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1.
Voltage-gated Ca2+ conductances were investigated with the whole-cell patch-clamp technique-either using Ca2+ or Ba2+ as charge carriers-in NB69 human neuroblastoma cells plated in “defined” serum-free (DM) and in “astroglial-conditioned” media (CM). Cells expressed the microtubule associated protein 1A when plated in both media, indicating neuronlike differentiation. Cells of similar sizes and shapes were selected for recordings. Different sets of voltage-gated Ca2+ current types were usually expressed in DM- and CM-plated cells. DM-plated cells exhibited a high-voltage-activated current (HVAC) in isolation, whereas 43% of the CM-plated cells also displayed the low-voltage-activated current (LVAC). The membrane surface density of the HVAC was about twofold higher in CM than in DM-plated cells and increased with plating time from 10 and 16pA/pF (days 1-4) to 24 and 37pA/pF (days 5-10) in DM- and CM-plated cells, respectively. However, the amplitude of the LVAC did not change significantly with culture age. In conclusion, NB69 cells expressed HVAC in isolation when plated in DM, whereas both HVAC and LVAC were present in many CM-plated cells, suggesting that the CM contained diffusible factors secreted by astroglial cells which: (1) could induce the appearance of the LVAC and (2) increased HVAC current expression. GLIA 20:70-78, 1997. © 1997 Wiley-Liss, Inc. 相似文献
2.
Whole cell patch-clamp techniques were used to study voltage-dependent sodium (Na+), calcium (Ca2+), and potassium (K+) conductances in acutely isolated neurons from cortical layer I of adult rats. Layer I cells were identified by means of γ-aminobutyric acid (GABA) immunocytochemistry. Positive stainings for the Ca2+-binding protein calretinin in a subset of cells, indicated the presence of Cajal–Retzius (C-R) cells. All investigated cells displayed a rather homogeneous profile of voltage-dependent membrane currents. A fast Na+ current activated at about −45 mV, was half-maximal steady-state inactivated at −66.6 mV, and recovery from inactivation followed a two-exponential process (τ1 = 8.4 ms and τ2 = 858.8 ms). Na+ currents declined rapidly with two voltage-dependent time constants, reaching baseline current after some tens of milliseconds. In a subset of cells (< 50%) a constant current level of < 65 pA remained at the end of a 90 ms step. A transient outward current (Ifast) activated ≈–40 mV, declined rapidly with a voltage-insensitive time constant (τ≈ 350 ms) and was relatively insensitive to tetraethylammonium (TEA, 20 mm ). Ifast was separated into two components based on their sensitivity to 4-aminopyridine (4-AP): one was blocked by low concentrations (40 μm ) and a second by high concentrations (6 mm ). After elimination of Ifast by a conditioning prepulse (50 ms to −50 mV), a slow K+ current (IKV) could be studied in isolation. IKV was only moderately affected by 4-AP (6 mm ), while TEA (20 mm ) blocked most (> 80%) of the current. IKV activated at about −40 mV, declined monoexponentially in a voltage-dependent manner (τ≈ 850 ms at −30 mV), and revealed an incomplete steady-state inactivation. In addition to Ifast and IKV, indications of a Ca2+-dependent outward current component were found. When Na+ currents, Ifast, and IKV were blocked by tetrodotoxin (TTX, 1 μm ), 4-AP (6 mm ) and TEA (20 mm ) an inward current carried by Ca2+ was found. Ca2+ currents activated at depolarized potentials at about −30 mV, were completely blocked by 50 μm cadmium (Cd2+), were sensitive to verapamil (≈ 40% block by 10 μm ), and were not affected by nickel (50 μm ). During current clamp recordings, isolated layer I neurons displayed fast spiking behaviour with short action potentials (≈ 2 ms, measured at half maximal amplitude) of relative small amplitude (≈ 83 mV, measured from the action potential threshold). 相似文献
3.
