Active control of spike-timing dependent synaptic plasticity in an electrosensory system.

A spike timing dependent learning rule is present at the synapse between parallel fibers and Purkinje-like medium ganglion cells in the electrosensory lobe of mormyrid electric fish. The synapse is depressed when a postsynaptic dendritic spike occurs within 50 ms of the onset of a parallel fiber excitatory postsynaptic potential, but is enhanced at all other timing relations. Operation of this learning rule results in the cancellation of predictable membrane potential changes, driving the cell towards a constant output frequency. But medium ganglion cells show a strong and predictable response to corollary discharge signals associated with the motor command that initiates the electric organ discharge. The modeling study presented here resolves this conflict by proposing an active control of dendritic spike threshold during the brief period of medium ganglion cell response.     

ICAM-2 expression mediates a membrane-actin link, confers a nonmetastatic phenotype and reflects favorable tumor stage or histology in neuroblastoma

The actin cytoskeleton is a primary determinant of tumor cell motility and metastatic potential. Motility and metastasis are thought to be regulated, in large part, by the interaction of membrane proteins with cytoplasmic linker proteins and of these linker proteins, in turn, with actin. However, complete membrane-to-actin linkages have been difficult to identify. We used co-immunoprecipitation and competitive peptide assays to show that intercellular adhesion molecule-2 (ICAM-2)/alpha-actinin/actin may comprise such a linkage in neuroblastoma cells. ICAM-2 expression limited the motility of these cells and redistributed actin fibers in vitro, and suppressed development of disseminated tumors in an in vivo model of metastatic neuroblastoma. Consistent with these observations, immunohistochemical analysis demonstrated ICAM-2 expression in primary neuroblastoma tumors exhibiting features that are associated with limited metastatic disease and more favorable clinical outcome. In neuroblastoma cell lines, ICAM-2 expression did not affect AKT activation, tumorigenic potential or chemosensitivity, as has been reported for some types of transfected cells. The observed ICAM-2-mediated suppression of metastatic phenotype is a novel function for this protein, and the interaction of ICAM-2/alpha-actinin/actin represents the first complete membrane-linker protein-actin linkage to impact tumor cell motility in vitro and metastatic potential in an in vivo model. Current work focuses on identifying specific protein domains critical to the regulation of neuroblastoma cell motility and metastasis and on determining if these domains represent exploitable therapeutic targets.     

Present State and Future Perspectives of Prostaglandins as a Differentiation Factor in Motor Neurons

Spinal motor neurons have the longest axons that innervate the skeletal muscles of the central nervous system. Motor neuron diseases caused by spinal motor neuron cell death are incurable due to the unique and irreplaceable nature of their neural circuits. Understanding the mechanisms of neurogenesis, neuritogenesis, and synaptogenesis in motor neurons will allow investigators to develop new in vitro models and regenerative therapies for motor neuron diseases. In particular, small molecules can directly reprogram and convert into neural stem cells and neurons, and promote neuron-like cell differentiation. Prostaglandins are known to have a role in the differentiation and tissue regeneration of several cell types and organs. However, the involvement of prostaglandins in the differentiation of motor neurons from neural stem cells is poorly understood. The general cell line used in research on motor neuron diseases is the mouse neuroblastoma and spinal motor neuron fusion cell line NSC-34. Recently, our laboratory reported that prostaglandin E₂ and prostaglandin D₂ enhanced the conversion of NSC-34 cells into motor neuron-like cells with neurite outgrowth. Moreover, we found that prostaglandin E₂-differentiated NSC-34 cells had physiological and electrophysiological properties of mature motor neurons. In this review article, we provide contemporary evidence on the effects of prostaglandins, particularly prostaglandin E₂ and prostaglandin D₂, on differentiation and neural conversion. We also discuss the potential of prostaglandins as candidates for the development of new therapeutic drugs for motor neuron diseases.

