Ethanol sensitivity of postsynaptic receptors in the abdominal ganglion ofAplysia californica

We examined the effects of ethanol on post-synaptic receptors in the abdominal ganglion ofAplysia california, including those sensitive to acetylcholine (ACh) and γ-aminobutyric acid (GABA). Current elicited by direct application of acetylcholine to the cell body was composed of two components; a fast component (peak current - ca. 800 ms) and a slower component (peak current - ca. 12.2 s). These two components were differentially sensitive to ethanol. The fast component, which represents a chloride conductance, was significantly reduced in ethanol concentrations as low as 50 mM, while the slow component of the response, carried by potassium ions, was unaffected by ethanol at concentrations as high as 200 mM. GABAergic transmission was less sensitive to ethanol, showing no change in 50 mM ethanol, and a slight, but statistically significant reduction of the response to GABA in 100 mM ethanol. These results suggest that the cholinergic systems warrants further study, since the receptors gating the fast response consistently show sensitivity in the clinically-relevant range. In addition, results obtained studying these receptors may bear on the question of ethanol's site of action, since the receptor-channel complexes associated with the fast and slow responses are differentially sensitive to ethanol, although they are found in the same cell, and therefore would share the same bulk lipid matrix.     

Microcystin-LR, a potent protein phosphatase inhibitor, prolongs the serotonin- and cAMP-induced currents in sensory neurons ofAplysia californica

Microcystin-LR (MCYST-LR), a hepatotoxin produced by cyanobacteria, inhibited purified protein phosphatases (PrPs) from rabbit skeletal muscle and the enzymes fromAplysia with an IC₅₀ of approximately 10⁻¹⁰ M. MCYST-LR also prolonged both serotonin- (5-HT) and cyclic adenosine monophosphate-induced inward currents in sensory neurons ofAplysia. These results, which are consistent with inhibition ofAplysia PrPs, indicate that MCYST-LR may be a useful probe to elucidate the function of PrPs in neural tissues.     

Identification of neurons contributing to presynaptic inhibition in Aplysia californica

Electrical stimulation of the connectives presynaptically inhibits the PSP from cell L10 to the left upper quadrant cells (LUQC)⁸. The present report decribes the properties of some of the individual neurons contributing to this response. Action potentials produced in a cluster of cells in the abdominal ganglion reduce the amplitude of the L10-LUQC PSP for periods greater than 30 sec. At least some of their inhibitory action is mediated by a slow hyperpolarization of L10 which results in a decreased transmitter release⁴. In other cases, however, the inhibition is produced with no significant alteration of L10 membrane potential, indicating that additional mechanisms may also be present. The neurons producing these effects are approximately 75 μm in diameter and are located on the left ventral surface of the ganglion. They have axons in the connectives and are thus activated by stimuli previously utilized to produce presynaptic inhibition. They appear to be some of the same cells that produce a slow inhibition of ink motoneuron L14; one of these has been identified as L32². The identification of these cells allows for the further biochemical, biophysical and morphological analysis of the events underlying presynaptic inhibition.     

A modification of the glyoxylic acid induced histofluorescence technique for demonstration of catecholamines and serotonin in tissues ofAplysia californica

A modified glyoxylic acid technique was used to examine central and peripheral nervous tissues inAplysia californica. In addition to confirming the distribution of catecholamines and serotonin in the central nervous system, the method demonstrated the presence of monoamines in the opaline gland and bag cell clusters where they may act as transmitters. In conjunction with electrophysiological techniques this method may be useful to identify other monoamine-containing neurons inAplysia.     

Properties of single potassium channels in cultured primary astrocytes

The patch-clamp technique was used to characterize the single channel ion currents in primary cultures of rat astrocytes. The most dominant channel type, which was found in over half of the inside-out membrane patches, was a potassium channel. The measured reversal potential was -67 mV, which is close to the calculated Nernst potential for potassium ions (-80 mV). These potassium channels activated with bursts of very brief openings. Once activated the channels did not inactivate. The measured probabilities of the channels to be closed showed at least 3 different modes of channel behaviour: one voltage-independent and two voltage-dependent modes. During each activity-mode a 'main' conductance level plus two other conductance levels were observed. In some recordings a pronounced outward rectification could also be seen.     

