Short-term action of insulin on Aplysia neurons: Generation of a possible novel modulator of ion channels

In mollusks as in other animals, peptides can act as hormones, growth factors, and neurotransmitters. The presence of insulin in vertebrate brain as well as its actions on nerve cells led us to examine the electrophysiological effects of the mammalian hormone on Aplysia neurons. Application of insulin extracellularly causes hyperpolarization of L14 and L10, identified neurons of the abdominal ganglion. This hyperpolarization is associated with a decreased membrane conductance that reverses at ?35 mV. We also injected inositol phosphate glycan (IPG) into the identified neurons. This complex sugar, which was purified from rat liver and which is a putative second messenger for insulin in nonneural vertebrate cells (Saltiel and Cuatrecasas, 1986; Saltiel, Osterman, and Darnell, 1988), causes hyperpolarization with decreased membrane conductance in L14 and L10 similar to the effects of insulin. Furthermore, exposure of isolated ganglia to insulin results in the generation of IPG with a compensating decrease in its glycosyl-phosphatidylinositol precursor. We suggest that, in addition to its other roles, insulin may function as a neuropeptide transmitter using IPG as a second messenger.     

The role of cAMP in generating excitatory serotonin responses of neurons in the edible snail

The neurons of the dorsal surface of snail Helix subesophageal ganglia respond similarly to the application of serotonin and the intracellular cAMP injection. These responses represent membrane depolarization. They increase in amplitude with membrane hyperpolarization and have a reverse potential between +10 and -30 mV. Presumably, these responses are associated with increased conductance for several ions. The values of the reverse potentials of serotonin and cAMP responses coincide in 7 out of 17 cells. Phosphodiesterase inhibitor theophylline caused a reversible increase in the amplitude and duration of both serotonin and cAMP responses and, used at a concentration of 1 mM, simulated them. The results obtained meet 2 out of 4 criteria demonstrating that cyclic nucleotides mediate a neurotransmitter response. It is suggested that cAMP may act as a second messenger in excitatory serotonin responses of snail Helix neurons.     

Properties of the slow excitatory postsynaptic potential in mammalian sympathetic ganglion cells

Two types of slow excitatory postsynaptic potentials (EPSPs) with different properties were found in neurons of the rabbit superior cervical sympathetic ganglion. In our group of neurons slow EPSPs increased during artificial hyperpolarization and decreased during depolarization of the membrane. The input resistance of the cells fell or remained unchanged during the development of slow EPSPs. In the second group of cells slow EPSPs increased during depolarization and decreased during hyperpolarization. The reversal potential of these responses, determined by extrapolation, was –78.9±3.6 mV. Depolarization responses to activation of muscarinic cholinergic receptors by acetylcholine or carbachol developed in 53% of neurons with an increase in input resistance and had a reversal potential of –83.2±6.7 mV. It is suggested that in cells of the first group the ionic mechanism of the slow EPSPs is similar to that of the fast EPSPs, whereas in cells of the second group its main component is a decrease in the potassium conductance of the membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 13, No. 4, pp. 371–379, July–August, 1981.     

一氧化氮对大鼠背根神经节神经元膜电位的作用

目的：观察一氧化氮对大鼠背根神经节神经元的作用及有关离子机制,并探讨一氧化氮在痛觉信息传递过程中的作用。方法：在分离的大鼠背根神经节标本上,应用细胞内记录技术,给予灌流一氧化氮供体硝普钠,观察硝普钠诱导的神经元膜反应。结果：大部分神经元对硝普钠敏感（79／102,77．45％）,滴加硝普钠（10～100mmol／L）后可引起浓度依赖性的超极化反应,剩余神经元没有反应。硝普钠（100mmol／L）可使神经元膜电导由（21．06±1．94）nS增加到（23．08±0．92）nS。L-NAME（非选择性一氧化氮合酶抑制剂,1mmol／L）、CdCl2（非选择性钙通道阻断剂,0．1mmol／L）、无Na^＋平衡盐液对硝普钠引起超极化反应无明显影响。四乙基碘化铵（非选择性钾通道阻断剂,10mmol／L）明显抑制硝普钠引起的超极化反应。结论：硝普钠在大鼠背根神经节神经元上可引起浓度依赖的超极化反应,且此超极化反应是钾电导介导的。     