     

Effect of electrical charges and fields on injury and viability of airborne bacteria

In this study, the effects of the electric charges and fields on the viability of airborne microorganisms were investigated. The electric charges of different magnitude and polarity were imparted on airborne microbial cells by a means of induction charging. The airborne microorganisms carrying different electric charge levels were then extracted by an electric mobility analyzer and collected using a microbial sampler. It was found that the viability of Pseudomonas fluorescens bacteria, used as a model for sensitive bacteria, carrying a net charge from 4100 negative to 30 positive elementary charges ranged between 40% and 60%; the viability of the cells carrying >2700 positive charges was below 1.5%. In contrast, the viability of the stress-resistant spores of Bacillus subtilis var. niger (used as simulant of anthrax-causing Bacillus anthracis spores when testing bioaerosol sensors in various studies), was not affected by the amount of electric charges on the spores. Because bacterial cells depend on their membrane potential for basic metabolic activities, drastic changes occurring in the membrane potential during aerosolization and the local electric fields induced by the imposed charges appeared to affect the sensitive cells' viability. These findings facilitate applications of electric charging for environmental control purposes involving sterilization of bacterial cells by imposing high electric charges on them. The findings from this study can also be used in the development of new bioaerosol sampling methods based on electrostatic principles.     

Myosin II distribution in neurons is consistent with a role in growth cone motility but not synaptic vesicle mobilization.

We have generated a polyclonal antibody against myosin II from a neuronally derived cell line in order to assess potential roles for myosin II in growth cone movement and synaptic transmission. The distribution of neuronal myosin II, in isolated cells as well as in tissues of the adult rat brain and spinal cord, was examined at the light microscopic and ultrastructural levels. In isolated neuroblastoma cells and dorsal root ganglion neurons, myosin II was found at the leading edge of growth cones, within neuritic processes and cell soma, and adjacent to the plasma membrane. The subcellular distribution of myosin II overlapped significantly with that of both actin and single-headed myosin I. These results implicate both myosin I and myosin II as molecular motors required for neurite elongation and growth cone motility. An exclusive postsynaptic distribution of myosin II in neurons of the mature central nervous system suggests that myosin II cannot play a role in the mobilization of synaptic vesicles, but could participate in synaptic plasticity.     

Faster voltage-dependent activation of Na+ channels in growth cones versus somata of neuroblastoma N1E-115 cells.

Kinetics of voltage-gated ionic channels fundamentally reflect the response of the channels to local electric fields. In this report cell-attached patch-clamp studies reveal that the voltage-dependent activation rate of sodium channels residing in the growth cone membrane differs from that of soma sodium channels in differentiating N1E-115 neuroblastoma cells. Because other electrophysiological properties of these channels do not differ, this finding may be a reflection of the difference in intramembrane electric field in these two regions of the cell. This represents a new mechanism for channels to attain a range of activities both within and between cells.     

A Few Comments on Electrostatic Interactions in Cell Physiology

The role of fixed charges present at the surface of biological membranes is usually described by the Gouy-Chapman-Grahame theory of the electric double-layer where the Grahame equation is applied independently on each side of the membrane and where the capacitive charges (linked to the transmembrane ionic currents) are disregarded. In this article, we generalize the Gouy-Chapman-Grahame theory by taking into account both intrinsic charges (resulting from the dissociation of membrane constituents) and capacitive charges, in the density value of the membrane surface charges. In the first part, we show that capacitive charges couple electrostatic potentials present on both sides of the membrane. The intensity of this coupling depends both on the value of the membrane specific capacitance and the transmembrane electric potential difference. In the second part, we suggest some physiological implications of membrane electric double-layers.     

