Two types of kainate response in cultured rat hippocampal neurons

1991 ◽  
Vol 66 (1) ◽  
pp. 2-11 ◽  
Author(s):  
S. Ozawa ◽  
M. Iino ◽  
K. Tsuzuki

1. Two different types of kainate response were recorded in cultured rat hippocampal neurons with the use of the whole-cell and outside-out configurations of the patch-clamp technique. 2. There was an outward rectification in the current-voltage (I-V) plot of the kainate-induced current (type I response) in relatively large neurons bearing a morphological resemblance to young pyramidal cells. In smaller neurons with elliptical somata and fine neurites, the kainate response was characterized by a remarkable inward rectification in the I-V plot of the kainate-induced current and a significant permeability to Ca2+ (type II response). 3. Both type I and type II responses were negligible below 2 microM and almost saturated at 500 microM kainate. The concentrations producing half-maximal responses and the Hill coefficients were 68 microM and 1.76 and 56 microM and 1.21 for type I and type II responses, respectively. Both responses were suppressed similarly by the non-N-methyl-D-aspartate (NMDA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). 4. The mean single-channel conductance (gamma) of the type II kainate response was estimated, from the relation between the whole-cell mean currents and current variances, to be 8.7 pS. The power spectrum for the current noise was fitted with the sum of two Lorentzians with cutoff frequencies (fc) of 61.1 +/- 1.4 and 327.8 +/- 10.5 Hz (n = 12).(ABSTRACT TRUNCATED AT 250 WORDS)

1992 ◽  
Vol 68 (4) ◽  
pp. 1359-1372 ◽  
Author(s):  
A. Kamondi ◽  
J. A. Williams ◽  
B. Hutcheon ◽  
P. B. Reiner

1. The whole-cell patch-clamp technique was used to study the membrane properties of identified cholinergic and noncholinergic laterodorsal tegmental neurons in slices of rat brain maintained in vitro. 2. On the basis of their expression of the transient outward potassium current IA and the transient inward calcium current IT, three classes of neurons were observed: type I neurons exhibited a large IT; type II neurons exhibited a prominent IA; and type III neurons exhibited both IA and IT. 3. Combining intracellular deposition of biocytin with NADPH diaphorase histochemistry revealed that the vast majority of type III neurons were cholinergic, whereas only a minority of type I and type II neurons were cholinergic. Thus mesopontine cholinergic neurons possess intrinsic ionic currents capable of inducing burst firing. 4. Delineation of the intrinsic membrane properties of identified mesopontine cholinergic neurons, in concert with recent results regarding the responses of these neurons to neurotransmitter agents, has led us to present a unifying and mechanistic hypothesis of brain stem cholinergic function in the control of behavioral states.


1995 ◽  
Vol 73 (2) ◽  
pp. 902-906 ◽  
Author(s):  
M. Martina ◽  
F. Strata ◽  
E. Cherubini

1. The patch-clamp technique was used to characterize, in acutely dissociated CA3 rat hippocampal neurons, the whole cell and single channel properties of a novel response to gamma-aminobutyric acid (GABA) present only during a restricted period of postnatal development. 2. At postnatal days 0-10 (P0-P10), both GABA (100 microM) and isoguvacine (50 microM) evoked at a holding potential of -50 mV, in symmetrical chloride solution, whole cell inward currents. Bicuculline blocked the response to isoguvacine but only reduced the response to GABA (from 512 +/- 137 pA to 60 +/- 13 pA, mean +/- SE). After P12, bicuculline abolished the response to GABA. 3. The bicuculline-insensitive GABA currents were Cl- mediated and antagonized by picrotoxin. The desensitization rate was slower than the conventional bicuculline-sensitive response. The peak to plateau ratio induced by 0.1 or 1 mM of GABA shifted from 4.6 +/- 0.4 and 17.7 +/- 2.6 to 1.5 +/- 0.1 and 3.1 +/- 0.5 in the absence or in the presence of bicuculline, respectively. The recovery from desensitization was significantly faster for the bicuculline-insensitive responses. 4. In excised outside-out patches, GABA (20 microM) activated, in the presence of bicuculline (100 microM), single channel currents having conductances of 14, 22, and 31 pS. These values were similar to those obtained in the same preparation, in the absence of bicuculline. 5. These findings suggest that this new receptor type, which mediates bicuculline-insensitive responses with slow kinetics, may potentiate the depolarizing action of GABA during a critical period of postnatal development and therefore play a crucial role in synaptogenesis.