Second messenger-controlled membrane conductance in locust (Locusta migratoria) olfactory neurons

The membrane conductance of olfactory neurons of Locusta migratoria was examined using the whole-cell configuration of the patch-clamp technique. Intracellular application of the second messenger inositol 1,4,5 trisphosphate (IP(3)) via a dual pipette technique elicited a clear increase in the membrane conductance. The IP(3)-induced conductance increased due to a rise in the extracellular concentration of calcium from 100&mgr;M to 4mM. Micromolar concentrations of ruthenium red partially blocked the IP(3)-induced increase in membrane conductance. Stimulating olfactory neurons with odour (hexenoic acid) resulted in an increase in the membrane conductance partially similar to that mediated IP(3). These findings suggest that stimulation with appropriate odours as well as intracellular application of IP(3) activate the same calcium-permeable ion channels in the plasma membrane of insect olfactory neurons.     

Cholinergic modulation of dopaminergic neurons in the mouse olfactory bulb

Considerable evidence exists for an extrinsic cholinergic influence in the maturation and function of the main olfactory bulb. In this study, we addressed the muscarinic modulation of dopaminergic neurons in this structure. We used different patch-clamp techniques to characterize the diverse roles of muscarinic agonists on identified dopaminergic neurons in a transgenic animal model expressing a reporter protein (green fluorescent protein) under the tyrosine hydroxylase promoter. Bath application of acetylcholine (1 mM) in slices and in enzymatically dissociated cells reduced the spontaneous firing of dopaminergic neurons recorded in cell-attached mode. In whole-cell configuration no effect of the agonist was observed, unless using the perforated patch technique, thus suggesting the involvement of a diffusible second messenger. The effect was mediated by metabotropic receptors as it was blocked by atropine and mimicked by the m2 agonist oxotremorine (10 muM). The reduction of periglomerular cell firing by muscarinic activation results from a membrane-potential hyperpolarization caused by activation of a potassium conductance. This modulation of dopaminergic interneurons may be important in the processing of sensory information and may be relevant to understand the mechanisms underlying the olfactory dysfunctions occurring in neurodegenerative diseases affecting the dopaminergic and/or cholinergic systems.     

Differences in the functional responses of two cell lines each expressing Pi-hydrolysis-coupled muscarinic receptors

Fluorescent oxonol dyes were used to measure changes in the membrane potential of two different cell lines each expressing Pi-hydrolysis coupled muscarinic receptors. Both SK-N-SH human neuroblastoma cells and m1-transfected A9 L cells express muscarinic receptors, which, when stimulated, elicit a large increase in intracellular calcium, and release of inositol phosphates. Despite the similarity in this second-messenger response, muscarinic stimulation resulted in a hyperpolarization in the transfected A9 L cells whereas a small depolarization was observed in the neuroblastoma cells. The carbachol-mediated hyperpolarization of the transfected A9 L cells could be mimicked by increasing intracellular calcium with the ionophore A23187, suggesting, that it may be mediated by calcium-activated potassium channels. Exposure of SK-N-SH cells to A23187, on the other hand, had no effect on the membrane potential. These studies demonstrate that the activation of a second messenger system does not solely dictate the electrophysiological response of a cell, but that other factors such as the expression of ion-channels is critical in the determination of that response.     

Electrophysiological study on the effects of leptin in rat dorsal motor nucleus of the vagus

Immunoreactivity of leptin receptor (Ob-R) has been detected in rat dorsal motor nucleus of the vagus (DMNV). Here, we confirmed the presence of Ob-R immunoreactivity on retrograde-labeled parasympathetic preganglionic neurons in the DMNV of neonatal rats. The present study investigated the effects of leptin on DMNV neurons, including parasympathetic preganglionic neurons, by using whole cell patch-clamp recording technique in brain stem slices of neonatal rats. Leptin (30-300 nM) induced membrane depolarization and hyperpolarization, respectively, in 14 and 15 out of 80 DMNV neurons tested. Both leptin-induced inward and outward currents persisted in the presence of TTX, indicating that leptin affected DNMV neurons postsynaptically. The current-voltage (I-V) curve of leptin-induced inward currents is characterized by negative slope conductance and has an average reversal potential of -90 +/- 3 mV. The reversal potential of the leptin-induced inward current was shifted to a more positive potential level in a high-potassium medium. These results indicate that a decrease in potassium conductance is likely the main ionic mechanism underlying the leptin-induced depolarization. On the other hand, the I-V curve of leptin-induced outward currents is characterized by positive slope conductance and has an average reversal potential of -88 +/- 3 mV, suggesting that an increase in potassium conductance may underlie leptin-induced hyperpolarization. Most of the leptin-responsive DMNV neurons were identified as being parasympathetic preganglionic neurons. These results suggest that the DMNV is one of the central target sites of leptin, and leptin can regulate parasympathetic outflow from the DMNV by directly acting on the parasympathetic preganglionic neurons of the DMNV.     