The effect of dendritic voltage-gated conductances on the neuronal impedance: a quantitative model

Neuronal impedance characterizes the magnitude and timing of the subthreshold response of a neuron to oscillatory input at a given frequency. It is known to be influenced by both the morphology of the neuron and the presence of voltage-gated conductances in the cell membrane. Most existing theoretical accounts of neuronal impedance considered the effects of voltage-gated conductances but neglected the spatial extent of the cell, while others examined spatially extended dendrites with a passive or spatially uniform quasi-active membrane. We derived an explicit mathematical expression for the somatic input impedance of a model neuron consisting of a somatic compartment coupled to an infinite dendritic cable which contained voltage-gated conductances, in the more general case of non-uniform dendritic membrane potential. The validity and generality of this model was verified through computer simulations of various model neurons. The analytical model was then applied to the analysis of experimental data from real CA1 pyramidal neurons. The model confirmed that the biophysical properties and predominantly dendritic localization of the hyperpolarization-activated cation current I (h) were important determinants of the impedance profile, but also predicted a significant contribution from a depolarization-activated fast inward current. Our calculations also implicated the interaction of I (h) with amplifying currents as the main factor governing the shape of the impedance-frequency profile in two types of hippocampal interneuron. Our results provide not only a theoretical advance in our understanding of the frequency-dependent behavior of nerve cells, but also a practical tool for the identification of candidate mechanisms that determine neuronal response properties.     

Intracellular injection of dopamine enhances acetylcholine responses of neuron R2 in the Aplysia abdominal ganglion

1. At different levels of the holding potential on neuron R2 membrane in the Aplysia depilans abdominal ganglion, dopamine injected intracellularly increases the amplitude of both inward and outward currents recorded in response to the application of acetylcholine (ACh) to the ganglion surface. 2. The addition of dopamine to the external perfused solution produces generation of inward currents and a decrease in the cell response to the ACh. 3. The enhancing effect of injected dopamine on ACh responses is retained after inhibition of acetylcholinesterase (AChE) by a specific organophosphorous inhibitor, compound Gd-42. 4. The modulating effect of injected dopamine on ACh responses is discussed in terms of the existence of intracellular receptors of neurotransmitters in the differentiated cells.     

Electrophysiological Properties of Motor Neurons in a Mouse Model of Severe Spinal Muscular Atrophy: In Vitro versus In Vivo Development

We examined the electrophysiological activity of motor neurons from the mouse model of severe spinal muscular atrophy (SMA) using two different methods: whole cell patch clamp of neurons cultured from day 13 embryos; and multi-electrode recording of ventral horns in spinal cord slices from pups on post-natal days 5 and 6. We used the MED64 multi-electrode array to record electrophysiological activity from motor neurons in slices from the lumbar spinal cord of SMA pups and their unaffected littermates. Recording simultaneously from up to 32 sites across the ventral horn, we observed a significant decrease in the number of active neurons in 5–6 day-old SMA pups compared to littermates. Ventral horn activity in control pups is significantly activated by serotonin and depressed by GABA, while these agents had much less effect on SMA slices. In contrast to the large differences observed in spinal cord, neurons cultured from SMA embryos for up to 21 days showed no significant differences in electrophysiological activity compared to littermates. No differences were observed in membrane potential, frequency of spiking and synaptic activity in cells from SMA embryos compared to controls. In addition, we observed no difference in cell survival between cells from SMA embryos and their unaffected littermates. Our results represent the first report on the electrophysiology of SMN-deficient motor neurons, and suggest that motor neuron development in vitro follows a different path than in vivo development, a path in which loss of SMN expression has little effect on motor neuron function and survival.     

Elastic, electrostatic and electrokinetic forces influencing membrane curvature

Many cellular and intracellular processes critically depend on membrane shape, but the shape generating mechanisms are still to be fully understood. In this study we evaluate how electrostatic/electrokinetic forces contribute to membrane curvature. Membrane bilayer had finite thickness and was either elastically anisotropic or anisotropic overall, but isotropic per sections (heads and tails). The physics of the situation was evaluated using a coupled system of elastic and electrostatic/electrokinetic (Poisson-Nernst-Planck) equations. The fixed charges present only on the upper membrane surface lead to the accumulation of counter-ions and depletion of co-ions that decay spatially very rapidly (Debye length<1nm), as does the potential and electric field. Spatially uneven electric field and the permittivity mismatch also induce charges at the membrane-solution interface, which are not fixed but influence the electrostatics nevertheless. Membrane bends due to - Coulomb force (caused by fixed membrane charges in the electric field) and the dielectric force (due to the non-uniform electric field and the permittivity mismatch between the membrane and the solution). Both act as membrane surface forces, and both depend supra-linearly on the fixed charge density. Regardless of sign of the fixed charges, the membrane bends toward the charged (upper) surface owing to the action of the Coulomb force, but this is opposed by the smaller dielectric force. The spontaneous membrane curvature becomes very pronounced at high fixed charge densities, leading to very small spontaneous radii (<50nm). In conclusion the electrostatic/electrokinetic forces contribute significantly to the membrane curvature.     