1989 ◽  
Vol 256 (6) ◽  
pp. G1028-G1035 ◽  
Author(s):  
R. M. Henderson ◽  
J. Graf ◽  
J. L. Boyer

The patch-clamp technique has been used to investigate single-channel and whole cell conductances in freshly isolated rat hepatocytes. Whole cell experiments, with high (144 mM) intracellular and extracellular potassium as the principal conductive species, show some variation between cells in the current-voltage relationship (mean whole-cell conductance at physiological potentials being 2.7 nS). This may suggest functional heterogeneity of cells. The most common finding is that the current-voltage relationship shows inward rectification. This is reflected in cell-attached single-channel recordings in which channels displaying strong inward rectification and K+ selectivity are seen. The channels show a mean inward conductance (with 144 mM potassium in the pipette) of 44 pS and an outward conductance of 23 pS. The open probability is not voltage dependent, and the channels do not exhibit calcium dependence. The channels are quite different from others described in hepatocytes, but they show marked similarities to channels recently described in renal epithelial cells. Current-voltage relationships in the whole cell mode exhibit an increase in slope conductance at large hyperpolarizing and depolarizing potentials.


1990 ◽  
Vol 64 (5) ◽  
pp. 1361-1367 ◽  
Author(s):  
S. Ozawa ◽  
M. Iino ◽  
K. Tsuzuki

1. The effects of increasing K+ concentration in Mg2(+)-free extracellular solution on N-methyl-D-aspartate (NMDA)-induced current were studied in cultured rat hippocampal neurons with the use of the whole-cell and outside-out configurations of the patch-clamp technique. 2. When the K+ concentration in the external solution was increased by replacement of Na+ with isomolar K+, the amplitude of the NMDA-induced current decreased in a concentration-dependent manner. The effect of K+ was almost saturated at 100 mM, when the NMDA response was reduced to 12% of that in K(+)-free, 150 mM Na+ solution. Increasing the external K+ concentration did not affect either the kainate- or quisqualate-induced current in these experimental conditions. 3. Increase in the external K+ concentration reduced the NMDA-induced current almost equally over the whole range of membrane potential tested (-60-30 mV). The reversal potential of the NMDA-induced current was not significantly shifted by the replacement of Na+ with K+. 4. A rise in the external K+ concentration to 100 mM did not reduce the single-channel conductance of the NMDA channel, whereas it reduced the mean open time to about two-thirds of that in the control external solution. 5. The suppressed activation of the NMDA receptor channel in high-K+ environments may have a functional significance to alleviate entry of toxic Ca2+ into neurons of the CNS in pathological conditions such as hypoxia and ischemia.