乙酰胆碱对蟾蜍背根神经节神经元膜电位的影响及离子机制

在离体灌流的蟾蜍背根神经节(DRG)标本上,用微电极进行胞内记录。在73个神经元中,依神经纤维的传导速度将神经元分为 A 型及 C 型,其中 A 型细胞67个,C 型6个,静息膜电位为-67.5±1.3mV((?)±SE)。当加4×10~(-4)—6×10~(-4)mol/L 乙酰胆碱(ACh),可观察到如下四种膜电位变化:1.超极化:幅值9.1±3.0mV((?)±SE,n=23);(2)去极化:幅值12.9±2.2mV((?)+SE,n=20);(3)双相反应(n=24):先超极化,后去极化,超极化幅值8.0±2.4mV((?)+SE),去极化幅值10.9±3.1mV((?)±SE);(4)无反应(n=6)。用阿托品(1.3×10~(-5)mol/L,n=23),或同时应用筒箭毒与六甲双铵(浓度均为1.4×10~(-5)mol/L,n=8)灌流,能分别阻断 ACh 引起的膜的超极化或去极化。ACh 引起超极化反应时膜电导平均增加13.8%,翻转电位值大约-96mV。四乙铵(TEA,20mmol/L)能使 ACh 的去极化幅值增加48.2±3.2%((?)±SE,n=6),超极化幅值减小79.4±4.3%((?)±SE,n=8)。MnCl_2(4mmol/L)使 ACh 的去极化及超极化幅值分别减小54.2±7.2%((?)±SE,n=5)及69.2±6.4%((?)±SE,n=14)。以上结果提示:ACh 引起的 DRG 神经细胞膜去极化反应由 N 型乙酰胆碱受体介导,而超极化反应由 Μ 型乙酰胆碱受体介导,前者可能包含了多种离子电导的改变,后者则可能与钾电导增加有关。     

Elementary Functional Properties of Single HCN2 Channels

Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels are tetramers that evoke rhythmic electrical activity in specialized neurons and cardiac cells. These channels are activated by hyperpolarizing voltage, and the second messenger cAMP can further enhance the activation. Despite the physiological importance of HCN channels, their elementary functional properties are still unclear. In this study, we expressed homotetrameric HCN2 channels in Xenopus oocytes and performed single-channel experiments in patches containing either one or multiple channels. We show that the single-channel conductance is as low as 1.67 pS and that channel activation is a one-step process. We also observed that the time between the hyperpolarizing stimulus and the first channel opening, the first latency, determines the activation process alone. Notably, at maximum hyperpolarization, saturating cAMP drives the channel to open for unusually long periods. In particular, at maximum activation by hyperpolarization and saturating cAMP, the open probability approaches unity. In contrast to other reports, no evidence of interchannel cooperativity was observed. In conclusion, single HCN2 channels operate only with an exceptionally low conductance, and both activating stimuli, voltage and cAMP, exclusively control the open probability.     