Action potential generation and Na+ currents were studied in B104 neuroblastoma cells in vitro using the whole-cell patch-clamp method in voltage-clamp and current-clamp mode. Action potential-like responses were elicited in 38 of 42 cells, with a threshold close to −55 mV for depolarizing stimuli, and −56 mV for anode-break stimuli. Response amplitudes were larger when cells were held at more negative prepulse potentials, and were well fit by a Boltzmann distribution with a midpoint of approx. −75 mV, close to theV1/2 for Na+ current steady-state inactivation in these cells. Cells displaying action potential-like responses exhibited a peak Na+ current density of 133 ± 0.14 pA/pF (range, 10.2–296.2 pA/pF) and a lowgK: gNa ratio (0.0067 ± 0.0023). Exposure to 0.1 mM Cd2+ did not block the generation of action potential-like responses in B104 cells, while 1 μM TTX abolished the responses. We conclude that low densities of Na+ channels ( < 3/μm2 and < 1/μm2 in some cells) can support the generation of action potential-like responses in B104 cells if they are held at hyperpolarized levels to remove inactivation. The low leak and K+ conductance of these cells may contribute to their ability to generate action potential-like responses under these circumstances. 相似文献
4.
Background Primary sensory neurons express several types of ion channels including transient receptor potential vanilloid 1 (TRPV1) and voltage‐gated Na+ channels. Our previous studies showed an increased excitability of bladder primary sensory and spinal neurons triggered by inflammation in the distal colon as a result of pelvic organ cross‐sensitization. The goal of this work was to determine the effects of TRPV1 receptor activation by potent agonists and/or colonic inflammation on voltage‐gated Na+ channels expressed in bladder sensory neurons. Methods Sprague–Dawley rats were treated with intracolonic saline (control), resiniferatoxin (RTX, 10?7 mol L?1), TNBS (colonic irritant) or double treatment (RTX followed by TNBS). Key Results TNBS‐induced colitis increased the amplitude of total Na+ current by two‐fold and of tetrodotoxin resistant (TTX‐R) Na+ current by 78% (P ≤ 0.05 to control) in lumbosacral bladder neurons during acute phase (3 days post‐TNBS). Instillation of RTX in the distal colon caused an enhancement in the amplitude of total Na+ current at ?20 mV from ?112.1 ± 18.7 pA/pF (control) to ?183.6 ± 27.8 pA/pF (3 days post‐RTX, P ≤ 0.05) without changes in TTX resistant component. The amplitude of net Na+ current was also increased by 119% at day 3 in the group with double treatment (RTX followed by TNBS, P ≤ 0.05 to control) which was significantly higher than in either group with a single treatment. Conclusions & Inferences These results provide evidence that colonic inflammation activates TRPV1 receptors at the peripheral sensory terminals leading to an up‐regulation of voltage gated Na+ channels on the cell soma of bladder sensory neurons. This mechanism may underlie the occurrence of peripheral cross‐sensitization in the pelvis and functional chronic pelvic pain. 相似文献
5.
D. H. Feldman J. S. Thinschmidt A. L. Peel R. L. Papke P. J. Reier 《Experimental neurology》1996,140(2):206-217
Multipotential progenitor cells grown from central nervous system (CNS) tissues in defined media supplemented with epidermal growth factor (EGF), when attached to a suitable substratum, differentiate to express neural and glial histochemical markers and morphologies. To assess the functional characteristics of such cells, expression of voltage-gated Na+and K+currents (INa, IK) was studied by whole-cell patch clamp methods in progenitors raised from postnatal rat forebrain. Undifferentiated cells were acutely dissociated from proliferative “spheres,” and differentiated cells were studied 1–25 days after plating spheres onto polylysine/laminin-treated coverslips.INaandIKwere detected together in 58%,INaalone in 11%, andIKalone in 19% of differentiated cells recorded with K+-containing pipettes. With internal Cs+(to isolateINa),INaup to 45 pA/pF was observed in some cells within 1 day after plating.INaranged up to 150 pA/pF subsequently. Overall, 84% of cells expressedINa, with an average of 38 pA/pF.INahad fast kinetics, as in neurons, but steady-state inactivation curves were strongly negative, resembling those of glialINa. Inward tail currents sensitive to [K+]outwere observed upon repolarization after the 10-ms test pulse with internal Cs+, indicating the expression of K+channels in 82% of cells. In contrast to the substantial currents observed in differentiating cells, little or noINaorIK-tail currents were detected in recordings from cells acutely dissociated from spheres. Thus, in the presence of EGF, ionic currents develop early during differentiation induced by attachment to an appropriate substratum. Cells switched from EGF to basic fibroblast growth factor (bFGF) when plated onto coverslips showed greatly reduced proliferation and developed less neuron-like morphologies than cells plated in the presence of EGF.INawas observed in only 53% of bFGF-treated cells, with an average of 9 pA/pF. Thus, in contrast to reports that bFGF promotes neuronal differentiation in some CNS progenitor populations, our EGF-generated postnatal rat CNS progenitors do not develop neuronal characteristics when switched to medium containing bFGF. Thus, differentiated CNS progenitors can express a mix of neuronal and glial molecular, morphological, and electrophysiological properties that can be modified by culture conditions. 相似文献
6.