Effects of α2-Adrenergic Agonists and Antagonists on Photoreceptor Membrane Currents

Type B photoreceptors of the nudibranch mollusc Hermissenda crassicornis receive excitatory synaptic potentials (EPSPs) whose frequency is controlled by potential changes of a neighboring cell known as the S optic ganglion cell which is thought to be electrically coupled to the presynaptic source of these EPSPs, the E optic ganglion cell. The frequency of the EPSPs increases when a conditioned stimulus (light) is paired with an unconditioned stimulus (rotation) during acquisition of a Pavlovian conditioned response. The results of the present study are consistent with an adrenergic origin for these EPSPs. Noradrenergic agonists (greater than 100 microM), norepinephrine and clonidine, only slightly depolarize the type B cell but clearly prolong its depolarizing response to light. Serotonin, by contrast, causes hyperpolarization of the type B cell's resting potential as well as after a light step. Clonidine reduces voltage-dependent outward K+ currents (IA, an early current, ICa2+-K+, a late Ca2+-dependent current) that control the type B cell's excitability (and thus its light response and membrane potential). These effects of clonidine are reduced or blocked by the alpha 2-receptor antagonist, yohimbine (0.5 microM), but not the alpha 1-blocker, prazosin. The same yohimbine concentration also blocked depolarizing synaptic excitation of the type B cell in response to depolarization of a simultaneously impaled S optic ganglion cell. Histochemical techniques (both the glyoxylic acid method of de la Torre and Surgeon and the formaldehyde-induced fluorescence or Falck-Hillarp method) demonstrated the presence of a biogenic amine(s) within a single neuron in each optic ganglion as well as three or four cells within the vicinity of previously identified visual interneurons. No serotonergic neurons were found within the optic ganglion or in proximity to visual interneurons. A clonidine-like synaptic effect on type B cells, therefore, could amplify conditioning-specific changes of membrane currents by increasing type B depolarization and possibly, as well, by elevating intracellular second messengers.     

A Neuron Model with Spatially Distributed Synaptic Input

I have assembled a neuron model simulating contiguous patches of nerve cell membrane. With this model I have examined the functional significance of different spatial and temporal distributions of synaptic inputs. The model consists of two terminal electronic analogue circuits with inputs controlled by a LINC computer. One terminal represents the inside of a membrane patch, the other represents the outside. Two circuit designs are used: one simulates spike-generating regions of the neuron, the other simulates subthreshold activity in inexcitable regions. To simulate a neuron, patches are assembled in various spatial arrangements by suitable connection to the “intracellular” nodes. Thus the relation of neuron geometry to aspects of spatiotemporal summation of synaptic inputs can be investigated readily. Performance of the model is assessed by comparison with results from microelectrode studies in the cochlear nucleus of the cat. In particular, the peristimulus time (PST) histogram and averaged membrane potential are used for quantitative comparison. The model suggests that the geometry of the neuron's receptive surface can account for a wide variety of physiologically observed behavior, particularly in response to dynamic stimuli.     

