The effects of midazolam on hippocampal dentate gyrus granule neurons from young and old Fischer 344 rats

The effects of midazolam (3 nM) perfusion on the membrane and synaptic properties of dentate gyrus granule neurons were examined in hippocampal slices obtained from young adult (4-6 months) and old (24-26 months) Fischer 344 rats. In young neurons, midazolam perfusion resulted in a hyperpolarization of the resting membrane potential with no apparent change in the input resistance. Midazolam perfusion also produced a significant increase in the amplitude of the post-spike train afterhyperpolarization (AHP). In neurons obtained from old animals, midazolam perfusion also produced a hyperpolarization of the resting membrane potential but did not significantly change the AHP. These effects may result from altered calcium homeostasis in neurons of the aged brain, and suggest that at least some of the direct actions of benzodiazepines on mammalian central neurons are altered during aging.     

Differential Sensitivity to Blockade by 4-Aminopyridine of Presynaptic Receptors Regulating [3H]Acetylcholine Release from Rat Hippocampus

The inhibitory effect of an adenosine analogue, R-N6-phenylisopropyl adenosine (R-PIA), of the cholinergic agonist carbachol, and of morphine on 3H efflux from [3H]choline-labeled field-stimulated rat hippocampal slices was compared with that produced by two inhibitors of N- and L-type Ca2+ channels, omega-conotoxin (CgTx; conotoxin GVIA) and cadmium chloride. 4-Aminopyridine (4-AP) caused a dose-dependent increase in evoked transmitter release, with a maximal effect (an almost threefold increase) at 100 microM. 4-AP (100 microM) did not affect the actions of CgTx, cadmium chloride, and R-PIA but almost abolished the effect of carbachol and morphine. The present results indicate that presynaptic muscarinic and opiate receptors reduce acetylcholine release by a mechanism that is somewhat different from that used by adenosine A1 receptors. Furthermore, the results indicate that presynaptic A1 receptors on hippocampal cholinergic neurons do not primarily regulate 4-AP-dependent potassium channels, but that they might act directly on a Ca2+ conductance.     

Inhibition in hippocampal pyramidal neurons during peripheral stimulation

Responses of hippocampal pyramidal neurons were investigated intracellularly in unanesthetized rabbits immobilized with tubocurarine. A single stimulus, applied to the sciatic nerve, evoked prolonged (up to 2.5 sec) hyperpolarization of the cell membrane, accompanied by inhibition of action potentials. The latent period of the evoked hyperpolarization was 48±16.4 msec, and its amplitude 2.5±1.9 mV. In some neurons the development of hyperpolarization potentials was preceded by excitation. The suggestion is made that hyperpolarization of the membrane of pyramidal cells during peripheral stimulation is manifested as an inhibitory postsynaptic potential (IPSP), generated with the participation of hippocampal interneurons. The possibility of prolonged tonic action of interneurons from outside as a cause of prolonged inhibition of the pyramidal neurons is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 1, No. 3, pp. 278–284, November–December, 1969.     

Actions of calcitonin gene-related peptide on rat sensory ganglion neurones

The membrane actions of calcitonin gene-related peptide (CGRP) and the effect of CGRP on the Ca-dependent action potential of rat dorsal root ganglion (DRG) neurons have been studied by means of an intracellular recording technique in isolated DRG of 2-3-week-old rats in vitro. Bath application of CGRP (10(-8)-10(-6) M for 1-5 min) elicited a slow reversible hyperpolarization and this hyperpolarizing effect was still observed in the medium containing TTX and TEA. However, about half of the large cells, classified by duration of action potential, were depolarized by CGRP. These membrane effects of CGRP were associated with an increase in membrane input resistance (about 20%). In addition, CGRP increased the duration of Ca-dependent action potentials. Our results are consistent with the role of CGRP as an excitatory neurotransmitter or neuromodulator in DRG-spinal cord.     