We studied several neurophysiological properties of in vitro maturing glycine receptors in mouse spinal cord neurons cultured for various times: 3–7 days (early), 10–12 days (intermediate), and 17–24 days (mature), using whole-cell and gramicidin-perforated techniques. The glycine-activated Cl− conductance increased about 6-fold during in vitro development, and the current density increased from 177 ± 42 pA/pF in early to 504 ± 74 pA/pF in mature neurons. The sensitivity to glycine increased transiently from 39 ± 2.8 μM in early neurons to 29 ± 1 μM in intermediate neurons. Using whole-cell recordings, we found that ECl did not change during development. With the gramicidin-perforated technique, on the other hand, ECl shifted from −27 to −52 mV with development. Thus, immature neurons were depolarized by the activation of glycine receptors, whereas mature neurons were hyperpolarized. The current decayed (desensitized) during the application of 500 μM glycine. The decay was single exponential and the time constant increased from 2,212 ± 139 msec in early neurons to 4,580 ± 1,071 msec in mature neurons. Picrotoxin (10 μM) inhibited the current to a larger extent in early neurons (46 ± 6% of control), and the sensitivity of these receptors to strychnine (IC50) increased from 23 ± 3 nM to 9 ± 1 nM in mature neurons. In conclusion, several properties of spinal glycine receptors changed during in vitro neuronal maturation. This indicates that, similar to GABAA receptors, the functions of these receptors are developmentally regulated. These changes should affect the excitability of spinal neurons as well as other maturation processes. Synapse 28:185–194, 1998. © 1998 Wiley-Liss, Inc. 相似文献
7.
Malignant hyperthermia-susceptible (MHS) pigs express a sarcoplasmic reticulum (SR) Ca2+-release channel mutation that results in lower than normal contractile thresholds in skeletal muscles. In adult MHS pig muscles the L-type calcium current (Is) is also reduced. We tested the hypothesis that there is a causal relationship between Is and the lower contractile threshold by recording Is from MHs and normal porcine myotubes using the whole cell patch-clamp technique. Current voltage relationships for both MHS and normal myotubes were similar, with peak Is between +20 and +30 mV. Maximum Is amplitudes were not different (normal: 4976 ± 566 pA; MHS: 6516 ± 1088 pA) nor was Is specific density (normal: 9.0 ± 0.8; MHS: 8.8 ± 1.1 pA/pF). In both MHS and normal myotubes, both the dihydropyridine antagonist PN200 –110 (200 nmol/L) and holding the membrane potential at −10 mV for 5 min decreased Is significantly (by more than 50%). There was no apparent direct relationship between the mutation in the SR Ca2+-release channel and lowered Is. We concluded that reduced Is in adult MHS pig muscles may be a secondary effect of the SR Ca2+-release channel mutation on muscle development. © 1996 John Wiley & Sons, Inc. 相似文献
8.