1993 ◽  
Vol 70 (3) ◽  
pp. 1076-1085 ◽  
Author(s):  
C. Rovira ◽  
Y. Ben-Ari

1. The effects of type I (BZ1) and type II (BZ2) benzodiazepine receptor ligands on monosynaptic gamma-aminobutyric acid (GABA)A-mediated inhibitory postsynaptic potentials (IPSPs) and on responses to exogenously applied GABA were studied using intracellular recordings from CA3 pyramidal cells of rat hippocampal slices taken at different postnatal stages [postnatal day 4 (P4)-P35)]. 2. The effects of midazolam, a BZ1 and BZ2 receptor agonist, were tested on the monosynaptic IPSPs at different stages. Monosynaptic, bicuculline-sensitive IPSPs were evoked by hilar stimulation in presence of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) antagonists [6-cyano-7-nitroquinoxaline-2,3-dione (10 microM) and D(-)2-amino-5-phosphonopentanoic acid (50 microM)]. Midazolam at 300 nM maximally increased the duration and amplitude of monosynaptic GABAA-mediated IPSPs in neurons from pups (P4-P6, n = 6) and young (P7-P12, n = 8) and adult (P25-P35, n = 9) rats. All the effects of midazolam on IPSPs were reversed by the antagonist Ro 15-1788 (10 microM). 3. The effect of midazolam was also tested on the response to exogenously applied GABA (5 mM) in the presence of tetrodotoxine [TTX (1 microM)]. In neurons from young rats (n = 9), midazolam (1 nM-1 microM) did not change the responses to exogenously applied GABA, whereas in adult rats (n = 8) midazolam maximally increased GABA currents at 30 nM. 4. The effect of zolpidem, a BZ1 receptor agonist, was tested on monosynaptic IPSPs and GABA currents at different stages. Zolpidem (10 nM-1 microM) was inactive in cells from young rats (n = 12). In neurons from adult rats, zolpidem maximally increased the duration and amplitude of the monosynaptic IPSPs at 300 nM (n = 5) and the amplitude of GABA current at 30-100 nM (n = 5). 5. Methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) (300 nM), an inverse agonist of BZ1 and BZ2 receptors, decreased the amplitude and duration of monosynaptic IPSPs in neurons from pups (n = 3) and young (n = 4) and adult (n = 5) rats. In all cases, full recovery was obtained after exposure to R0 15-1788 (10 microM). DMCM (300 nM-10 microM) failed to reduce GABA responses in cells from young (n = 3) or adult (n = 7) rats. 6. Results indicate that the regulation by benzodiazepine of GABAA-mediated IPSPs varies with the developmental stage.(ABSTRACT TRUNCATED AT 400 WORDS)


2004 ◽  
Vol 19 (2) ◽  
pp. 155-169 ◽  
Author(s):  
Manning J. Correia ◽  
Thomas G. Wood ◽  
Deborah Prusak ◽  
Tianxiang Weng ◽  
Katherine J. Rennie ◽  
...  

A fast inwardly rectifying current has been observed in some of the sensory cells (hair cells) of the inner ear of several species. While the current was presumed to be an IKir current, contradictory evidence existed as to whether the cloned channel actually belonged to the Kir2.0 subfamily of potassium inward rectifiers. In this paper, we report for the first time converging evidence from electrophysiological, biochemical, immunohistochemical, and genetic studies that show that the Kir2.1 channel carries the fast inwardly rectifying currents found in pigeon vestibular hair cells. Following cytoplasm extraction from single type II and multiple pigeon vestibular hair cells, mRNA was reverse transcribed, amplified, and sequenced. The open reading frame (ORF), consisting of a 1,284-bp nucleotide sequence, showed 94, 85, and 83% identity with Kir2.1 subunit sequences from chick lens, Kir2 sequences from human heart, and a mouse macrophage cell line, respectively. Phylogenetic analyses revealed that pKir2.1 formed an immediate node with hKir2.1 but not with hKir2.2–2.4. Hair cells (type I and type II) and supporting cells in the sensory epithelium reacted positively with a Kir2.1 antibody. The whole cell current recorded in oocytes and CHO cells, transfected with pigeon hair cell Kir2.1 (pKir2.1), demonstrated blockage by Ba2+ and sensitivity to changing K+ concentration. The mean single-channel linear slope conductance in transfected CHO cells was 29 pS. The open dwell time was long (∼300 ms at −100 mV), and the closed dwell time was short (∼34 ms at −100 mV). Multistates ranging from 3–6 were noted in some single-channel responses. All of the above features have been described for other Kir2.1 channels. Current clamp studies of native pigeon vestibular hair cells illustrated possible physiological roles of the channel and showed that blockage of the channel by Ba2+ depolarized the resting membrane potential by ∼30 mV. Negative currents hyperpolarized the membrane ∼20 mV before block but ∼60 mV following block. RT-PCR studies revealed that the pKir2.1 channels found in pigeon vestibular hair cells were also present in pigeon vestibular nerve, vestibular ganglion, lens, neck muscle, brain (brain stem, cerebellum and optic tectum), liver, and heart.