钾通道阻断剂4-氨基吡啶诱导海马CA1锥体神经元钙瞬变

目的 研究4-氨基吡啶(4-AP)诱导的急性脑片海马CA1锥体神经元钙瞬变现象,探讨钾通道功能与钙瞬变的关系及可能机制.方法 荧光探针标记正常大鼠急性脑片海马神经元.共聚焦显微镜技术进行钙成像,观察不同浓度4-AP及细胞灌流液条件对神经元钙瞬变的影响.结果 低浓度(<15 mmol/L)4-AP诱导的钙瞬变峰值与剂量呈线性相关(r2=0.910,P=0.000),高浓度(20～80 mmol/L)4-AP诱导的钙瞬变峰值随浓度增高而下降.在无钙灌流液条件下,4-AP诱导的钙瞬变峰值水平下降,达峰时间延长,与含钙灌流液比较差异有统计学意义(P<0.05).结论 4-AP可诱导急性脑片海马CA1锥体神经元的钙瞬变,其机制包括细胞外钙内流与钙库钙释放.

Abstract：

Objective To investigate the calcium transient of CA1 pyramidal neurons induced by potassium blocker 4-aminopyridine (4-AP) in acute hippocampal slices to explore the relation between potassium channel function and calcium transient, and their mechanism. Methods Fluorescent probe was employed to mark the hippocampai neurons in acute brain slices of rats; confocal microscopy was used to perform calcium imaging to observe the influences of different concentrations of 4-AP and perfusate with/without calcium on calcium transient of CA1 pyramidal neurons. Results The response of [Ca2+]I to lower concentration of 4-AP (<15 mmol/L) was in a dose-dependent manner (r2=0.910, P=0.000); the higher the concentration of 4-AP (20-80 mmol/L), the lower the peak level of calcium transient. The latency and amplitude of calcium transient induced by 4-AP were obviously reduced when the extracellular condition was switched to an absence of calcium, which was significantly different as compared with that with calcium (P<0.05). Conclusion Blockade of potassium channels with 4-AP can increase [Ca2+]I in the hippocampal pyramidal neurons of acute slices. The increase of [Ca2+]1 to 4-AP could be ascribe to calcium release from intracellular stores and calcium influx from extracellular matrix.     

Caffeine modulates potassium currents in Drosophila neurons

We investigated the effects of caffeine on the delayed-rectifier potassium current (IK(DR)) which is important in repolarizing the membrane potential, and the transient A-type potassium current (IK(A)) which regulates neuronal firing threshold and the rate of repetitive action potentials. The whole-cell patch-clamp technique was used to measure the currents from cultured Drosophila neurons derived from embryonic neuroblasts. The currents were measured from neurons before and after the application of 1mM caffeine to the external saline of the same neuron. IK(DR) measured in the caffeine-containing solution (470+/-36 pA, n=18), was smaller than that measured in the control 6K/0Ca Tris solution (745+/-51 pA, n=18). IK(A) measured in the caffeine-containing solution (17+/-2 pA, n=16) was smaller than that measured in the control 6K/0Ca Tris solution (35+/-4 pA, n=16). These results indicate that caffeine reduces IK(DR) and IK(A) amplitudes and possibly leads to increased action potential frequency and enhanced neuronal excitability.     

Pentylenetetrazole-induced changes of the single potassium channel in primary cultured cerebral cortical neurons

To elucidate the behavior of the single ionic channels of cerebral cortical neurons during bursting activity, the effects of pentylenetetrazole (PTZ) on a single potassium channel of primary cultured cerebral cortical neurons from mice were examined. All of the examined 10-day-old primary cultured neurons of the cerebral cortex showed clear bursting activity after extracellular application of PTZ using whole-cell patch-clamp recording. Less than half of the examined single potassium channels, both outward and inward, of the 2- and 3-day-old as well as 6–10-day-old primary cultured cerebral cortical neurons showed the bursting-type open-close state by application of PTZ. The PTZ-sensitive single potassium channels were found in the voltage-dependent as well as calcium-activated channels.     

