A Model of Information Carried over a Neuron Soma

A series of cellular automata models of amino acid side chains on a neuron soma membrane have been created to simulate their hydropathic influences on adjacent water molecules. The presence of pathways, referred to as water wires, is identified. These pathways are invoked as passage ways across a neuron soma of proton hopping carrying the information from dendrites to the axon hillock.     

Inhibitory Miniature Potentials in the Stretch Receptor Neurons of Crayfish

Intracellular microelectrodes inserted into the soma of crayfish stretch receptor neurons record frequent fluctuations of the membrane potential. Time course, amplitude, and interval distribution indicate that they are miniature potentials. At the average resting potential the polarity of the miniature potentials depends on the anion used in the microelectrode: KCl electrodes record depolarizing, K citrate or K₂SO₄ electrodes, hyperpolarizing miniature potentials. The inhibitory postsynaptic potentials (i.p.s.p.'s) show a similar polarity change. The reversal potentials of i.p.s.p.'s and miniature potentials are equal and within 10 mv of the resting potential, more negative with K citrate (or K₂SO₄), less negative with KCl electrodes. Reversal can be accomplished by changing the membrane potential by stretching or by current passing. Injection of Cl^- into the soma or replacement of external Cl by propionate results in an abrupt increase of the amplitude of the miniature potentials lasting for several minutes. The miniature potentials like the i.p.s.p.'s are reversibly abolished by the application of picrotoxin and γ-aminobutyric acid. They are not affected by tetrodotoxin, nor by acetylocholine, eserine, or atropine. It is concluded that the miniature potentials represent a spontaneous quantal release of transmitter substance from inhibitory nerve terminals, and that the transmitter substance predominantly increases the Cl^- permeability of the postsynaptic membrane. The effect of the spontaneously released transmitter on the behavior of the receptor neuron is considerable. The membrane conductance is increased by up to 36% and the excitability is correspondingly depressed.     

Spike Potentials Recorded from the Insect Photoreceptor

Slow and spike potentials were recorded from single cells in the receptor layer of the compound eye of the drone of the honeybee. From electron microscopic observation of the drone ommatidium, it was concluded that the response had been recorded from the retinula cell. The following hypothesis is suggested for the initiation of spike potentials in the drone compound eye: Photic stimulation results in a decrease in the resistance of all or part of the retinula cell membrane, giving rise to the retinal action potential. The retinal action potential causes outflow of the current through the proximal process of the cell. This depolarizing current initiates spike potentials in the proximal process or axon of the retinula cell which are recorded across the soma membrane of the retinula cell.     

Site of Origin and Propagation of Spike in the Giant Neuron of Aplysia

The form and time sequence of spikes generated by orthodromic, antidromic, and direct stimulation and during spontaneous activity have been studied with intracellular electrodes simultaneously introduced in the soma and in different parts of the axon of the giant nerve cell of Aplysia. Evidence was obtained that under normal conditions of excitability, the spike originates at some distance from the soma in an axonal region with a higher excitability surpassing that of the surrounding membranes. Between the trigger zone and the soma is situated a region of transitional excitability where the conduction of the spike towards the soma may be blocked at a functionally determined and variable locus. The cell body is electrically excitable, but has the highest threshold of all parts of the neuron. The inactivation or even the removal of the cell body does not suppress synaptic transmission.     

Computer Simulation of After-Inhibition in Crayfish Slowly Adapting Stretch Receptor Neuron

A theoretical model is described which is able to mimic the responses of slowly adapting stretch receptor neurons of crayfish to applied currents. Its principal feature is postspike inhibition, in which each nerve impulses produces a small inhibitory current that decays with a simple exponential time-course that is long compared with normal interspike intervals. The model was simulated with both an analogue and a digital computer. Parameters for particular model neurons were determined both by an analysis of experimental data obtained from adaptation to constant injected currents, and by matching computer output to the data. Parameter values estimated by the two techniques agreed within ±10%. Model parameters determined from adaptation data successfully predicted the magnitude and time-course of posttetanic hyperpolarization (PTH) in the stretch receptor neuron. In addition, model neurons were able to reproduce posttetanic depression (PTD) as seen in stretch receptor cells.     