2000 ◽  
Vol 84 (5) ◽  
pp. 2204-2216 ◽  
Author(s):  
Marc L. Belleau ◽  
Richard A. Warren

We have studied the postnatal development of the physiological characteristics of nucleus accumbens (nAcb) neurons in slices from postnatal day 1 ( P1) to P49 rats using the whole cell patch-clamp technique. The majority of neurons (102/108) were physiologically identified as medium spiny (MS) projection neurons, and only these were subjected to detailed analysis. The remaining neurons displayed characteristics suggesting that they were not MS neurons. Around the time of birth and during the first postnatal weeks, the membrane and firing characteristics of MS neurons were quite different from those observed later. These characteristics changed rapidly during the first 3 postnatal weeks, at which point they began to resemble those found in adults. Both whole cell membrane resistance and membrane time constant decreased more than fourfold during the period studied. The resting membrane potential (RMP) also changed significantly from an average of −50 mV around birth to less than −80 mV by the end of the third postnatal week. During the first postnatal week, the current-voltage relationship of all encountered MS neurons was linear over a wide range of membrane potentials above and below RMP. Through the second postnatal week, the proportion of neurons displaying inward rectification in the hyperpolarized range increased steadily and after P15, all recorded MS neurons displayed significant inward rectification. At all ages, inward rectification was blocked by extracellular cesium and tetra-ethyl ammonium and was not changed by 4-aminopyridine; this shows that inward rectification was mediated by the same currents in young and mature MS neurons. MS neurons fired single and repetitive Na+/K+ action potentials as early as P1. Spike threshold and amplitude remained constant throughout development in contrast to spike duration, which decreased significantly over the same period. Depolarizing current pulses from rest showed that immature MS neurons fired action potentials more easily than their older counterparts. Taken together, the results from the present study suggest that young and adult nAcb MS neurons integrate excitatory synaptic inputs differently because of differences in their membrane and firing properties. These findings provide important insights into signal processing within nAcb during this critical period of development.


1993 ◽  
Vol 177 (1) ◽  
pp. 201-221 ◽  
Author(s):  
H. A. Pearson ◽  
G. Lees ◽  
D. Wray

1. Using the patch-clamp technique, Ca2+ channel currents were recorded from neurones freshly isolated from the thoracic ganglia of the desert locust Schistocerca gregaria. 2. In solutions containing 10 mmol l-1 Ba2+ we observed high-voltage-activated whole-cell inward currents with sustained and transient components, both of which had similar steady-state inactivation properties. 3. Substitution of Ca2+ for Ba2+ was found to reduce whole-cell currents, whereas removal of monovalent cations had no effect. 4. Cd2+ (1 mmol l-1) completely blocked the whole-cell current, but at 10 micromolar preferentially inhibited the sustained component without affecting the transient component. 5. Verapamil (1 micromolar) inhibited both current components but appeared to be more selective for the sustained component, whereas nitrendipine (1 micromolar) had no effect on either component. 6. A single-channel recording suggested that the transient component was carried by a low- conductance channel. 7. Certain compounds with insecticidal action (ryanodine, S-bioallethrin, deltamethrin and avermectin) did not affect calcium channel currents in these cells. 8. These data suggest that there are two types of Ca2+ channels present in locust neurones. These channel types have properties differing from the T-, L- and N-type channels found in vertebrates and, furthermore, were not targets for the insecticides we tested.


1997 ◽  
Vol 78 (5) ◽  
pp. 2592-2601 ◽  
Author(s):  
Shun-Ichi Itazawa ◽  
Tadashi Isa ◽  
Seiji Ozawa