钾通道阻断剂4-氨基吡啶诱导海马CA1锥体神经元钙瞬变

目的 研究4-氨基吡啶(4-AP)诱导的急性脑片海马CA1锥体神经元钙瞬变现象,探讨钾通道功能与钙瞬变的关系及可能机制.方法 荧光探针标记正常大鼠急性脑片海马神经元.共聚焦显微镜技术进行钙成像,观察不同浓度4-AP及细胞灌流液条件对神经元钙瞬变的影响.结果 低浓度(<15 mmol/L)4-AP诱导的钙瞬变峰值与剂量呈线性相关(r2=0.910,P=0.000),高浓度(20～80 mmol/L)4-AP诱导的钙瞬变峰值随浓度增高而下降.在无钙灌流液条件下,4-AP诱导的钙瞬变峰值水平下降,达峰时间延长,与含钙灌流液比较差异有统计学意义(P<0.05).结论 4-AP可诱导急性脑片海马CA1锥体神经元的钙瞬变,其机制包括细胞外钙内流与钙库钙释放.     

钾通道阻断剂4-氨基吡啶诱导海马CA1锥体神经元钙瞬变

目的 研究4-氨基吡啶(4-AP)诱导的急性脑片海马CA1锥体神经元钙瞬变现象,探讨钾通道功能与钙瞬变的关系及可能机制.方法 荧光探针标记正常大鼠急性脑片海马神经元.共聚焦显微镜技术进行钙成像,观察不同浓度4-AP及细胞灌流液条件对神经元钙瞬变的影响.结果 低浓度(<15 mmol/L)4-AP诱导的钙瞬变峰值与剂量呈线性相关(r2=0.910,P=0.000),高浓度(20～80 mmol/L)4-AP诱导的钙瞬变峰值随浓度增高而下降.在无钙灌流液条件下,4-AP诱导的钙瞬变峰值水平下降,达峰时间延长,与含钙灌流液比较差异有统计学意义(P<0.05).结论 4-AP可诱导急性脑片海马CA1锥体神经元的钙瞬变,其机制包括细胞外钙内流与钙库钙释放.     

Phencyclidine blocks two potassium currents in spinal neurons in cell culture

We studied the effects of phencyclidine (PCP) on the transient and delayed outward K+ currents recorded from spinal cord neurons grown (10-20 days) in cell culture. Sodium channels were blocked with tetrodotoxin (1 microM) and solutions containing low calcium concentrations in the presence of Mg2+ or Co2+ (5 mM) were used to reduce Ca2+ currents. PCP decreased the amplitude and prolonged the decay phase of the action potentials recorded at a holding potential of -70 mV. PCP (0.1-0.5 mM) was more effective than tetraethylammonium (TEA) or 4-aminopyridine (4-AP) in reducing both transient and delayed currents. The amplitude of the transient current during control experiments was always larger than that of the delayed current. It appeared that 4-AP (5 mM) was more potent in blocking the transient current, while TEA (10 mM) modified the delayed current more effectively. Both currents were also reduced by about 10% when the cell soma was perfused with Co2+. This suggested that a small fraction of the total outward current is a Ca2+-activated K+ current. The PCP-induced blockade of K+ currents in central neurons coupled with the profound synaptic effects of the drug may provide the basis for explaining the psychopathology of this hallucinogenic agent.     

Paraoxon block of chloride conductance in cell R2 of Aplysia californica

In the presence of paraoxon, the amplitudes of chloride currents activated by acetylcholine (ACh) or gamma-aminobutyric acid (GABA) were reduced in cell R2 of Aplysia californica. IC₅₀ values were 12 and 9.7 μM for ACh and GABA responses, respectively. Paraoxon did not affect resting membrane potential, input resistance, or chloride reversal potential. Both the slopes and maxima of ACh and GABA concentration-response curves were reduced by paraoxon, suggesting that paraoxon antagonism of these responses is not competitive. The antagonism of ACh and GABA responses by paraoxon was not related to inhibition of acetylcholinesterase.     