Apple procyanidins induce hyperpolarization of rat aorta endothelial cells via activation of K+ channels

Apple procyanidins (AP), one of the polyphenol-rich compounds, showed an endothelial-dependent vasorelaxation in rat aorta, but the mechanisms of beneficial effects are still unclear. The present study was designed to clarify the potential role of AP in rat aorta endothelial cells (RAECs). The treatment of RAECs with AP (1-10 μg/ml) resulted in a dose-dependent hyperpolarization with a maximum effect at 10 μg/ml, and for this reason, AP (10 μg/ml) was used in all the following experiments. AP-induced hyperpolarization was significantly inhibited by pretreatment of nonspecific K(+) inhibitor, tetraethyl ammonium chloride or specific K(+) channel inhibitors, iberiotoxin, glibenclamide, 4-aminopyridine and BaCl(2), as well as by high KCl or Ca(2+)-free solution. AP-induced hyperpolarization was also proved using 64-channel multielectrode dish system that can monitor a direct and real-time change of membrane potential. Furthermore, AP treatment caused a significant increase of nitric oxide (NO) production and cyclic guanosine monophosphate levels via endothelial NO synthase messenger RNA expression. The NO production was inhibited by N(G)-monoethyl-l-arginine or Ca(2+)-free solution and was completely abolished by their combination. Also, AP inhibited endothelial proliferation, while the effect was significantly abolished by N(G)-monoethyl-l-arginine or tetraethyl ammonium chloride. These findings suggest that AP induces both hyperpolarization of RAECs via multiple activation of K(+) channels and activation of NO/cyclic guanosine monophosphate pathway via increasing NO production or is responsible for antiangiogenic effect. Diminishment of hyperpolarization as well as NO production of AP in Ca(2+)-free solution implicated that AP would play a crucial role in promoting Ca(2+) influx into endothelial cells so as to promote both actions.     

Diverse effects of insulin-induced hyperpolarization on 3-O-methyl-D-glucose (3-O-MG) transport in frog skeletal muscles

It has been suggested that the insulin-induced hyperpolarization might be a mediator of the stimulatory action of insulin on glucose transport. The purpose of the present study was to investigate the relationship between the insulin-induced hyperpolarization and the stimulatory action of insulin on glucose transport in skeletal muscle. Satorius muscles dissected from bullfrogs (Rana catesbeiana) were used. Insulin induced a hyperpolarization of the membrane and an increase in the 3-O-Methyl-D-glucose (3-O-MG) uptake and extrusion. In the presence of valinomycin, insulin had no significant effect on the membrane potential. Insulin still had the stimulatory action on both the 3-O-MG uptake and extrusion even in the presence of valinomycin, under whose condition insulin had no significant effect on the membrane potential. The magnitude of the stimulatory action of insulin on the 3-O-MG uptake in the presence of valinomycin was smaller than that in the absence of valinomycin. The magnitude of the stimulatory action of insulin on the 3-O-MG extrusion was, on the contrary, larger than that in the absence of valinomycin. The abolishment of the insulin-induced hyperpolarization decreased the 3-O-MG uptake and increased the 3-O-MG extrusion. The observation in the present study concludes that insulin has two different actions on glucose transport. One of them is developed through the insulin-induced hyperpolarization, which increases the 3-O-MG uptake and decreases the 3-O-MG extrusion. The other action is irrelevant of the insulin-induced hyperpolarization and stimulates both the 3-O-MG uptake and extrusion.     