Itazawa, Shun-Ichi, Tadashi Isa, and Seiji Ozawa. Inwardly rectifying and Ca2+-permeable AMPA-type glutamate receptor channels in rat neocortical neurons. J. Neurophysiol. 78: 2592–2605, 1997. Current-voltage ( I-V) relations and Ca2+ permeability of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)type glutamate receptor channels were investigated in neurons of rat neocortex by using the whole cell patch-clamp technique in brain slices. To activate AMPA receptor channels, kainate was used as a nondesensitizing agonist. A patch pipette was filled with solution containing 100 μM spermine to maintain the inward rectification of Ca2+-permeable AMPA receptor channels. Three types of responses to kainate were observed: type I response with outwardly rectifying I-V relation, type II response with I-V relation of marked inward rectification, and intermediate response with I-V relation of weaker inward rectification. Neurons with type I, type II and intermediate I-V relations were referred to as type I, type II, and intermediate neurons, respectively. Of a total of 223 recorded cells, 90 (40.4%) were type I, 129 (57.8%) intermediate, and 4 (1.8%) type II neurons. Properties of AMPA receptor channels were examined in the former two types of neurons. The value of PCa:PCs, the ratio of the permeability coefficients of Ca2+ and Cs+, was estimated from the reversal potentials of kainate responses in the outside-out patches bathed in Na+-free solution containing 100 mM Ca2+ according to the constant-field equation. They ranged from 0.05 to 0.10 (0.08 ± 0.02, mean ± SD, n = 8) for type I neurons and from 0.14 to 1.29 (0.60 ± 0.37, n = 11) for the intermediate neurons. There was a close correlation between the inward rectification and the Ca2+ permeability in AMPA receptor channels in these neurons. Intermediate neurons stained with biocytin were nonpyramidal cells with ellipsoidal-shaped somata. Type I neurons had either triangular- or ellipsoidal-shaped somata. Excitatory postsynaptic currents (EPSCs) recorded in both type I and intermediate neurons had 6-cyano-7-nitroquinoxaline-2,3-dione-sensitive fast and d−2-amino-5-phosphonovalerate-sensitiveslow components. The I-V relation of the fast component exhibited inward rectification in the intermediate neuron, whereas that in the type I neuron showed slight outward rectification. The fast component of EPSCs in the intermediate neuron was suppressed more prominently (to 56 ± 15% of the control, n = 12) than that in the type I neuron (to 78 ± 6% of the control, n = 6) by bath application of 1 mM spermine. These results indicate that inwardly rectifying and Ca2+-permeable AMPA receptor channels are expressed in a population of neurons of rat neocortex and are involved in excitatory synaptic transmission.


1998 ◽  
Vol 274 (4) ◽  
pp. L475-L484 ◽  
Author(s):  
Lucky Jain ◽  
Xi-Juan Chen ◽  
Lou Ann Brown ◽  
Douglas C. Eaton

We used the patch-clamp technique to study the effect of nitric oxide (NO) on a cation channel in rat type II pneumocytes [alveolar type II (AT II) cells]. Single-channel recordings from the apical surface of AT II cells in primary culture showed a predominant cation channel with a conductance of 20.6 ± 1.1 (SE) pS ( n = 9 cell-attached patches) and Na+-to-K+selectivity of 0.97 ± 0.07 ( n = 7 cell-attached patches). An NO donor, S-nitrosoglutathione (GSNO; 100 μM), inhibited the basal cation-channel activity by 43% [open probability ( P o), control 0.28 ± 0.05 vs. GSNO 0.16 ± 0.03; P < 0.001; n = 16 cell-attached patches], with no significant change in the conductance. GSNO reduced the P o by reducing channel mean open and increasing mean closed times. GSNO inhibition was reversed by washout. The inhibitory effect of NO was confirmed by using a second donor of NO, S-nitroso- N-acetylpenicillamine (100 μM; P o, control 0.53 ± 0.05 vs. S-nitroso- N-acetylpenicillamine 0.31 ± 0.04; −42%; P < 0.05; n = 5 cell-attached patches). The GSNO effect was blocked by methylene blue (a blocker of guanylyl cyclase; 100 μM), suggesting a role for cGMP. The permeable analog of cGMP, 8-bromo-cGMP (8-BrcGMP; 1 mM), inhibited the cation channel in a manner similar to GSNO ( P o, control 0.38 ± 0.06 vs. 8-BrcGMP 0.09 ± 0.02; P < 0.05; n = 7 cell-attached patches). Pretreatment of cells with 1 μM KT-5823 (a blocker of protein kinase G) abolished the inhibitory effect of GSNO. The NO inhibition of channels was not due to changes in cell viability. Intracellular cGMP was found to be elevated in AT II cells treated with NO (control 13.4 ± 3.6 vs. GSNO 25.4 ± 4.1 fmol/ml; P < 0.05; n = 6 cell-attached patches). We conclude that NO suppresses the activity of an Na+-permeant cation channel on the apical surface of AT II cells. This action appears to be mediated by a cGMP-dependent protein kinase.


Sign in / Sign up

Export Citation Format

Share Document