An Analysis of Field Potentials During Different Tailflip Behaviours in Crayfish

Crayfish tailflips have been intensively studied to reveal the decision-making processes and neural organisation underlying a stereotyped escape behaviour. Three behaviours mediated by different neural pathways have been well described: medial giant, lateral giant and non-giant tailflips. It has proved difficult to distinguish between the three without invasive or restrictive experimental manipulation. We report unambiguous differences between the signals generated by the crayfish Cherax destructor during the three types of tailflip when recorded by bath electrodes placed in the holding aquarium. Using our ability to distinguish between the different behaviours in freely moving animals we examined the relationship between the type of tailflip evoked by stimulation to different parts of the body. The transition zone between medial and lateral giant tailflips is the thoracic-abdominal border but it is not absolute and some stimuli produce responses that cannot be unambiguously assigned to either behavioural category. We examined the latency between stimulation at different points down the length of the body and the appearance of the electrical signal accompanying escape for both medial and lateral tailflips. We used two methods to estimate the proportion of the latency accounted for by giant fibre conduction velocity. The results support current views of the differences between the activation sites of the two giant fibre systems and suggest why stimulation in the transition zone results in ambiguous outcomes.     

Automated and Accurate Detection of Soma Location and Surface Morphology in Large-Scale 3D Neuron Images

Automated and accurate localization and morphometry of somas in 3D neuron images is essential for quantitative studies of neural networks in the brain. However, previous methods are limited in obtaining the location and surface morphology of somas with variable size and uneven staining in large-scale 3D neuron images. In this work, we proposed a method for automated soma locating in large-scale 3D neuron images that contain relatively sparse soma distributions. This method involves three steps: (i) deblocking the image with overlap between adjacent sub-stacks; (ii) locating the somas in each small sub-stack using multi-scale morphological close and adaptive thresholds; and (iii) fusion of the repeatedly located somas in all sub-stacks. We also describe a new method for the accurate detection of the surface morphology of somas containing hollowness; this was achieved by improving the classical Rayburst Sampling with a new gradient-based criteria. Three 3D neuron image stacks of different sizes were used to quantitatively validate our methods. For the soma localization algorithm, the average recall and precision were greater than 93% and 96%, respectively. For the soma surface detection algorithm, the overlap of the volumes created by automatic detection of soma surfaces and manually segmenting soma volumes was more than 84% for 89% of all correctly detected somas. Our method for locating somas can reveal the soma distributions in large-scale neural networks more efficiently. The method for soma surface detection will serve as a valuable tool for systematic studies of neuron types based on neuron structure.     

The Action Potentials and Currents on the I-V Plane in the Molluscan Neuron

The action potentials and the corresponding transmembrane currents, directly recorded in the F1 neuron of Helix aspersa by the Self-clamp Technique, were plotted on the I-V plane to represent the real electrical cycle of the cell membrane during activity. The membrane electrical cycle, experimentally obtained, agreed in several aspects with a similar cycle obtained from calculated data on the giant axon of Loligo, but not for the sign, with the consequence of a different localization, as far as voltage and time are concerned, of the negative impedance period. The negative impedance proved to be −614 ± 181 Ω cm² and corresponded to the late phase of the repolarization after the action potential peak. A constant positive impedance was found of 522 ± 131 Ω cm² during the ascending tract of the action potential. These two results are in contrast with previous analyses. The simultaneous availability of the conjugate voltage and current directly measured signals led to the immediate representation of the membrane total conductance in its real time course during activity, in agreement with the Hodgkin and Huxley predictive model. The peak conductance was 1.9 ± 0.7 mmho/cm² in this preparation. The electrical work spent to sustain a single active event proved to be 70 ± 19 nJ/cm². A vectorial representation of the membrane electrical activity is proposed to describe analytically the characteristic behaviour of excitable cells, as well as a new method that utilizes the only action potential to measure the threshold potential in spontaneously discharging cells. The proposed new experimental protocol, based on the use of the Self-clamp Technique, proved to be faster, easier, more productive when compared with the conventional methods; it could be used advantageously in the electrophysiological studies on excitable cells both to define the basic conditions of the investigated preparation and to directly evaluate the effects of subsequent pharmacological stimulations.     