Effects of polymethylene derivatives of 4-aminopyridine on functional properties of hippocampal neurons

The effects of amiridin (9-amino-2,3,5,6,7,8-hexahydro-IH-cyclopenta(b) quinoline) and tacrine (1,2,3,4-tetrahydro-9-aminoacridine) on Schaffer collaterals--CAI field potentials were compared in rat hippocampal slice preparations. Similar dose-dependent increase in pop-spike amplitude was observed during slice perfusion with low concentrations of amiridin (5-50 microM) or tacrine (0.5-10 microM). This facilitation was not always fully reversible. The effect was accompanied by slight decrease in pop-EPSP amplitude suggesting membrane depolarization as a possible mechanism of pop-spike facilitation. Further increase in drug concentrations led to the depression and full blockade of pop-spike, that was associated with significant decrease in the pop-EPSP and fiber potential amplitudes. In contrast structurally related 4-aminopyridine evoked dose-dependent increase in both pop-EPSP and pop-spike amplitudes with all the concentrations tested (0.05-1000 microM), this facilitation was transformed into epileptiform response with 4-aminopyridine concentration about 500 microM. Possible mechanisms of drug actions on hippocampal neuron reactivity are discussed. It is suggested that amiridin might turn to be as effective as tacrine in symptomatic treatment of Alzheimer disease.     

大鼠海马脑中枝形吊灯样中间神经元的形态与电生理学特征

在大鼠海马脑片标本上,用微电极记录并标记了４个枝形吊灯样中间,约占所记录到的３９个中间神经元的１０％,与蓝状中间神经元显著不同之处是,这４个神经元均对细胞内去极化电流表现出不同程度的放电频率适应现象,用细胞内注射ｂｉｏｃｙｔｉｎ的方法证实,蓝状中间神经元轴突终末主要分布在锥体在颗粒细胞层,与这些细胞胞体形成接触,而４个枝形吊灯样中间神经元的轴突终末增高度选择性分布在锥体和颗粒细胞轴突始段区,上述结     

去甲肾上腺素引起蟾蜍背根神经节神经细胞膜电位变化的离子机制

本文应用细胞内记录方法,对去甲肾上腺素(NA)引起蟾蜍背根神经节(DRG)神经细胞膜电位去极化或超极化反应时的膜电导及翻转电位值进行了测量,并观察了钾和钙离子通道阻断剂灌流DRG对NA引起膜电位反应的影响。当NA引起去极化反应时,15个细胞的膜电导减小32.6％。少数细胞膜电导开始增加,继而减小(n=4)。NA超极化反应时膜电导增加13.2％(n=8)。NA去极化反应的翻转电位值为-88.5±0.9mV((?)±SE,n=4),NA超极化反应在膜电位处于-89至-92mV时消失。 钾通道阻断剂四乙铵可使NA去极化幅值增加73.7±11.9％((?)±SE,n=7),并使NA超极化幅值减小40.5％(n=4)。细胞内注入氯化铯使苯肾上腺素去极化幅值增加34.5％(n=4)。钙通道阻断剂氯化锰使NA去极化及超极化反应分别减小50.5±9.9％((?)±SE,n=10)和89.5±4.9％((?)±SE,n=7)。结果提示,NA引起DRG神经细胞膜电位的去极化或超极化反应,可能与膜的钾及钙通道活动的改变有关。     

Purification of hepoxilin epoxide hydrolase from rat liver

Hepoxilin epoxide hydrolase activity was demonstrated in rat liver cytosol using as substrate [1-14C] hepoxilin A3, a recently described hydroxy epoxide derivative of arachidonic acid. The enzyme was isolated and purified to apparent homogeneity using conventional chromatographic procedures resulting in 41-fold purification. The protein eluted during isoelectric focusing at a pI in the 5.3-5.4 range. The specific activity of the purified protein was 1.2 ng/microgram protein/20 min at 37 degrees C. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under denaturing conditions, a molecular mass value of 53 kDa was observed. Using native polyacrylamide gel electrophoresis, enzyme activity corresponded to the main protein band. The purified protein used hepoxilin A3 as preferred substrate converting it to trioxilin A3. The enzyme was marginally active toward other epoxides such as leukotriene A4 and styrene oxide. The Mr, pI, and substrate specificity of the hepoxilin epoxide hydrolase indicate that this enzyme is different from the recently reported leukotriene A4 hydrolase from human erythrocytes and rat and human neutrophils and constitutes a hitherto undescribed form of epoxide hydrolase with specificity toward hepoxilin A3. Tissue screening for enzyme activity revealed that this enzyme is ubiquitous in the rat.     