Endogenous voltage-gated potassium currents were investigated in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells using whole-cell voltage clamp recording. Depolarizing voltage steps from −70 mV triggered an outwardly rectified current in nontransfected HEK293 cells. This current had an amplitude of 296 pA at +40 mV and a current density of 19.2 pA/pF. The outward current was eliminated by replacing internal K+ with Cs+ and suppressed by the K+ channel blockers tetraethylammonium and 4-aminopyridine. Raising external K+ attenuated the outward current and shifted the reversal potential towards positive potentials as predicted by the Nernst equation. The current had a fast activation phase but inactivated slowly. These features implicate delayed rectifier (IK)-like channels as mediators of the observed current, which was comparable in size to IK currents in many other cells. A small native inward rectifier current but no transient outward current IA, the M current IM, or Ca2+-dependent K+ currents were detected in HEK293 cells. In contrast to these findings in HEK293 cells, little or no IK-like current was detected in CHO cells. The difference in endogenous voltage-activated currents in HEK293 and CHO cells suggest that CHO cell lines are a preferred system for exogenous K+ channel expression. J. Neurosci. Res. 52:612–617, 1998. © 1998 Wiley-Liss, Inc. 相似文献
9.
《Brain research. Developmental brain research》1994,77(1):89-95
Using whole-cell patch-clamp techniques in situ (whole-tissue and tissue slices), we have studied two aspects of rat telencephalic cell development during the period of embryogenesis starting at E12. The first aspect was related to junctional coupling as revealed by low input resistance, intercellular dye spread and pharmacologic blockade. Coupling appeared to decrease with time, both in extent and occurrence. The second aspect dealt with cell excitability as revealed by voltage-dependent Na current (INa) expression. Immature action potentials and their underlying INas were present in a small proportion of E12 cells. These currents were blocked 36% and 78% by 10−7 M and 10−6 M tetrodotoxin (TTX), respectively. From then onward, INas got larger and more prevalent while no obvious changes in kinetics were observed. At E21, INas were abolished by 10−7 M TTX and channel density apparently was sufficient to support overshooting yet still immature action potentials. 相似文献
10.
Whole-cell voltage-gated currents were recorded from bipolar cells in the zebrafish retinal slice. Two physiological populations of bipolar cells were identified. In the first, depolarizing voltage steps elicited a rapidly activating A-current that reached peak amplitude ≤ 5 ms of step onset. IA was antagonized by external tetraethylammonium or 4-aminopyridine, and by intracellular caesium. The second population expressed a delayed rectifying potassium current (IK) that reached peak amplitude ≥ 10 ms after step onset and did not inactivate. IK was antagonized by internal caesium and external tetraethylammonium. Bipolar cells expressing IK also expressed a time-dependent h-current at membrane potentials < – 50 mV. Ih was sensitive to external caesium and barium, and was also reduced by Na+-free Ringer. In both groups, a calcium current (ICa) and a calcium-dependent potassium current (IK(Ca)) were identified. Depolarizing voltage steps > – 50 mV activated ICa, which reached peak amplitude between – 20 and – 10 mV. ICa was eliminated in Ca+2-free Ringer and blocked by cadmium and cobalt, but not tetrodotoxin. In most cells, ICa was transient, activating rapidly at – 50 mV. This current was antagonized by nickel. The remaining bipolar cells expressed a nifedipine-sensitive sustained current that activated between – 40 and – 30 mV, with both slower kinetics and smaller amplitude than transient ICa. IK(Ca) was elicited by membrane depolarizations > – 20 mV. Bipolar cells in the zebrafish retinal slice preparation express an array of voltage-gated currents which contribute to non-linear I–V characteristics. The zebrafish retinal slice preparation is well-suited to patch clamp analyses of membrane mechanisms and provides a suitable model for studying genetic defects in visual system development. 相似文献
11.