Voltage and frequency dependence of prestin-associated charge transfer

Membrane protein prestin is a critical component of the motor complex that generates forces and dimensional changes in cells in response to changes in the cell membrane potential. In its native cochlear outer hair cell, prestin is crucial to the amplification and frequency selectivity of the mammalian ear up to frequencies of tens of kHz. Other cells transfected with prestin acquire voltage-dependent properties similar to those of the native cell. The protein performance is critically dependent on chloride ions, and intrinsic protein charges also play a role. We propose an electro-diffusion model to reveal the frequency and voltage dependence of electric charge transfer by prestin. The movement of the combined charge (i.e., anion and protein charges) across the membrane is described with a Fokker–Planck equation coupled to a kinetic equation that describes the binding of chloride ions to prestin. We found a voltage- and frequency-dependent phase shift between the transferred charge and the applied electric field that determines capacitive and resistive components of the transferred charge. The phase shift monotonically decreases from zero to −90° as a function of frequency. The capacitive component as a function of voltage is bell-shaped, and decreases with frequency. The resistive component is bell-shaped for both voltage and frequency. The capacitive and resistive components are similar to experimental measurements of charge transfer at high frequencies. The revealed nature of the transferred charge can help reconcile the high-frequency electrical and mechanical observations associated with prestin, and it is important for further analysis of the structure and function of this protein.     

Potential clamp of isolated dialyzed neuron: minimalization of the effect of series resistance

A modification of the technique of intracellular dialysis of isolated single excitable cells, such as rat spinal ganglion neuron, suitable for potential clamping of its somatic membrane is described. The advantage of the new modification is the substantial reduction of the effect of inherent resistance in series (RS) to the membrane resistance (RM) on precision of potential clamping. This is attained by reversal of cell position in the perfusion pipette resulting in an approximately tenfold reduction in the area of active membrane. The resistance of this area proportionally increased while RS remained unchanged. Hence the error in potential fixation, which is inversely proportional to the ratio RM/RS, is by approximately one order smaller with the new technique than with the original one. An essential step in the new technique is the osmotic expansion of the cell to improve the contact of the cell with the perfusion pipette in the pore and to facilitate disruption of the appropriate part of the membrane. All features and advantages of the technique of intracellular dialysis, such as simplicity, the possibility to easily change ionic composition of media, and/or to apply drugs to any side of the membrane in the same cell, etc., have been maintained.     

Purkyn?'s description of pressure phosphenes and modern neurophysiological studies on the generation of phosphenes by eyeball deformation

(a) When a subject indents one of his eyeballs in total darkness, he immediately perceives light extending slowly across the whole visual field of the indented eye. The appearance and the time course of these pressure or deformation phosphenes are described. (b) With simultaneous binocular indentation of the eyeballs a flickering patterned phosphene is observed. (c) A short history of the research on pressure phosphenes and its consequences for the theories of vision is presented. (d) Purkyn?'s observations of monocular deformation phosphenes are described. He repeatedly noted patterned light structures, which most observers only perceive with simultaneous binocular eyeball deformation. It is suggested that Purkyn?'s deviating observations were caused by amblyopia of one eye. (e) The neurophysiological basis of the monocular pressure phosphenes was investigated by means of microelectrode recordings from single optic tract fibers. The activity of single retinal ganglion cells (on-center, off-center neurons, latency class I [Y-neurons] or latency class II [X-neurons]), was recorded in anaesthetized cats. Eyeball deformation in total darkness led to an activation of the on-center ganglion cells, while the off-center ganglion cells were inhibited. The latency and strength of this activation or inhibition varied considerably between different neurons, but were fairly constant in the same neuron when the eyeball indentation was repeated after a pause of 1-3 min. The latency and strength of neuronal activation or inhibition seemed to be dependent mainly upon the neuron location relative to the point of eyeball indentation. Some on-center neurons also exhibited a short activation at "deformation off". (f) The antagonistic response type of on-center and off-center ganglion cells was also observed when the eyeball was deformed as a hydrostatic open system and the intraocular pressure was kept at 25 mm Hg basic pressure. (g) Dark adaptation up to 45 min affected the deformation responses of retinal neurons only to a small degree, if at all. This corresponds to the observation that deformation phosphenes in a human observer changed little during the course of dark adaptation. (h) We assume that the activation of on-center and inhibition of off-center ganglion cells by eyeball deformation are caused by retinal stretching, which also leads to horizontal cell stretch. Stretching the horizontal cell membrane probably generates an increase in membrane sodium conductivity and a depolarization of the membrane potential. This depolarization of the horizontal cell membrane potential is transmitted either directly or indirectly (via receptor synapses) from the horizontal to the bipolar cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Recapitulation of spinal motor neuron-specific disease phenotypes in a human cell model of spinal muscular atrophy