Membrane potential measurements during rat liver regeneration

Membrane potential was measured in perfused rat liver and was shown to increase from ?33 ± 1.0 mV in livers from normal rats to ?50 ± 1.1 mV in livers from rats 12 hr after partial hepatectomy. The hyperpolarization of the membrane in regenerating liver was no longer evident after perfusion with 1 mM ouabain for 5 min. Ouabain had a small (4 mV) depolarizing effect on membrane potential in normal liver. The potential measured in normal and regenerating liver decreased as a function of the external potassium concentration above 5 mM; however, the potential was more electronegative in regenerating liver compared to normal liver at all values of external potassium concentration, and the differences in potential between the two kinds of cells did not decrease at higher concentrations of external potassium. Thus, a plot of membrane potential vs external potassium concentration resulted in approximately parallel curves for the two different cell types. We conclude that hyperpolarization of the liver cell membrane is an early event during rat liver regeneration and results from an electrogenic Na-K pump.     

Acetylcholine-induced responses in the salivary gland cells ofHelisoma trivolvis

This study describes the actions of acetylcholine (ACh) on the salivary gland cells of Helisoma. Perfusion of the salivary gland cells with ACh produces a long-lasting depolarization accompanied by an increase in the input conductance of the gland cells. The depolarization is often followed by a long-lasting hyperpolarization. Carbamylcholine, tetramethylammonium, and choline also produce depolarizing responses. Nicotine and pilocarpine produce only a small depolarization in the gland cells. The following cholinergic antagonists are effective in blocking the gland-cell response to ACh: tetraethylammonium, atropine, hexamethonium, d-tubocurarine, and strychnine. A new preparation, the "isolated acinus," was utilized to obtain the reversal potential of the ACh response. The mean reversal potential in 10 preparations was -7 +/- 8 mV. The depolarizing phase of the response is dependent on the presence of both external calcium and external sodium ions. The long-lasting hyperpolarization is produced by the activity of an electrogenic sodium-potassium pump. The properties of the acetylcholine receptors on the salivary gland cells of Helisoma are compared with those described in other gastropod preparations.     

Serotonin agonist and antagonist actions in hippocampal CA1 neurons

The actions of serotonin (5-HT) and its putative agonists and antagonists were examined in vitro on hippocampal CA1 neurons using intracellular recordings, demonstrating that the cellular pharmacological effects can not necessarily be predicted from binding characteristics alone. The first response following 5-HT application was often a long-lasting (several minutes) hyperpolarization associated with decreased input resistance. Subsequent 5-HT applications caused only brief hyperpolarizations (30-120 s) and associated decreased input resistance, often followed by membrane depolarization. The post-spike train afterhyperpolarization (AHP) was prolonged for several minutes following the 5-HT induced hyperpolarization. 5-HT1 agonists (8-hydroxy-2-(di-n-propylamino)tetralin, 5-methoxytryptamine, MK-212) caused a prolonged hyperpolarization, decreased input resistance, and enhancement of the AHP. 5-HT applied following agonist application elicited only short-lasting hyperpolarizations. The 5-HT2 antagonists, cyproheptadine and mianserin, and a nonspecific 5-HT antagonist, methysergide, also caused a prolonged hyperpolarization with decreased input resistance. Spiperone, a nonspecific 5-HT antagonist, and ritanserin, a putative specific 5-HT2 receptor antagonist, depolarized CA1 neurons with little or no change in input resistance. The 5-HT-induced short-lasting hyperpolarization was not affected by drop application of 5-HT antagonists, except for methysergide, but perfusion of methysergide, ritanserin, and spiperone attenuated this response. The long-lasting 5-HT hyperpolarization might be mediated by 5-HT1A receptor activation, and the short-lasting hyperpolarization by another serotonergic receptor subtype.     

