Genes for the GABAA receptor subunit types and their expression.
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Animals↗
Biomedical subjects
R Shingai
Publications and source records attributed to R Shingai.
Effect of nebracetam on nicotinic and muscarinic acetylcholine receptors expressed in Xenopus oocyte by injecting exogenous mRNA.
Using voltage- and current-clamp methods the effects of nebracetam 4-aminomethyl-1-benzylpyrrolidine-2-one hemifumarate, WEB 1881 FU, CAS 118607-07-1), a new agent with nootropic property, on the nicotinic (nAChRs) and muscarinic acetylcholine receptors (mAChRs) were studied, which were expressed in Xenopus oocytes by injecting E. electricus mRNA and rat brain mRNA, respectively. Simultaneous application of nebracetam (0.03-2 mmol/l) with acetylcholine (ACh) (0.01-1 mmol/l) inhibited the ACh-responses of both nAChRs and mAChRs, whereas preapplication of these concentrations of nebracetam for 30 s to 1 min potentiated such inhibition. A simple competitive inhibition model for the effects of both drugs simultaneously applied yielded the inhibition constant, K1 of 0.419 and 0.212 mmol/l for nAChRs and mAChRs, respectively, indicating that the action on mAChRs is a little more potent than on nAChRs. Nebracetam induced a concentration-dependent slight increase in inward currents on mAChRs but not on nAChRs. It is suggested that the direct effects of nebracetam on nAChRs and mAChRS, which were induced only by a rather high concentration, as compared with the clinically expected plasma level, may be a contributing factor to the clinical effectiveness of the drug only if there is some critical change in the sensitivity to the drug.
Acetylcholine↗
Effects of subunit types of the cloned GABAA receptor on the response to a neurosteroid.
Combinations of cloned GABAA receptor subtypes, having the subunit combinations alpha i + beta 1 or alpha i + beta 1 + gamma 2 (i = 1, 2, 3), were expressed in Xenopus oocytes. The endogenous steroid 3 alpha-hydroxy-5 alpha-pregnan-20-one potentiates GABA currents induced therein by GABA. This potentiation was greater in the alpha 1 + beta and alpha 3 + beta 1 than in the alpha 2 + beta 1 combinations. The presence of the gamma 2-subunit increased the steroid potency in alpha 1 + beta 1 and alpha 2 + beta 1, but the combination alpha 3 + beta 1 + gamma 2 became much less steroid-sensitive. It is concluded that the steroid modification of the GABAA receptor is strongly influenced by the alpha- and the gamma 2-subunit types.
Animals↗
Intracellular cAMP regulates the response to NMDA in hippocampal neurons.
We have studied the effects of intracellular cyclic AMP (cAMP) on the response to N-methyl-D-aspartate (NMDA) in hippocampal cultured neurons by loading them with 2'-o-dibutyryladenosine 3', 5'-cyclic monophosphate (dcAMP) and have obtained evidence for regulation of Ca2+ release from intracellular stores by cAMP. Extracellular Ringer's solution was either Ca(2+)-containing or Ca(2+)-free. A brief initial stimulation with NMDA (10-100 microM, 12 s) was required before the neurons were loaded with dcAMP to potentiate the changes in intracellular Ca2+ concentration induced by the second stimulus of NMDA. Forskolin (10 microM) mimicked dcAMP, but to a lesser extent. A phorbol ester 12-o-tetradecanoylphorbol-13-acetate (100 nM) inhibited the effect of dcAMP.
Animals↗
Hippocampal neurotrophic factors influence the perikaryal size of septal acetylcholinesterase-containing neurons in culture.
The septal neurons were cultured under the following conditions: (1) treated with 7S nerve growth factor (NGF) (50 ng/ml); (2) grown with hippocampal cell-conditioned medium supernatant; (3) cocultivated with hippocampal cells; (4) cocultivated with cerebellar cells; (5) no treatment. Acetylcholinesterase histochemistry was used to identify cholinergic cells after pretreatment with diisopropylfluorophosphate. The mean values of the perikaryal major axis and minor axis at day 14 of culture were significantly larger in septal cells cocultivated with hippocampal cells than in septal cells grown under other conditions. NGF-treated septal cells showed a smaller, but significant, increase in the mean value of the major axis of these neurons.
Acetylcholinesterase↗
Acetylcholinesterase-containing neurons in the striatum, septum and hippocampus of the rat in embryonic culture and adult in situ.