GABA-mediated synaptic inhibition of projection neurons in the antennal lobes of the sphinx moth,Manduca sexta

Responses of neurons in the antennal lobe (AL) of the moth Manduca sexta to stimulation of the ipsilateral antenna by odors consist of excitatory and inhibitory synaptic potentials. Stimulation of primary afferent fibers by electrical shock of the antennal nerve causes a characteristic IPSP-EPSP synaptic response in AL projection neurons. The IPSP in projection neurons reverses below the resting potential, is sensitive to changes in external and internal chloride concentration, and thus is apparently mediated by an increase in chloride conductance. The IPSP is reversibly blocked by 100 microM picrotoxin or bicuculline. Many AL neurons respond to application of GABA with a strong hyperpolarization and an inhibition of spontaneous spiking activity. GABA responses are associated with an increase in neuronal input conductance and a reversal potential below the resting potential. Application of GABA blocks inhibitory synaptic inputs and reduces or blocks excitatory inputs. EPSPs can be protected from depression by application of GABA. Muscimol, a GABA analog that mimics GABA responses at GABAA receptors but not at GABAB receptors in the vertebrate CNS, inhibits many AL neurons in the moth.     

血管升压素对大鼠背根神经节神经元膜的作用

为研究血管升压素(arginine vasopressin,AVP)对大鼠背根神经节(dorsal root ganglion,DRG)神经元的作用及其机制,用细胞内微电极记录技术记录离体灌流DRG神经元的膜电位。结果如下:(1)在受检的120个细胞中,大多数(81.67％)在滴加AVP后产生明显的超极化反应。(2)滴加AVP(10μmol/L)后膜电导增加约19.34％(P<0.05)。(3)灌流平衡液巾的NaCl以氯化胆碱(CH-Cl)置代和用Cd2+阻断Ca2+通道后,AVP引起超极化反应的幅值均无明显变化(P>0.05),而加入K+通道阻断剂四乙铵(TEA)后,AVP引起的超极化反应幅值明显减小(P<0.05)。(4)AVP引起的超极化反应可被AVP V.受体拈抗剂阻断。结果捉示,AVP可使DRG大多数神经元膜产生超极化,DRG神经元膜上存在AVP V,受体,且AVP引起的超极化反应是通过神经元膜上AVP V.受体介导的K+外流所致.AVP可能参与了初级感觉信息传入的调制。     

Glycosyl phosphatidylinositol (GPI)/inositolphosphate glycan (GPI): An intracellular signalling system involved in the control of thyroid cell proliferation

In porcine thyrocytes, TSH alone does not induce cell growth. Recently, it has been demonstrated that acute stimulation by TSH of porcine thyrocytes leads to release an inositolphosphate glycan (IPG) described as a putative second messenger for various growth factors in different cell types. IPG isolated from porcine thyrocytes induces proliferation of fibroblasts EGFR T17 and porcine thyrocytes. In porcine thyrocytes we have confirmed that cell growth requires the presence of both TSH and insulin. This effect is reproduced by 8-bromo cyclic AMP suggesting a mediation by intracellular cyclic AMP. Cooperative effects between 8-bromo cyclic AMP and IPG have also been evidenced and are in favour of a crosstalk between distinct signalling pathways.     

Purification and characterization of insulin-mimetic inositol phosphoglycan-like molecules from grass pea (Lathyrus sativus) seeds.

BACKGROUND: Signal transduction through the hydrolysis of glycosyl-phosphatidylinositol (GPI) leading to the release of the water-soluble inositol phosphoglycan (IPG) molecules has been demonstrated to be important for mediating some of the actions of insulin and insulin-like growth factor-I (IGF-I). MATERIALS AND METHODS: In the present study, GPI from grass pea (Lathyrus sativus) seeds has been purified and partially characterized on the basis of its chromatographic properties and its compositional analysis. RESULTS: The results indicate that it shows similarities to GPI previously isolated from other sources such as rat liver. IPG was generated from L. sativus seed GPI by hydrolysis with a GPI-specific phospholipase D (GPI-PLD). This IPG inhibited protein kinase A (PKA) in an in vitro assay, caused cell proliferation in explanted cochleovestibular ganglia (CVG), and decreased 8-Br-cAMP-induced phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression in cultured hepatoma cells. CONCLUSIONS: Our data indicate that L. sativus seed IPG possess insulin-mimetic activities. This may explain why L. sativus seeds have been used in some traditional medicines to ameliorate diabetic symptoms.     