Use of the single electrode voltage clamp to perform noise and relaxation studies of acetylcholine-activated channels in Aplysia neurons

The single electrode voltage clamp has been used to perform fluctuation analysis ("noise" analysis) and relaxation experiments in order to study the average lifetime and conductance of ACh-activated sodium channels in Aplysia neurons. Measured values of average channel lifetime, which is approximately 20 msec at --80 mV and 11 degrees C, and elementary conductance, which is approximately 8 pS, are consistent with previously published results using two electrode clamping. The frequency response of the clamp was evaluated to determine its capabilities and limitations for the study of membrane currents. Sinusoidal currents injected into a voltage-clamped model membrane to simulate the frequency components of membrane noise are accurately reproduced at frequencies up to 500 Hz. Following a voltage clamp command, the new membrane potential is established in less than 2 msec, and current relaxations recorded after that time can be used to determine average channel lifetime. Since the frequency response of the clamp is much greater than the average lifetime of ACh-activated channels in Aplysia neurons, the single electrode voltage clamp is comparable to conventional two-electrode systems for investigating the properties of these channels, and may also be useful in other systems in which the time course of membrane currents is much slower than the frequency response of the clamp.     

An arginine vasotocin-like neuropeptide is present in the nervous system of the marine mollusc Aplysia californica

Radioimmunoassays and high pressure liquid chromotography have been used to demonstrate the presence of an arginine-vasotocin-like peptide (AVT) in the anterior ganglia of Aplysia. Previously, AVT, using similar methods, was found to be present only in vertebrates. AVT when perfused over the abdominal ganglion (10⁻⁶–10⁻¹²M) was found to increase the bursting activity of R₁₅, to decrease the bursting activity of L₃–L₆ and to increase the CNS's suppressive influence over the gill withdrawal reflex evoked by siphon stimulation. The AVT present in the nervous system of Aplysia may mediate long-term suppression of gill reflex behaviors induced by factors such as satiation and, as well, regulate the activity of certain neurosecretory neurons.     

Distribution of AT4 receptors in the Macaca fascicularis brain

Angiotensin IV (Val Tyr lie His Pro Phe), administered centrally, increases memory retrieval and induces c fos expression in the hippocampus and piriform cortex. Angiotensin IV binds to a high affinity site that is quite distinct in pharmacology and distribution from the angiotensin II AT₁ and AT₂ receptors and is known as the AT₄ receptor. These observations suggest that the AT₄ receptor may have multiple central effects. The present study uses in vitro receptor autoradiography, and employs [¹²⁵I]angiotensin IV to map AT₄ receptors in the macaca fascicularis brain. The distribution of the AT₄ receptor is remarkable in that its distribution extends throughout several neural systems. Most striking is its localization in motor nuclei and motor associated regions. These include the ventral horn spinal motor neurons, all cranial motor nuclei including the oculomotor, abducens, facial and hypoglossal nuclei, and the dorsal motor nucleus of the vagus. Receptors are also present in the vestibular, reticular and inferior olivary nuclei, the granular layer of the cerebellum, and the Betz cells of the motor cortex. Moderate AT₄ receptor density is seen in all cerebellar nuclei, ventral thalamic nuclei and the substantia nigra pars compacta, with lower receptor density observed in the caudate nucleus and putamen. Abundant AT₄ receptors are also found in areas associated with cholinergic nuclei and their projections, including the nucleus basalis of Meynert, ventral limb of the diagonal band and the hippocampus, somatic motor nuclei and autonomic preganglionic motor nuclei. AT₄ receptors are also observed in sensory regions, with moderate levels in spinal trigeminal, gracile, cuneate and thalamic ventral posterior nuclei, and the somatosensory cortex. The abundance of the AT₄ receptor in motor and cholinergic neurons, and to a lesser extent, in sensory neurons, suggests multiple roles for the AT₄ receptor in the primate brain.     