Neurons of the rat brain, of either adult in situ or embryonic culture, have been studied by using a sensitive method for acetylcholinesterase (AChE) histochemistry. In the culture system, incubated for 6-18 days, AChE-positive neurons were found in tissues originating from the striatum and septum, but not in those from the hippocampus. These positive somata were morphometrically analyzed in terms of the cell size, i.e. the lengths of the major axis (Lmax) and the minor axis (Lmin) in cultured dishes of the striatum and septum; the mean Lmax was 20 and 22 microns, respectively. In in situ adult brain sections, a similar morphometric examination of AChE-positive neurons gave comparable results to those obtained in the culture system. An evaluation of both in vitro and in vivo through the histogramatical analysis revealed that the striatum contained more than two populations of AChE-positive cells differing in cell size. In contrast, a major single peak of Lmax was detected in the histogram of the septum. In both cases of striatum and septum in in situ adult brain, sagittal sections show larger size of Lmax, indicating that AChE-positive neurons are arranged in the sagittal direction. In studies on electrophysiological properties of large striatal cells in culture, both acetylcholine and glutamate induced changes in the membrane potential and/or the frequency of excitatory postsynaptic potential, while dopamine induced much smaller responses.
Acetylcholinesterase↗
The dose-response relationship for N-methyl-D-aspartate currents in cultured rat septal neurons: effects of magnesium ions and 2-amino-5-phosphonovaleric acid.
Dose-response relations of the peak ionic current induced by N-methyl-D-aspartate (NMDA) under Mg-free vs normal saline (1.2 mM Mg) conditions were obtained in cultured neurons dissociated from septa of fetal rat brains using the single electrode voltage clamp method. The effect of D,L-2-amino-5-phosphonovalerate (APV) on the dose-response relationship was also studied. In the Mg-free condition, the response to NMDA increased progressively with dose up to 0.2 mM, at which point the response saturated up to 1 mM NMDA (ED50 was about 70 microM). At larger doses (1-3 mM) the response increased sharply. In normal saline the response increased up to doses of 10 mM, and there was no apparent plateau. In a comparison between the depressant effects of APV and Mg ions on the peak current value, the depression caused by APV (10-30 microM) at higher NMDA concentrations was larger than that caused by Mg.
2-Amino-5-phosphonovalerate↗
Sequence and functional expression of a single alpha subunit of an insect nicotinic acetylcholine receptor.
We report the isolation and sequence of a cDNA clone that encodes a locust (Schistocerca gregaria) nervous system nicotinic acetylcholine receptor (AChR) subunit (alpha L1). The calculated molecular weight of the unglycosylated polypeptide, which contains in the proposed extracellular domain two adjacent cysteine residues which are characteristic of alpha (ligand binding) subunits, is 60,641 daltons. Injection into Xenopus oocytes, of RNA synthesized from this clone in vitro, results in expression of functional nicotinic receptors in the oocyte membrane. In these, nicotine opens a cation channel; the receptors are blocked by both alpha-bungarotoxin (alpha-Bgt) and kappa-bungarotoxin (kappa-Bgt). Reversible block of the expressed insect AChR by mecamylamine, d-tubocurarine, tetraethylammonium, bicuculline and strychnine has also been observed. These data are entirely consistent with previously reported electrophysiological studies on in vivo insect nicotinic receptors and also with biochemical studies on an alpha-Bgt affinity purified locust AChR. Thus, a functional receptor exhibiting the characteristic pharmacology of an in vivo insect nicotinic AChR can be expressed in Xenopus oocytes by injection with a single subunit RNA.
Amino Acid Sequence↗
Junctions form between catfish horizontal cells in culture.
Cone horizontal cells from the catfish retina extend out processes after a few days in culture that sometimes contact adjacent cone horizontal cells. Two types of specialized junctions were observed by electron microscopy along the newly formed contact areas. One junctional type consisted of prominent electron-dense material along and just under the plasma membrane of one or both of the contacting elements. Sometimes vesicle clusters were associated with these junctions. The other type of junction showed some electron-dense material along the membranes of both processes and patchy areas of close membrane apposition resembling gap junctions. In about half of the cases tested, electrical coupling was detected between cone horizontal cells that had made contact in culture. In no case was the coupling as tight as is typically found between horizontal cells that had formed gap junctions in vivo.