Action of excitatory amino acids and their antagonists on hippocampal neurons

Intracellular recordings were obtained from guinea pig hippocampal neurons maintained in vitro. Current- and voltage-clamp techniques were used to study the effect of microiontophoresis of excitatory amino acid agonists. Modification of agonist responses by bath application of known concentrations of antagonist agents was also examined. All agonists used, glutamate, aspartate, N-methyl-D-aspartic acid (NMDA), and quisqualate, depolarized hippocampal neurons and caused repetitive firing. NMDA was also noted to induce burst-firing in some neurons. Quisqualate and NMDA were more potent than glutamate or aspartate. In slices perfused with a nominally calcium-free saline containing tetrodotoxin and manganese, quisqualate application produced a depolarization associated with a conductance increase. Under those conditions, NMDA-induced depolarizations caused apparent decreases as well as increases in conductance. The apparent decreases in conductance were observed in the voltage range of -40 to -70 mV, whereas increases in conductance were observed at membrane potentials more positive than -35 mV. Under voltage-clamp conditions, quisqualate produced an inward current whose amplitude increased with hyperpolarization and decreased upon depolarization, reversing near 0 mV. The conductance change induced by quisqualate was independent of voltage. NMDA application resulted in an inward current that was maximal around the resting potential and decreased with both hyperpolarization and depolarization. Response reversal was not observed with hyperpolarization to -100 mV but was apparent with depolarization beyond 0 mV. Conductance changes induced by NMDA were voltage dependent, and the application of this agent was associated with the appearance of a region of negative slope conductance in the current-voltage relationship. Apparent decreases in conductance in response to NMDA were reduced when the extracellular magnesium concentration was lowered. Response amplitudes were not affected. The NMDA receptor antagonist DL-2-amino-5-phosphonovalerate (DL-APV) was a potent and selective blocker of NMDA responses, whereas the antagonist DL-2-amino-4-phosphonobutyric acid (DL-APB) was less potent and did not select between NMDA and quisqualate responses. Analysis of iontophoretic dose-response curves indicated that DL-APV was a competitive antagonist. The results of these experiments indicate that hippocampal CA1 pyramidal neurons possess separate receptors for quisqualate and NMDA, with different pharmacological and electrophysiological profiles.     

Odorant-induced hyperpolarization and suppression of cAMP-activated current in newt olfactory receptor neurons

Although many studies have reported that odorants can elicit inhibitory responses as well as excitatory responses in vertebrate olfactory receptor neurons, the cellular mechanisms that underlie this inhibition are unclear. Here we examine the inhibitory effect of odorants on newt olfactory receptor neurons using whole cell patch clamp recording. At high concentrations, odorant stimulation decreased the membrane conductance and inhibited depolarization. Various odorants (anisole, isoamyl acetate, cineole, limonene and isovaleric acid) suppressed the depolarizing current in a dose-dependent manner. Furthermore, one odorant could suppress the depolarization caused by another odorant. The depolarization caused by isoamyl acetate was inhibited by anisole in cells that were excited by isoamyl acetate but not by anisole. Odorants were able to hyperpolarize cells that were depolarized by cAMP-induced conductance. Given that this inhibitory effect of odorants can affect excitation caused by other odorants, we suggest that it might play a role in coding odorants in olfactory receptor neurons.     

Locomotor reactions and excitability of neurons during the blockade of dopamine by haloperidol in vertebrate and invertebrate animals

Two groups of rats with different level of motor activities: high- and low-active animals, were distinguished. The blockade of dopamine receptors by haloperidol led to depression of locomotor activity in both groups of rats; in grape snails, haloperidol caused a decrease of the velocity of locomotor responses. In was found that within 5 minutes of intravenous injection of haloperidol the excitability of spinal centers of rats decreased; but in 30 minutes in started restoring. Chronic application of the preparation depressed the effect of posttetanic potentiation of H-response in gastrocnemius muscle of spinal rats. In command neurons of grape snail, chronic injections of haloperidol causes a significant hyperpolarization shift of membrane potential and an increase of threshold of the generation of action potential. It was shown that the selective pharmacological inhibition of dopaminergic system of the brain led to a decrease of excitability in some determined neurons of the snail and spinal motor centers of rats, as well as inhibited the locomotor responses both in vertebrate and in invertebrate animals.     

Ionic mechanism of 5-hydroxytryptamine induced hyperpolarization and inhibitory junctional potential in body wall muscle cells of Hirudo medicinalis.