Molecular size and hydrophobicity as factors which determine the efficacy of the blocking action of amino-adamantane derivatives on NMDA channels

Neurons isolated from the CA-1 region of rat hippocampal slices by the "vibrodissociation" method were voltage-clamped in the whole cell configuration. The currents through NMDA channels were recorded in response to rapid application (solution exchange time <30 ms) of 100 microM aspartate (ASP) in a Mg2+-free solution in the presence of 3 microM glycine. When added to the ASP solution, amantadine as well as other amino-adamantane derivatives (AAD) produced an open-channel blockade of NMDA channels. Membrane hyperpolarization enhanced the AAD block. The affinity between NMDA channels and AAD was different for various AAD. The analysis of the experimental data led us to conclude that this affinity depended both on the molecular size of the blocker (calculated using HyperChem molecular modeling program) and on the blocker's hydrophobicity (calculated according to Hansch and Leo, 1979). The affinity between NMDA channels and AAD diminished with an increase in molecular size and raised with an increase in blocker's hydrophobicity. We propose an empirical equation which describes the dependence of affinity on the size and hydrophobicity of the blocker. The estimated critical diameter of the NMDA channel pore where the AAD blocking site is located proved to be about 17 A.     

Bending the Primary Cilium Opens Ca2+-sensitive Intermediate-Conductance K+ Channels in MDCK Cells

Increasing tubular fluid flow rate has previously been shown to induce K+ secretion in mammalian cortical collecting duct. The mechanism responsible was examined in the present study using MDCK cells as a model. The change in membrane potential difference (EM) of MDCK cells was measured with a fluorescent voltage-sensitive dye, DiBAC4(3), when the cell's primary cilium was continuously bent with a micropipette or by the flow of perfusate. Bending the cilium produced a hyperpolarization of the membrane that lagged behind the increase in intracellular Ca2+ concentration by an average of 36 seconds. Gd3+, an inhibitor of the flow-induced Ca2+ increase, prevented the hyperpolarization. Blocking K+ channels with Ba2+ reduced the flow-induced hyperpolarization, implying that it resulted from activation of Ca2+-sensitive K+ channels. Further studies demonstrated that the hyperpolarization was diminished by the blocker of Ca2+-activated K+ channels, charybdotoxin, whereas iberiotoxin or apamin had no effect, results consistent with the activation of intermediate-conductance Ca2+-sensitive K+ channels. RT-PCR analysis and sequencing confirmed the presence of intermediate-conductance K+ channels in MDCK cells. We conclude that the increase in intracellular Ca2+ associated with bending of the primary cilium is the cause of the hyperpolarization and increased K+ conductance in MDCK cells.     

Exendin-4, a new peptide from Heloderma suspectum venom, potentiates cholecystokinin-induced amylase release from rat pancreatic acini.

We examined the actions of exendin-4, a new peptide isolated from Heloderma suspectum venom, on dispersed acini from rat pancreas. Exendin-4 caused a 3-fold increase in cAMP but did not alter cellular calcium concentration. Exendin-4-induced increases in cAMP were inhibited by an exendin-receptor antagonist, exendin (9-39)NH2, but not by VIP-receptor antagonists. Whereas up to 1 microM exendin-4 alone did not alter amylase release, potentiation of enzyme release was observed when the peptide (greater than 30 pM) was combined with cholecystokinin. Potentiation of amylase release was also observed when exendin-4 was combined with carbamylcholine, bombesin or a calcium ionophore, A23187. These results indicate that stimulation of exendin receptors on rat pancreatic acini causes an increase in cellular cAMP. Although this increase in cAMP alone does not result in amylase release, combination of exendin-4 with agents that increase cell calcium results in potentiation of amylase release.     

Developmental patterns and plasticities: the hippocampal model.