Animals↗
Some properties of membrane current fluctuations induced by kainate, quisqualate, and NMDA in cultured septal neurons of rat.
Ionic currents induced by glutamate, kainate, quisqualate, and N-methyl-D-aspartate (NMDA) in cultured septal neurons were analyzed by fluctuations analysis. The power spectrum (PWS) of NMDA current fluctuations always fitted a single Lorentzian. PWSs of the other agonists fitted the sum of two Lorentzians; however, the slopes of PWSs became larger and the PWSs became closer to single Lorentzians as the number of drug application increased. This may be explained in such a way that, in multiple conductance channels activated by these agonists, the high frequency component decreases the gating activity in later recordings, whereas the low frequency component keeps its gating kinetics.
Animals↗
Muscarinic cholinergic receptor binding and electrophysiological response to acetylcholine in cultured rat caudate putamen nucleus neurons.
Binding sites with specificity for N-[3H]methylscopolamine ([3H]NMS) are present in homogenates of primary culture neurons from the rat caudate putamen nucleus (CPN). Specific binding is saturable with respect to [3H]NMS. The Scatchard analysis indicates a dissociation constant of 0.6 nM and a density of 7.0 X 10(3) per cell, or 78 fmol/mg protein. In electrophysiological measurements, excitatory postsynaptic currents (EPSCs) in larger cells were increased by pressure application of acetylcholine under voltage clamp condition. The EPSCs may come from neighboring smaller cells with muscarinic acetylcholine receptors.
Acetylcholine↗
Single inward rectifier channels in horizontal cells.
The ion channels responsible for inward rectification in horizontal cells were studied using the patch clamp technique applied to isolated cells from goldfish retina. Inward currents recorded from these cells were identified as due to the opening of inward rectifier channels based on their ion selectivity, channel gating behavior, and the effects of external blocking ions. The single channel conductance was 20 pS in 125 mM external K+. The null current potential shifted with changes in the K+ concentration as expected for a channel permeable to K+, and the channel appeared to have little permeability to Na+. The probability of a channel being in an open state increased as the membrane was hyperpolarized from the K+ equilibrium potential (0 to -10 mV) over potentials ranging to -80 mV, in the presence of external Na+. The closing rate was insensitive to membrane potential in the presence of external Na+. The opening rate of the channel increased as the membrane was hyperpolarized. The increase in the probability of a channel being open at negative potentials was therefore caused by the voltage sensitivity of the rate of channel opening.
Animals↗
Voltage-clamp analysis of currents produced by glutamate and some glutamate analogues on horizontal cells isolated from the catfish retina.
Horizontal cells isolated from the catfish retina were exposed to radiolabeled glutamate, glycine, gamma-aminobutyric acid (GABA), and sucrose to determine if the enzymatic dissociation procedure altered the high-affinity uptake mechanism for GABA and generally reduced membrane selectivity. As in the intact retina, isolated cells could transport GABA but not the other substances. The horizontal cells were voltage clamped using a single low-resistance patch-type electrode. The acidic amino acid L-glutamate, and its analogues kainate and quisqualate, were applied to the cell by pressure ejection from a nearby pipette. All three agonists produced inward currents that reversed near O mV. Quisqualate produced a current with a similar time course as glutamate, but the time course of the response to kainate was faster. The agonists N-methyl-D-aspartate and L-aspartate had little effect on the membrane conductance. The current-to-voltage (I-V) relationship for all three agonists was nonlinear when the membrane potential was hyperpolarized. The nonlinearity was, at least in part, a result of the decreased response to the three agonists. Removal of Mg did not alter this nonlinear relationship. When the inward potassium rectifier was blocked with 100 microM Ba, the response to glutamate was increased compared with the control experiment before block by barium; however, the I-V relationship was still highly nonlinear. Thus glutamate block of the inward potassium current cannot account entirely for the nonlinear I-V. The increase in membrane permeability to specific ions in the presence of an agonist was determined by ion substitution experiments and measuring the shift in the reversal potential. The three agonists appear to increase the membrane permeability to cations but not to anions. The amino acid antagonists cis-2,3-piperidine dicarboxylic acid (PDA) and D-glutamyl glycine (DGG) were bath applied to test their ability to block the depolarizing effects of glutamate. DGG had no measureable effect at 100 microM concentration, whereas PDA reversibly reduced the glutamate response at 1 mM concentration although block was incomplete. Isolated horizontal cells responded to bath-applied glutamate in concentrations of 10-500 microM. In concentrations of glutamate greater than 50 microM, when the membrane potential was held at the resting potential, the inward current reached a maximum followed by a decrease to a steady-state level. This apparent time-dependent desensitization at high agonist concentrations was at least partially removed when Mg was removed from the bathing solution.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Excitable properties and voltage-sensitive ion conductances of horizontal cells isolated from catfish (Ictalurus punctatus) retina.