Five-hydroxytryptamine (5-HT) causes a hyperpolarization and increased conductance of the leech body wall muscle cell membrane. If 5-HT is applied in the absence of the Cl minus ion, the response appears as a depolarization, whereas if 5-HT is applied in the absence of the K+ ion, the response is a hyperpolarization. In both cases, the conductance of the muscle cell membrane is increased. Stimulation of the peripheral nerve to the body wall muscle produces a complex junctional potential in muscle cells. Exposing the muscle to d-tubocurarine (d-TC) eliminates the excitatory component (EJP) of the complex potential. The inhibitory potential (IJP) that remains has an equilibrium potential at approximately 65 mV. Furthermore, this IJP appears as a depolarization when the nerve is stimulated in the presence of d-TC and low CL minus, whereas this is not the case if the nerve is stimulated in the presence of d-TC and low K+. The drugs BOL-148 and cyproheptadine block the IJP's in the body wall muscle. These data are interpreted as indicating that 5-HT acts on leech body wall muscle cells by increasing the conductance to the Cl minus ion and that the IJP's caused by nerve stimulation are probably the result of 5-HT release at nerve terminals. As a final point, it has been shown that the inhibition by 5-HT of the spontaneous EJP's that occur on the leech body wall muscle results from an inhibition of central neurons and not from any direct effect on the muscle cell or on peripheral synapses.     

IPG (inositolphosphate glycan) as a cellular signal for TGF-β1 modulation of chondrocyte cell cycle

The knowledge of transforming growth factor (TGF)-β receptors has greatly progressed in the recent years. TGF-β receptors type I and II have been implicated in the modulation of cell proliferation, whereas type III (betaglycan) may act as a component presenting TGF-β to its signaling receptors. In addition, four other proteins that bind TGF-β1 or TGF-β2 have been recently identified in some cell lines, three being anchored to the membrane through a glycosylphosphatidylinositol (GPI). Despite this knowledge, the molecular mechanism of signal transduction through the TGF-β receptors remain an enigma. TGF-β family does not signal via any of the classical pathways. As GPI anchors of membrane proteins have been implicated in the transduction of some hormonal effects, we investigated the putative role of GPI in signaling the TGF-β effects on the proliferation of rabbit articular chondrocytes (RAC). We previously showed that TGF-β1 increased DNA replication rate of RAC, with a recruitment of cells in G2/M followed by a subsequent mitosis wave. Here, we find that the factor causes specific GPI hydrolysis, with correlated increase of inositolphosphate glycan (IPG). This effect was specifically inhibited by antibodies that bind TGF-β1. Using [³H]-inositol labeling and Triton X-114 extraction, we demonstrate that a hydrophobic material from the membrane is cleaved by treatment of cell cultures with phosphatidylinositol specific phospholipase C (PI-PLC) or by exposure to TGF-β, supporting that a PI-anchored molecule gives rise to IPG by TGF-β-induced hydrolysis. The biological relevance of this hydrolysis was demonstrated by the enhancing effect of purified IPG on the DNA synthesis rate, which mimicked the TGF-β action. These results demonstrate that IPG could be an early messenger in the cellular signaling that mediates the effect of TGF-β on RAC growth. © 1993 Wiley-Liss, Inc.     

Elementary Functional Properties of Single HCN2 Channels

Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels are tetramers that evoke rhythmic electrical activity in specialized neurons and cardiac cells. These channels are activated by hyperpolarizing voltage, and the second messenger cAMP can further enhance the activation. Despite the physiological importance of HCN channels, their elementary functional properties are still unclear. In this study, we expressed homotetrameric HCN2 channels in Xenopus oocytes and performed single-channel experiments in patches containing either one or multiple channels. We show that the single-channel conductance is as low as 1.67 pS and that channel activation is a one-step process. We also observed that the time between the hyperpolarizing stimulus and the first channel opening, the first latency, determines the activation process alone. Notably, at maximum hyperpolarization, saturating cAMP drives the channel to open for unusually long periods. In particular, at maximum activation by hyperpolarization and saturating cAMP, the open probability approaches unity. In contrast to other reports, no evidence of interchannel cooperativity was observed. In conclusion, single HCN2 channels operate only with an exceptionally low conductance, and both activating stimuli, voltage and cAMP, exclusively control the open probability.