at half maximum amplitude was longer in younger age groups. Both the maximum rate of rise and the maximum rate of fall in the action potential increased during the first 3 weeks postnatal. Electrophysiologically more than half neurons were type III. In summary, it is suggested that developmental changes in electrophysiological properties in rNTS occur during the first three weeks in rats.
Subject(s)
Animals , Humans , Rats , Action Potentials , Brain Stem , Membrane Potentials , Neurons , Patch-Clamp Techniques , Solitary NucleusABSTRACT
Objective To study the changes of cell motility of outer hair cells in the cochlear of guinea pigs under treatment of sodium nitroprusside(SNP). Methods By using the whole cell patch clamp recording technique in normal external and internal cell solution, the cell motility of outer hair cells under stimulation of voltage was observed in different concentration of SNP. Results Sodium nitroprusside apparently inhibited outer hair cells' motility when SNP concentration was higher than 100 ?Mol/L ( P
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AIM:To investigate the inhibitory effect of dihydro-?-erythroidine on nicotine induced-current in cultured superior cervical ganglion of neonatal rats.METHODS:Pneumatic pressure administration of drug and whole-cell recording techniques were performed to compare induced-current amplitude.RESULTS:Dihydro-?-erythroidine competitively antagonized nicotinic effect,and EC50 was about 0.015mmol/L.CONCLUSION:Dihydro-?-erythroidine is a competitive antagonist of nicotinic receptors in sympathetic neurons.