The postnatal development of spontaneous GABAergic transmission between cerebellar Golgi cells and granule cells was investigated with voltage-clamp recording from rat cerebellar slices, in symmetrical Cl-conditions. Between postnatal days 7 and 14 (P7–14), bicuculline-and TTX (tetrodotoxin)-sensitive spontaneous inhibitory postsynaptic currents (sIPSCs), occurred at high frequency in 56% of granule cells. Between P10 and P14, sIPSCs were superimposed on tonic current of-12 ± 1.8 pA at -70 mV, that was accompanied by noise with variance of 17 ± 3 pA2. Both the current and noise were inhibited by bicuculline. TTX blocked the bicuculline-sensitive current and noise by?60%. Between P18 and P25, sIPSCs were less frequent; all cells showed tonic, bicuculline-sensitive currents, but these were partially inhibited by TTX (?35%). Between P40 and P53, slPSCs were rare; tonic, bicuculline-sensitive currents and noise were greater in amplitude, with mean values of-17 pA and 22 pA2 at-70 mV, they were present in all cells but they were not inhibited by TTX. Glycine receptor channels that were expressed in immature, but not adult cells, did not mediate spontaneous currents. Our results indicate that spontaneous transmission onto cerebellar granule cells in immature animals consists primarily of action potential-dependent, phasic release of vesicular GABA. This generates GABAA receptor-mediated slPSCs. The effects of GABA transporter blockers suggest that it also produces the TTX-sensitive current-noise, as GABA spills out of synapses to activate extrasynaptic receptors or receptors in neighbouring synapses. In older animals, action potential-independent release of transmitter is predominant and results in tonic activation of GABAA receptors. This does not appear to be spontaneous vesicular release of GABA. Neither does it appear to be reversed uptake of GABA, although further work is required to rule out these possibilities. 相似文献
12.
Stephan Wierschke Thomas‐Nicolas Lehmann Christoph Dehnicke Peter Horn Robert Nitsch Rudolf A. Deisz 《Epilepsia》2010,51(3):404-414
Purpose: Hyperpolarization‐activated cation currents (IH) play a pivotal role in the control of neuronal excitability. In animal models of epilepsy both increases and decreases of IH have been reported. We, therefore, characterized properties of IH in human epileptogenic neocortex. Methods: Layer II/III neurons in slices from epilepsy surgery tissues and rat cortex were investigated with whole‐cell patch‐clamp recordings. Results: A total of 484 neurons from 96 temporal lobe epilepsy (TLE) tissues and 32 neurons from 8 frontal lobe epilepsy (FLE) tissues were recorded. Voltage‐clamp recordings revealed on hyperpolarizing command steps two time‐ and voltage‐dependent inward currents, namely a fast, Ba2+‐sensitive current (KIR) and a slowly activating current, namely consisting of two kinetically distinct components sensitive to the established IH blocker ZD7288. Only, the fast component (IH(fast)) of TLE neurons was on average smaller and activated more slowly (density 2.7 ± 1.6 pA/pF; tau 38.4 ± 34.0 ms) than in FLE neurons (4.7 ± 2.3 pA/pF; 16.6 ± 7.9 ms; p < 0.001 for both). Within the TLE tissues the IH(fast) density (averaged per patient) was smaller in cases with numerous annual grand mal seizures (GM; 2.2 ± 0.6 pA/pF) compared to those with few GM (2.8 ± 1.0 pA/pF; p = 0.0184). A similar difference was obtained in the case of complex partial seizures (CPS; many CPS 2.2 ± 0.6 pA/pF; few CPS 2.9 ± 1.0 pA/pF, p = 0.0037). Discussion: The biophysical properties of IH in cortices from TLE, FLE, and rat tissue suggest a deficit of HCN1 subunits in the human epileptogenic neocortex, which in turn may increase excitability and probability of seizure activity. 相似文献
13.