Slow and Spike Potentials Recorded from Retinula Cells of the Honeybee Drone in Response to Light

Responses to light recorded by means of intracellular microelectrodes in isolated heads kept in oxygenated Ringer solution consist of a slow depolarization. Light adaptation increases the rates of depolarization and repolarization and decreases the amplitude of the response. Qualitatively these changes are similar to those observed in Limulus by Fuortes and Hodgkin. They are rapidly reversible during dark adaptation. In retinula cells of the drone eye a large single spike is recorded superimposed on the rising phase of the slow potential. The spike is a regenerative phenomenon; it can be triggered with electric current and is markedly reduced, sometimes abolished by tetrodotoxin. In rare cases cells were found which responded to light with a train of spikes. This behavior was only found under "unusual" experimental conditions; i.e., towards the end of a long experiment, during impalement, or at the beginning of responses to steps of strongly light-adapted preparations.     

The Effects of Various Ions on Resting and Spike Potentials of Barnacle Muscle Fibers

Effects of monovalent cations and some anions on the electrical properties of the barnacle muscle fiber membrane were studied when the intra- or extracellular concentrations of those ions were altered by longitudinal intra-cellular injection. The resting potential of the normal fiber decreases linearly with increase of logarithm of [K⁺]_out and the decrement for a tenfold increase in [K⁺]_out is 58 mv when the product, [K⁺]_out ·[Cl^-]_out, is kept constant. It also decreases with decreasing [K⁺]_in but is always less than expected theoretically. The deviation becomes larger as [K⁺]_in increases and the resting potential finally starts to decrease with increasing [K⁺]_in for [K⁺]_in > 250 mM. When the internal K⁺ concentration is decreased the overshoot of the spike potential increases and the time course of the spike potential becomes more prolonged. In substituting for the internal K⁺, Na⁺ and sucrose affect the resting and spike potentials similarly. Some organic cations (guanidine, choline, tris, and TMA) behave like sucrose while some other organic cations (TEA, TPA, and TBA) have a specific effect and prolong the spike potential if they are applied intracellularly or extracellularly. In all cases the active membrane potential increases linearly with the logarithm of [Ca⁺⁺]_out/[K⁺]_in and the increment is about 29 mv for tenfold increase in this ratio. The fiber membrane is permeable to Cl^- and other smaller anions (Br^- and I^-) but not to acetate^- and larger anions (citrate^-, sulfate^-, and methanesulfonate^-).     

Calelectrin, a Calcium-Dependent Membrane-Binding Protein Associated with Secretory Granules in Torpedo Cholinergic Electromotor Nerve Endings and Rat Adrenal Medulla

Calelectrin, a calcium-dependent membrane-binding protein of subunit molecular weight 32,000 has been isolated from the electric organ of Torpedo, and shown to occur in cholinergic neurones and in bovine adrenal medulla. In this study a monospecific antiserum against the Torpedo protein has been used to study the localization of calelectrin in the rat adrenal gland. The cortex was not stained, whereas in the medulla the cytoplasm of the chromaffin cells was stained in a particulate manner. An identical staining pattern was obtained with an antiserum against the chromaffin granule enzyme dopamine beta-hydroxylase, although the two antisera did not cross-react with the same antigen. The purified protein aggregates bovine chromaffin granule membranes and cholinergic synaptic vesicles and also self aggregates in a calcium-dependent manner. Negative staining results demonstrate that calcium induces a transformation of the purified protein from circular structures 30-80 nm in diameter into a highly aggregated structure. Calelectrin may have a structural or regulatory role in the intracellular organization of secretory cells.     

Synapse-to-Nucleus Communication through NFAT Is Mediated by L-type Ca2+ Channel Ca2+ Spike Propagation to the Soma

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Modulation of Activity of One Neuron by Subthreshold Slow Potentials in Another in Lobster Cardiac Ganglion

A direct demonstration is given of interaction between specific neurons without impulses, via graded slow potentials electrotonically spread from one cell to another. Repetitive polarizing or depolarizing current pulses of 50 to 200 msec. and subthreshold intensity were passed through an intracellular electrode in the soma of a follower cell of the isolated ganglion. When the frequency is near the natural rhythm of impulse bursts corresponding to heart beats and arising in a pacemaker cell 5 to 10 mm. posteriorly, the bursts rapidly become synchronized with the pulses. The effect disappears upon withdrawing the intracellular electrode. Brief pulses or full spikes in the follower are not effective. Hyperpolarizing long pulses attract the burst to a fixed period after the end of the pulse, depolarizations after the beginning of the pulse. The natural rhythm promptly reappears when the pulses are stopped and occasionally breaks through during weak repetitive pulses. Current pulses in postsynaptic cells also alter the threshold of a presynaptic neuron to externally applied stimuli. Some kind of direct, low resistance pathway for electrotonic spread, discriminating against spikes because of their brevity, is inferred, providing a basis for subthreshold interaction which is specific and not by way of a field effect. Due to the sensitivity of modulation of ongoing rhythms, electrotonic currents can be effective even after decrementing over several millimeters.     