External horizontal cells were enzymatically dissociated from intact catfish (Ictalurus punctatus) retina and pipetted onto a small chamber attached to the stage of an inverted phase-contrast microscope. Individual horizontal cells were recognized by their large size and restricted dendritic arborization. Low-resistance (3-12 M omega) patch-type electrodes were used to record intracellular potentials and to pass current across the cell membrane under either current or voltage-clamp conditions. The average resting potential of isolated horizontal cells was -67 V + 6.9 mV (mean +/- SD, n = 40). At the resting potential, the cell membrane appears to be mainly permeable to K. A depolarizing current step evoked an action potential in the cell. The maximum rate of rise of the action potential (dV/dt) in normal physiological solution was 6.5 +/- 1.8 V/s (means +/- SD, n = 24) and was reduced to 1.2 +/- 0.39 V/s (means +/- SD, n = 9) in 1-10 micron tetrodotoxin (TTX) and 3.2 +/- 1.4 V/s (means +/- SD, n = 6) in Ca-free solution. The maximum dV/dt was reduced in 10 mM extracellular K concentration [K]o to about half of that seen in standard saline, and values in 30 or 80 mM [K]o were similar to that measured in TTX. Following an action potential, the membrane potential reached a plateau potential of + 17.4 +/- 8.1 mV (means +/- SD, n = 17) and remained depolarized for variable periods of time lasting from less than a second to a few minutes. When the plateau potential was long lasting, the cell repolarized slowly and upon reaching zero rapidly repolarized to the original resting potential. The duration of the plateau potential decreased or was absent in saline containing one of the following calcium channel antagonists: La, Cd, Co, or Ni. The voltage-clamp technique was used to identify the membrane currents responsible for the membrane potential changes seen under current clamp. Experiments were carried out using either a single or two individual electrodes. Fast and steady-state inward currents were recorded from isolated horizontal cells in the voltage range between -20 and +20 mV. These currents were a result of increased membrane conductance to both Na and Ca ions. The Na channels are inactivated at depolarized potentials and are TTX sensitive. Ca channels are partially inactivated at depolarized potentials. The Ca conductance is decreased by Cd, Co, Ni, and La. Ba can substitute for Ca in the channel.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Excitatory effects of L-glutamate and some analogs on isolated horizontal cells from the catfish retina.
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Animals↗
A comparison of spatio-temporal receptive fields of ganglion cells in the retinas of the tadpole and adult frog.
Receptive fields formed by ganglion cells were measured simultaneously in time and space in the adult and tadpole retinas. The spatio-temporal receptive-fields (STRFs) were measured by cross-correlating the spatio-temporal white-noise stimulus with the cells' spike discharges. Crosscorrelation was made photographically to extract the first order STRF kernel (approximately linear component of the STRFs). The time-course of STRF formed by the frog ganglion cells was twice as fast as that of the tadpole cells. The STRF formed by the tadpole ganglion cells was either a center-brightening or a center-dimming type whereas in the frog there was another class of cells which produced either complex STRFs or did not show any linear component in their STRFs. Size of the STRFs in frog and tadpole was similar in both frog and tadpole retinas.
Action Potentials↗
Sodium and calcium currents measured in isolated catfish horizontal cells under voltage clamp.
A low resistance suction microelectrode was used to record intracellularly from enzymatically dissociated horizontal cells obtained from catfish retina (Ictalurus punctatus). This microelectrode was connected to a voltage clamp circuit and the transmembrane currents were recorded during depolarizing and hyperpolarizing clamp pulses. Two fast transient inward currents were recorded, one sensitive to tetrodotoxin and one insensitive to this drug. A slower and persistent tetrodotoxin insensitive inward current was also recorded. The results indicate that separate membrane conductances exist for both sodium and calcium ions. We suggest that the action potential recorded from isolated horizontal cells is generated by both sodium and calcium currents.
Animals↗
Cellular analysis on localization of lens forming potency in the newt iris epithelium.
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Animals↗