Carbachol Potentiates Q Current and Activates a Calcium-dependent Non-specific Conductance in Rat Hippocampus In Vitro 总被引:9,自引:0,他引:9
Intracellular recordings were made from CA1 neurons in rat hippocampal slices maintained in vitro. When Na+ currents were blocked with tetrodotoxin and K+ conductances known to be sensitive to suppression by muscarinic agonists were blocked by 2 mM Ba2+, CA1 cells were depolarized by carbachol (3 – 10 μM) with an attendant conductance increase, whereas prior to Ba2+ the agonist produced a decrease or no change in conductance. Under voltage clamp at ~–60 mV and in the presence of tetrodotoxin and Ba2+, carbachol (3 – 10 μM) induced a variable-latency biphasic inward current of up to 380 pA associated with a conductance increase of ~50%. The first phase was associated with an increase (more than 2-fold) of the Cs+-sensitive, hyperpolarization-activated cationic current, IQ. Carbachol also accelerated the kinetics of IQ at – 100 mV with an average 24% reduction in its activation time constant. The second phase reflected an additional inward current that was Cs+-resistant, displayed little apparent voltage sensitivity and had a mean extrapolated reversal potential, determined in the presence of external Cs+ (>5 mM), of ~–20 mV. In a small proportion of cells the second phase of inward current was followed (or overlapped) by an outward current, also associated with a conductance increase, which reversed at ~–70 mV. These carbachol actions were prevented by extracellular 300 μM Cd2+ and 2 mM Mn2+, by high levels (>5 mM) of extracellular Mg2+ or Ca2+, and by omission of Ca2+ or reduction of extracellular Na+ to 25 mM by substitution of NaCl with Tris or N-methyl-d -glucamine. Carbachol action was not mimicked by oxotremorine (≤60 μM), but was irreversibly blocked by this drug. Likewise, atropine (100 nM) irreversibly and gallamine (10 μM) reversibly antagonized carbachol's action. The action of carbachol was blocked shortly after prior exposure of slices to 2 – 5 mM caffeine. Chronic or acute incubation of slices with 2 mM Li+ potentiated (between 1- and 2-fold) carbachol responses. The data indicate that muscarinic activation increases cationic flux by a calcium-dependent potentiation of IQ and activation of a non-selective conductance. The probability that inositol phospholipid metabolism is involved in triggering these events is discussed. 相似文献
14.
Ethanol at concentration of 200 mM induces anesthesia in experimental animals and depresses neurotransmission in isolated spinal cords. To determine whether actions on primary afferent nerve terminals contribute to ethanol's depressant effects on spinal cord, a study was undertaken to test whether ethanol blocks sodium currents (INa) in dorsal root ganglion neurons (DRGn). Whole-cell patch clamp was used to examine INa in DRGn isolated from 1- to 15-day-old rats. At a holding potential of −80 mV ethanol (200 mM) decreased peak tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) INa by 19.0% ± 2.7 (mean ± SEM) and 8.5% ± 2.2, respectively. Maximal available INa was reduced to 82 ± 4% (TTX-R) and 93 ± 1% (TTX-S) of control. Steady-state inactivation curves were shifted in the hyperpolarizing direction by 2.1 ± 0.2 mV (TTX-R) and 1.1 ± 0.1 mV (TTX-S). At prepulse potentials of −30 mV (TTX-R) and −70 mV (TTX-S), these shifts contributed an additional 17 ± 1% (TTX-R) and 7 ± 1% (TTX-S) reduction in available INa. Ethanol thus selectively induced both voltage-independent and voltage-dependent block of TTX-R INa in DRGn. Because DRGn TTX-R sodium channels are associated with small-diameter primary afferent fibers, these results are consistent with a role for ethanol actions on sodium channels in depression of nociceptive-related neurotransmission in spinal cord. J. Neurosci. Res. 54:433–443, 1998. © 1998 Wiley-Liss, Inc. 相似文献
15.
Developmental regulation of Na+ and K+ conductances in glial cells of mouse hippocampal brain slices
Klaus Kressin Elena Kuprijanova Ronald Jabs Gerald Seifert Christian Steinhuser 《Glia》1995,15(2):173-187
The relative contribution of voltage activated Na+ and K+ currents to the whole cell current pattern of hippocampal glial cells was analyzed and compared during different stages of postnatal maturation. The patch-clamp technique was applied to identified cells in thin brain slices obtained from animals between postnatal day 5 and 35 (p5-35). We focused on a subpopulation of glial cells in the CA1 stratum radiatum which most probably represents a pool of immature astrocytes, termed “complex” cells. These cells could not be labelled by 01/04 antibodies, but some of the older cells were positively stained for glial fibrillary acidic protein (GFAP). In the early postnatal days, the current pattern of the “complex” cells was dominated by two types of K+ outward currents: a delayed rectifier and a transient component. In addition, all cells expressed significant tetrodotoxin (TTX)-sensitive Na+ currents. During maturation, the contribution of delayed rectifier and A-type currents significantly decreased. Furthermore, almost all cells after p20 lacked Na+ currents. This down-regulation of voltage gated Na+ and K+ outward currents was accompanied by a substantial increase in passive and inward rectifier K+ conductances. We found increasing evidence of electrical coupling between the “complex” cells with continued development. It is concluded that these developmental changes in the electrophysiological properties of “complex” glial cells could be jointly responsible for the well known impaired K+ homeostasis in the early postnatal hippocampus. © 1995 Wiley-Liss, Inc. 相似文献
16.