Monoamine-containing neurons in the aplysia brain

The localization of monoamine-containing neurons in the CNS of Aplysia depilans has been studied by fluorescent histochemistry (the glyoxylic acid condensation method) and microspectrofluorimetry. Yellow fluorescent nerve cells and fibers show the emission maximum at 515–520 nm which corresponds to that of serotonin fluorophore in a model system. Green fluorescent nerve cells have the emission maximum at 485 nm which corresponds to that of catecholamine. Central catecholamine-containing neurons were found in cerebral, buccal, pedal and unpaired abdominal ganglia. The majority of them were revealed in cerebral ganglia (about 40). Serotonin-containing neurons are abundant in cerebral and pedal ganglia. More than 30 serotonin-containing nerve cells were localized in cerebral ganglia. In the right pedal ganglion approximately 100 neurons were revealed; in the left one about 150. In the abdominal ganglion all nerve cells of this chemical type (except one) are located in the right hemiganglion. The results are summarized in corresponding schemes.     

缺氧对大鼠大脑皮层神经元钙激活性钾通道的影响

目的　研究缺氧对大鼠大脑皮层神经元钙激活性钾 (Kca)通道的影响 ,以揭示神经元抗缺血损伤的电生理机制。方法　在不同缺氧条件下 ,应用膜片钳技术记录大脑皮层神经元上Kca通道电流活动。电流信号经放大、滤波及A/D、D/A转换后输入微机进行采样和储存。实验数据应用PClamp(6 .0 .2 )软件进行分析处理。结果　缺氧对通道的开放概率 (Po)及平均开放时间 (To)有明显影响 ,在缺氧实验早期通道Po明显增加 ,其中 10 μmol·L-1NaCN缺氧组其增加程度大于 2 0 μmol·L-1和 30 μmol·L-1NaCN缺氧组 (P <0 .0 5 )。而在缺氧实验后期通道Po和To明显降低 ,其中 30 μmol·L-1NaCN缺氧组其降低程度大于 2 0 μmol·L-1和 10 μmol·L-1NaCN缺氧组 (P <0 .0 5 )。结论　缺氧早期大脑皮层神经元Kca通道激活 ,产生超极化电位 ,从而稳定细胞膜 ,降低细胞兴奋性 ,延缓缺氧除极的发生 ,这可能是神经元自身的一种代偿作用     

蛛网膜下腔出血后脑血管痉挛中钾通道的研究进展

脑血管痉挛（cerebral vasospasm，cvs）是蛛网膜下腔出血（subarachnoid hemorrhage，SAH）后高致死率、致残率的主要原因之一，发生率约为45％。过去数十年来国内外学者们做了大量动物实验和临床试验，从细胞、分子水平对CVS的发病机制进行了论述，然而至今仍未完全阐明。目前，普遍认为SAH后CVS是由多因素所致，可能与内皮的损伤、平滑肌细胞内离子泵功能的紊乱和蛋白激酶（PKC）的激活、血管壁炎症反应以及血管细胞的增殖等有关。二十世纪八十年代以来，随着分子生物学技术的发展，以及电压钳和单细胞通道记录等新技术的应用，医学界对钾通道的认识逐渐深入，在钾离子生理药理的研究领域特别是钾通道开放剂的开发项目上屡有重大突破，而后者也有望成为继钙通道阻滞剂后又一通过作用于离子通道而改善血管痉挛的临床用药。本文结合国内外最新相关文献报道，主要就SAH后钾通道的生理状态与CVS之间关系的研究进展做一综述。     

Nitric oxide activates a potassium current in olfactory receptor neurons from Caudiverbera caudiverbera and Xenopus laevis

The putative role of nitric oxide (NO) in the physiology of olfactory receptor neurons (ORNs) is controversial. Here we report that pulses of NO caused an outward current in voltage-clamped isolated olfactory neurons. The I–V relation of this effect, its sensitivity to charybdotoxin and its dependence on external potassium suggest that NO activates a K⁺-conductance. As blockers of soluble guanylyl cyclases failed to affect the current, we conclude that NO opens K⁺-channels in a cGMP-independent manner.