Spider toxin (JSTX) on the glutamate synapse

A new neurotoxin (JSTX) was separated from spider (Nephila clavata, Joro spider) venom. JSTX irreversibly suppressed the excitatory postsynaptic potential (EPSP) and the glutamate potential in the lobster neuromuscular junction with high degree of specificity. The threshold concentration for suppressing EPSPs corresponds to a small fraction of the toxin in a venom gland, roughly estimated as low as 10(-10) M/l. 10(-10) M/l. In the giant synapse of squid stellate ganglion JSTX suppressed EPSPs without affecting the antidromic response. Glutamate-induced membrane depolarization was blocked by JSTX. In mammalian brain slice preparation, JSTX suppressed the orthodromic spike response but failed to affect on the antidromic spike in the hippocampal pyramidal neuron of CA1 and CA3 region. The above results strongly support the view that the squid giant synapse and synapses in the hippocampal pyramidal neuron are mediated by glutamate.     

Optimal stimulus shapes for neuronal excitation

An important problem in neuronal computation is to discern how features of stimuli control the timing of action potentials. One aspect of this problem is to determine how an action potential, or spike, can be elicited with the least energy cost, e.g., a minimal amount of applied current. Here we show in the Hodgkin & Huxley model of the action potential and in experiments on squid giant axons that: 1) spike generation in a neuron can be highly discriminatory for stimulus shape and 2) the optimal stimulus shape is dependent upon inputs to the neuron. We show how polarity and time course of post-synaptic currents determine which of these optimal stimulus shapes best excites the neuron. These results are obtained mathematically using the calculus of variations and experimentally using a stochastic search methodology. Our findings reveal a surprising complexity of computation at the single cell level that may be relevant for understanding optimization of signaling in neurons and neuronal networks.     

Kinesin mRNA Is Present in the Squid Giant Axon

Abstract: Recently, we reported the construction of a cDNA library encoding a heterogeneous population of polyadenylated mRNAs present in the squid giant axon. The nucleic acid sequencing of several randomly selected clones led to the identification of cDNAs encoding β-actin and β-tubulin, two relatively abundant axonal mRNA species. To continue characterization of this unique mRNA population, the axonal cDNA library was screened with a cDNA probe encoding the carboxy terminus of the squid kinesin heavy chain. The sequencing of several positive clones unambiguously identified axonal kinesin cDNA clones. The axonal localization of kinesin mRNA was subsequently verified by in situ hybridization histochemistry. In addition, the presence of kinesin RNA sequences in the axoplasmic polyribosome fraction was demonstrated using PCR methodology. In contrast to these findings, mRNA encoding the squid sodium channel was not detected in axoplasmic RNA, although these sequences were relatively abundant in the giant fiber lobe. Taken together, these findings demonstrate that kinesin mRNA is a component of a select group of mRNAs present in the squid giant axon, and suggest that kinesin may be synthesized locally in this model invertebrate motor neuron.     

Ricardo Miledi and the calcium hypothesis of neurotransmitter release

Ricardo Miledi has made significant contributions to our basic understanding of how synapses work. Here I discuss aspects of Miledi's research that helped to establish the requirement of presynaptic calcium for neurotransmitter release, from his earliest scientific studies to his classic experiments in the squid giant synapse.     

Cytochemical localization of cholinesterase activity at the giant synapse of the squid.

The giant synapse of squid stellate ganglion is a chemical synapse where the transmitter substance is not known. The components of the ACh-system are present in squid nervous tissue in large quantities. However externally applied cholinergic drugs have no effect on junctional transmission. Using the Copper thiocholine method for electron microscopic cytochemistry the reaction product was found at the axolemmal surface, in the cisternae of the endoplasmic reticulum of neurons and occasionally between the infoldings of the sheat cells surounding the axons. Abundant deposits of end product are observed in the extracellular space in the proximity to junctional region. However, the localization of the cytochemical end product at the junctional region proper was observed frequently, but not consistently. Radiometric measurements of enzyme activity have revealed that neither specific inhibitors nor specific substrates generaly used for differentiation of cholinesterases in mammalian nervous tissue can be employed for differentiation of squid enzymes. Considering the permeability barriers imposed for external acetylcholine by cytoplasmic processes and the high enzyme activity of structures surrounding the giant synapse, the possibility that acetylcholine may still be a candidate for the missing transmitter is discussed.     

Some observations on the fine structure of the giant synapse in the stellate ganglk of the squid,Doryteuphis bleekeri

Summary The fine structure of the synapse between the second-order giant fibre and the third order-giant fibre of the squid Doryteuphis bleekeri was studied by means of electron microscope. In the synaptic region, the two giant fibres are arranged side by side. Many small processes from the third-order giant fibre penetrate the common sheath which separats the adjacent giant axons making synaptic contact with the second order giant axon.The contact surface consists of opposing two plasma membranes of adjacent axons separated by a narrow space of 20–30 m in width. The synaptic membranes are more electron dense and thicker than the other part of the axon membrane. The synaptic vesicles are concentrated exclusively in the presynaptic axon.The fine structural differences between giant synapse in the stellate ganglion of the squid and the giant-to-motor giant synapse of the crayfish were discussed.This work was supported by Grant Number B-3348 from the National Institutes of Health, United States Public Health Service, Department of Health, Education and Welfare.     