Differential Kv1.3, KCa3.1, and Kir2.1 expression in “classically” and “alternatively” activated microglia
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Hai M. Nguyen Eva M. Grössinger Makoto Horiuchi Kyle W. Davis Lee‐Way Jin Izumi Maezawa Heike Wulff 《Glia》2017,65(1):106-121
Microglia are highly plastic cells that can assume different phenotypes in response to microenvironmental signals. Lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ) promote differentiation into classically activated M1‐like microglia, which produce high levels of pro‐inflammatory cytokines and nitric oxide and are thought to contribute to neurological damage in ischemic stroke and Alzheimer's disease. IL‐4 in contrast induces a phenotype associated with anti‐inflammatory effects and tissue repair. We here investigated whether these microglia subsets vary in their K+ channel expression by differentiating neonatal mouse microglia into M(LPS) and M(IL‐4) microglia and studying their K+ channel expression by whole‐cell patch‐clamp, quantitative PCR and immunohistochemistry. We identified three major types of K+ channels based on their biophysical and pharmacological fingerprints: a use‐dependent, outwardly rectifying current sensitive to the KV1.3 blockers PAP‐1 and ShK‐186, an inwardly rectifying Ba2+‐sensitive Kir2.1 current, and a Ca2+‐activated, TRAM‐34‐sensitive KCa3.1 current. Both KV1.3 and KCa3.1 blockers inhibited pro‐inflammatory cytokine production and iNOS and COX2 expression demonstrating that KV1.3 and KCa3.1 play important roles in microglia activation. Following differentiation with LPS or a combination of LPS and IFN‐γ microglia exhibited high KV1.3 current densities (~50 pA/pF at 40 mV) and virtually no KCa3.1 and Kir currents, while microglia differentiated with IL‐4 exhibited large Kir2.1 currents (~ 10 pA/pF at ?120 mV). KCa3.1 currents were generally low but moderately increased following stimulation with IFN‐γ or ATP (~10 pS/pF). This differential K+ channel expression pattern suggests that KV1.3 and KCa3.1 inhibitors could be used to inhibit detrimental neuroinflammatory microglia functions. GLIA 2016;65:106–121 相似文献
17.
The pattern of development and regulation of the apamin receptor (afterhyperpolarization channel) was studied in cultures of skeletal muscle prepared from 1–2-day-old rat pups. Expression was measured by the specific binding of 125I-apamin. Apamin binding was virtually undetectable until the time of fusion (3–4 days in culture) of single myoblasts into myotubes. Mature myotubes (5–7 days in vitro) displayed a Bmax of 7.4 fmol/mg protein and a Kd of 376 pmol/L. When studied in mature muscle cells, apamin binding was found to increase twofold in response to tetrodotoxin (TTX) and elevated K0, which resulted in decreased Nai. In contrast, treatments causing an increase in Nai, such as monensin and veratridine, caused a decrease in apamin binding. The increase in apamin binding following TTX treatment was due mainly to synthesis of new channels, as the effect was blocked by cycloheximide. Alterations in cytosolic Ca2+ by calcium ionophore or Ca-channel blockers were without effect on apamin-sensitive channel expression. We conclude that afterhyperpolarization channel expression is regulated by the level of intracellular Na+ ions. © 1996 John Wiley & Sons, Inc. 相似文献
18.