真核表达MERS-CoV刺突蛋白亚单位的信号肽序列优化研究

中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus,MERS-CoV)的刺突蛋白(Spike,S)亚单位1(S1)是引起宿主免疫反应和产生中和抗体的主要靶抗原,也是疫苗研发和病原检测的重要靶标,选用适宜的真核表达系统高效表达S1蛋白是进行相关研究的基础。为确定MERS-CoV S1在哺乳动物细胞中高效分泌性表达的信号肽序列,构建了含高斯荧光素酶(Gaussia luciferase,GLuc)、人组织纤溶酶原激活剂(Tissue plasminogen activator,tPA)及小鼠免疫球蛋白G的2a亚型(Mouse immunoglobular G subtype 2a,MIgG2a)7个信号肽(原始序列和改造序列)序列的MERS-CoV S1表达质粒,瞬时转染细胞后,通过Western Blot检测并比较细胞培养上清和裂解液中S1的表达水平及分泌表达效率(条带密度灰度扫描比),并对哺乳动物细胞表达的S1蛋白的纯度与抗原特性进行了分析。结果表明7种信号肽在293T、BHK21和ExpiCHO-S^TM三种细胞系统中介导MERS-CoV S1的高效分泌表达的效率各有不同,其中tPA-1信号肽介导S1抗原在ExpiCHO-S^TM中具有较高的分泌表达效率与产量,纯化的S1蛋白保持了较好的抗原性。本研究为进一步研发基于MERS-CoV S1的亚单位疫苗及免疫学检测试剂奠定了基础。     

Effects of Sodium, Potassium, and Calcium Ions on Slow and Spike Potentials in Single Photoreceptor Cells

The influence of changes in the ionic composition of the bathing medium on responses of the retinula cell of the honeybee drone to light was examined by means of intracellular microelectrodes. The resting potential of the cell was influenced mainly by the concentration of K. The peak of the receptor potential (the transient), which in a normal solution and with strong light approaches zero membrane potential, overshot this level in a K-rich solution. An increase in the concentration of K also raised the level of the steady-state phase of the receptor potential (the plateau). The amplitude of the receptor potential was decreased and the spike potential rapidly abolished when Na was replaced by either sucrose, choline, or Tris. In a Ca-free solution the amplitude of the response and especially that of the plateau, was increased. An increase in Ca had the opposite effects. All these changes were reversible. An attempt was made to interpret the receptor and spike potentials in terms of passive movements of Na and K across the membrane of the retinula cell. The major difficulty encountered was to find an explanation for the persistence of an appreciable fraction of the transient and the plateau in preparations kept up to 12 hr in a solution in which all the Na had been replaced by choline, Tris, or sucrose.     

Membrane Potentials,Synaptic Responses,Neuronal Circuitry,Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell''s membrane potential and which we address in these experiments are: (1) Ion concentration of K⁺ on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.     

Cytotoxicity and Calcium-Dependent Antigen of Yersinia

The relationship between invasiveness and calcium dependency was examined in various strains of Yersinia enterocolitica and Y. pseudotuberculosis by using established cell lines. Infection with calcium-dependent bacteria resulted in the formation of microvilli and the adherence of bacteria on the cell surface, and the adherent bacteria were ingested 1.5 hr after infection. Morphological changes in the cells became visible 2 to 3 hr after infection, and intracellular multiplication of the ingested bacteria was noted. When the cells were incubated with bacteria at 37 C for 1.5 hr and then at 25 C, however, the morphological changes in the infected cells were not observed. No isogenic strains that had lost calcium dependency for growth at 37 C were able to elicit the morphological changes in the cells, though they possessed the ability to adhere to and penetrate the cells. The antigen(s) supposedly related to cytotoxicity of the calcium-dependent Yersinia was sought by using antibodies prepared against calcium-dependent bacteria and then absorbed with calcium-independent bacteria and with calcium-independent bacterial cytosol. Double diffusion tests between the antisera and bacterial cytosol extracts revealed the presence of an antigen which was a cytoplasmic substance common to all calcium-dependent but not calcium-independent strains of Y. enterocolitica and Y. pseudotuberculosis.