Because developmental activity-dependent synaptic plasticity has been hypothesized to participate in network refinement, leading to the precise mapping of synaptic contacts constituting a functional brain, it is important to investigate the spatio-temporal structure of immature network activities. This article is briefly reviewing 15 years of studies on the immature rat hippocampus which, together with recent results obtained from awake rat pups, represent an important step toward the understanding of spontaneous patterns of activity and their potential implication in network maturation. Due to synergistic excitatory actions of GABA and glutamate receptor mediated signals during early postnatal life, spontaneous patterns of hippocampal activity that have been characterized both in vitro and in vivo are likely to provide hebbian modulation of developing glutamatergic and GABAergic synapses. Together with studies on trophic actions of these transmitters, study of the immature hippocampal network patterns and plasticities allows for multiple technical and conceptual approaches and represents an interesting experimental model for development studies.     

Effect of changes in pH of the medium on electrical activity of the Ranvier node

The effect of changes in pH of the medium from 4 to 10 on the action potential and its first derivative was studied at the original resting potential and during hyperpolarization of the membrane in experiments on single nodes of Ranvier. Raising the pH of the medium from 7 to 9 led to a decrease in amplitude of the action potential and of its derivative (V_max). During hyperpolarization of the membrane these parameters were fully restored. Lowering the pH of the solution led to an increase in the action potential and a decrease in V_max. During hyperpolarization of the membrane the action potential and its derivative were not completely restored. Under the influence of solutions with low and high pH values the duration of the action potential was increased. Changes in the action potential and in V_max with an increase in pH can be attributed to increased inactivation of the sodium permeability of the membrane, and in solutions with low pH to a decrease in the maximal sodium permeability and to weakening of its inactivation.A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 6, No. 2, pp. 205–210, March–April, 1974.     

Relationship between ionic surroundings and insulin actions on glucose transport and Na,K-pump in muscles

1. It is well known that insulin has various effects on glucose transport and the Na,K-pump in muscles. It is also known to have some effects on the membrane potential--in general, insulin induces a hyperpolarization of the membrane in muscles. Furthermore, it is suggested that the actions of insulin are modified by changes in ionic surroundings. 2. In this review article, the actions of ionic surroundings and insulin on glucose transport in muscles are discussed; in particular, the effects of changes in extracellular and/or intracellular concentrations of Na, K, Ca and H ions will be mentioned. 3. The actions of ionic surroundings and insulin on the Na,K-pump in muscles are discussed; in particular, the effects of changes in extracellular an/or intracellular concentrations of Na, K, Ca and H ions will be examined. 4. The relationship between the actions of ionic surroundings and insulin are discussed. 5. In particular, the effects of changes in ionic surroundings on the insulin-induced hyperpolarization of the membrane are discussed by relating it to the Na,K-pump function. The relationship between the insulin-induced change in membrane potential and glucose transport will be also mentioned.     

A 4-AP-sensitive current is enhanced by chronic carbon monoxide exposure in coronary artery myocytes

A physiological role of carbon monoxide has been suggested for coronary myocytes; however, direct evidence is lacking. The objective of this study was to test the effect of chronic carbon monoxide exposure on the K(+) currents of the coronary myocytes. The effect of 3-wk chronic exposure to carbon monoxide was assessed on K(+) currents in isolated rat left coronary myocytes by the use of the patch-clamp technique in the whole cell configuration. Moreover, membrane potential studies were performed on coronary artery rings using intracellular microelectrodes, and coronary blood flow in isolated heart preparation was recorded. Carbon monoxide did not change the amplitude of global whole cell K(+) current, but it did increase the component sensitive to 1 mM 4-aminopyridine. Carbon monoxide exposure hyperpolarized coronary artery segments by approximately 10 mV and, therefore, increased their sensitivity to 4-aminopyridine. This effect was associated with an enhancement of coronary blood flow. We conclude that chronic carbon monoxide increases a 4-aminopyridine-sensitive current in isolated coronary myocytes. This mechanism could, in part, contribute to hyperpolarization and to increased coronary blood flow observed with carbon monoxide.