Establishing human cell models of spinal muscular atrophy (SMA) to mimic motor neuron-specific phenotypes holds the key to understanding the pathogenesis of this devastating disease. Here, we developed a closely representative cell model of SMA by knocking down the disease-determining gene, survival motor neuron (SMN), in human embryonic stem cells (hESCs). Our study with this cell model demonstrated that knocking down of SMN does not interfere with neural induction or the initial specification of spinal motor neurons. Notably, the axonal outgrowth of spinal motor neurons was significantly impaired and these disease-mimicking neurons subsequently degenerated. Furthermore, these disease phenotypes were caused by SMN-full length (SMN-FL) but not SMN-Δ7 (lacking exon 7) knockdown, and were specific to spinal motor neurons. Restoring the expression of SMN-FL completely ameliorated all of the disease phenotypes, including specific axonal defects and motor neuron loss. Finally, knockdown of SMN-FL led to excessive mitochondrial oxidative stress in human motor neuron progenitors. The involvement of oxidative stress in the degeneration of spinal motor neurons in the SMA cell model was further confirmed by the administration of N-acetylcysteine, a potent antioxidant, which prevented disease-related apoptosis and subsequent motor neuron death. Thus, we report here the successful establishment of an hESC-based SMA model, which exhibits disease gene isoform specificity, cell type specificity, and phenotype reversibility. Our model provides a unique paradigm for studying how motor neurons specifically degenerate and highlights the potential importance of antioxidants for the treatment of SMA.     

Optical recording of the electrical activity of synaptically interacting Aplysia neurons in culture using potentiometric probes.

We used multiple-site optical recording methods, in conjunction with impermeant molecular probes of the cell membrane potential, to record the electrical activity of model neural circuits in vitro. Our system consisted of co-cultured pairs of left upper quadrant neurons from the abdominal ganglion of the marine gastropod Aplysia. These neurons interact via inhibitory synapses in vitro. Photodynamic damage to the neurons was essentially eliminated over the time course of the measurements, approximately less than 30 s, by removing oxygen from the recording solution and replacing it with argon. This procedure did not affect the synaptic interactions. We observed repetitive spiking activity in single-trace optical recordings with a maximum signal-to-noise ratio per detector of approximately 50. Individual optical signals that corresponded to either the activity of the presynaptic neuron or that of the postsynaptic neuron were clearly identified. This allowed us to monitor the activity of synaptically interacting neurons, observed as a reduction of the firing rate of the postsynaptic cell after activity of the presynaptic cell. Our results demonstrate that optical methods are appropriate for recording prolonged, asynchronous activity from synaptically interacting neurons in culture.     

Morphological and electrophysiological changes produced by electrical stimulation in cultured neuroblastoma cells.

Electric fields, which were equivalent to those generated by medical devices, were applied to cultured neuroblastoma cells (mouse and human) to test for morphological damage and to establish damage thresholds. Each of two methods of applying fields permitted flow of electrical current and minimized exposure of cells to electrode-breakdown products. One method consisted of a pair of parallel wires in a Petri dish by which current was delivered within a fixed volume of flowing tissue-culture media. With the other method, the cells were held in a confined geometrical chamber and current was applied via agar bridges. Under a given set of stimulation parameters, damage was found to be variable from cell to cell. By changing the strength of the electric field (frequency and duration of stimulation held constant), thresholds of several V/cm were found above which cell damage could be reliably produced. Depending on the intensity of the field, damage took the form of cell lysis or damage to neurites. Intracellular recordings from the mouse neuroblastoma cells revealed a correlation between a decline in resting transmembrane potential and stimulus intensity. Human neuroblastoma cells were less susceptible to damage than were the mouse neuroblastoma cells, given the same strength of applied electric fields.