Oscillatory firing activity in cerebellar Purkinje cells (PCs) can be maintained by intrinsic ionic conductances in the apparent absence of excitatory and inhibitory synaptic input as demonstrated by application of TTX or antagonists of amino acid-mediated transmission or both. Bursting activity in these cells is associated with a region of ZSR (zero slope resistance, the beginning part of a negative slope resistance region) of the whole cell quasi-steady-state I–V relationship. Blockade of Na+ current by TTX unmasked the ZSR region in all PCs tested. Based on current and voltage clamp experiments, hyperpolarization-activated cation current (Ih) participates in the rhythmic firing activity by influencing the amplitude and duration of the interburst interval and the resultant pattern of the burst generation. Blockade of Ih with cesium (Cs+) retards the membrane rebound from the after-hyperpolarization and results in longer and more negative hyperpolarization between bursts. However, Cs+ did not affect the presence and characteristic of the ZSR region of the whole cell quasi-steady-state I–V curve. 相似文献
19.
We have previously shown that (1) removal of extracellular sodium (Na+) reduces the anoxia-induced depolarization in neurons in brain-slice preparations and (2) amiloride, which blocks Na+-dependent exchangers, prevents anoxic injury in cultured neocortical neurons. Since anoxia-induced depolarization has been linked to neuronal injury, we have examined in this study the role of Na+-dependent exchangers and voltage-gated Na+ channels in the maintenance of membrane properties of CA1 neurons at rest and during acute hypoxia. We recorded intracellularly from CA1 neurons in hippocampal slices, monitored Vm and measured input resistance (Rm) with periodic injections of negative current. We found that tetrodotoxin (TTX, 1 μM) hyperpolarized CA1 neurons at rest and significantly attenuated both the rate of depolarization (ΔVm/dt) and the rate of decline of Rm (ΔRm/dt) by about 60% during the early phase of hypoxia. The effect of TTX was dose-dependent. Amiloride (1 mM) decreased Vm and increased Rm in the resting condition but changed little the effect of hypoxia on neuronal function. Benzamil and 5-(N-ethyl-N-isopropyl)-2′,4′-amiloride (EIPA), two specific inhibitors of Na+ dependent exchangers, were similar to amiloride in their effect. We conclude that neuronal membrane properties are better maintained during anoxia by reducing the activity of TTX-sensitive channels and not by the action of Na+-dependent exchangers. 相似文献
20.
The expression and properties of voltage-gated Na(+) currents in cardiac dorsal root ganglion (DRG) neurons were assessed in this study. Cardiac DRG neurons were labelled by injecting the Fast Blue fluorescent tracer into the pericardium. Recordings were performed from 138 cells. Voltage-dependent Na(+) currents were found in 115 neurons. There were 109 neurons in which both tetrodotoxin-sensitive (TTX-S, blocked by 1 microM of TTX) and tetrodotoxin-resistant (TTX-R, insensitive to 1 microM of TTX) Na(+) currents were present. Five cells expressed TTX-R current only and one cell only the TTX-S current. The kinetic properties of Na(+) currents and action potential waveform parameters were measured in neurons with cell membrane capacitance ranging from 15 to 75 pF. The densities of TTX-R (110.0 pA/pF) and TTX-S (126.1 pA/pF) currents were not significantly different. Current threshold was significantly higher for TTX-R (-34 mV) than for TTX-S (-40.4 mV) currents. V(1/2) of activation for TTX-S current (-19.6 mV) was significantly more negative than for TTX-R current (-9.2 mV), but k factors did not differ significantly. V(1/2) and the k constant for inactivation for TTX-S currents were -35.6 and -5.7 mV, respectively. These values were significantly lower than those recorded for TTX-R current for which V(1/2) and k were -62.3 and -7.7 mV, respectively. The action potential threshold was lower, the 10-90% rise time and potential width were shorter before than after the application of TTX. Based on this we drew the conclusion that action potential recorded before adding tetrodotoxin was mainly TTX-S current dependent, while the action potential recorded after the application of toxin was TTX-R current dependent. We also found 23 cells with mean membrane capacitance ranging from 12 to 35 pF (the smallest labelled DRG cells found in this study) that did not express the Na(+) current. The function of these cells is unclear. We conclude that the overwhelming majority of cardiac dorsal root ganglion neurons in which voltage-dependent Na(+) currents were present, exhibited both TTX-S and TTX-R Na(+) currents with remarkably similar expression and kinetic properties. 相似文献