The Green’s function formalism as a bridge between single- and multi-compartmental modeling

Neurons are spatially extended structures that receive and process inputs on their dendrites. It is generally accepted that neuronal computations arise from the active integration of synaptic inputs along a dendrite between the input location and the location of spike generation in the axon initial segment. However, many application such as simulations of brain networks use point-neurons—neurons without a morphological component—as computational units to keep the conceptual complexity and computational costs low. Inevitably, these applications thus omit a fundamental property of neuronal computation. In this work, we present an approach to model an artificial synapse that mimics dendritic processing without the need to explicitly simulate dendritic dynamics. The model synapse employs an analytic solution for the cable equation to compute the neuron’s membrane potential following dendritic inputs. Green’s function formalism is used to derive the closed version of the cable equation. We show that by using this synapse model, point-neurons can achieve results that were previously limited to the realms of multi-compartmental models. Moreover, a computational advantage is achieved when only a small number of simulated synapses impinge on a morphologically elaborate neuron. Opportunities and limitations are discussed.     

Theoretical simulation of the calcium action potential in squid giant synapse: The plateau termination

A biophysical model is proposed for the simulation of the experimentally observed calcium action potential at the squid giant synapse. It is observed that while Ca activation at the synapse is responsible for the generation of the upstroke of the action potential, a repolarizing process needs to be invoked to simulate the plateau termination and other long-time effects. Out of the likely candidates, the Ca-activated K current has been chosen as the most plausible repolarizing process. The model can reproduce all the observed features of calcium action potential excepting its behaviour after repetitive stimulation.     

Virtual NEURON: a strategy for merged biochemical and electrophysiological modeling

Because of its highly branched dendrite, the Purkinje neuron requires significant computational resources if coupled electrical and biochemical activity are to be simulated. To address this challenge, we developed a scheme for reducing the geometric complexity; while preserving the essential features of activity in both the soma and a remote dendritic spine. We merged our previously published biochemical model of calcium dynamics and lipid signaling in the Purkinje neuron, developed in the Virtual Cell modeling and simulation environment, with an electrophysiological model based on a Purkinje neuron model available in NEURON. A novel reduction method was applied to the Purkinje neuron geometry to obtain a model with fewer compartments that is tractable in Virtual Cell. Most of the dendritic tree was subject to reduction, but we retained the neuron’s explicit electrical and geometric features along a specified path from spine to soma. Further, unlike previous simplification methods, the dendrites that branch off along the preserved explicit path are retained as reduced branches. We conserved axial resistivity and adjusted passive properties and active channel conductances for the reduction in surface area, and cytosolic calcium for the reduction in volume. Rallpacks are used to validate the reduction algorithm and show that it can be generalized to other complex neuronal geometries. For the Purkinje cell, we found that current injections at the soma were able to produce similar trains of action potentials and membrane potential propagation in the full and reduced models in NEURON; the reduced model produces identical spiking patterns in NEURON and Virtual Cell. Importantly, our reduced model can simulate communication between the soma and a distal spine; an alpha function applied at the spine to represent synaptic stimulation gave similar results in the full and reduced models for potential changes associated with both the spine and the soma. Finally, we combined phosphoinositol signaling and electrophysiology in the reduced model in Virtual Cell. Thus, a strategy has been developed to combine electrophysiology and biochemistry as a step toward merging neuronal and systems biology modeling.     

Morphological and physiological properties of the giant interneuron of the hermit crab (Pagurus pollicaris)

The physiological and morphological properties of the giant interneurons in the hermit crab Pagurus pollicaris are described. The cell bodies are located anteriorly in the supraesophageal ganglion, close to the mid-line. Each cell sends a neurite posteriorly and then laterally, so that they cross over in the center of the ganglion. Each axon then branches: one branch runs laterally while the other travels posteriorly and leaves the ganglion in the circumesophageal connective on the side contralateral to the cell body. The giant axons travel in the circumesophageal connectives and through the thoracic and abdominal ganglia without branching. Each giant axon makes synaptic contact with its ipsilateral giant abdominal flexor motor neuron and with a second flexor motor neuron that has its axon in the contralateral third root. In the supraesophageal ganglion there is a bidirectional synapse between the two giant interneurons. Intracellular recordings from the giant axons show that there is a delay of 0.5 to 0.75 ms that cannot be accounted for by spike propagation along the axons, and may be accounted for by a chemical synapse between the giant interneurons.     

Differential Effects of Human L1CAM Mutations on Complementing Guidance and Synaptic Defects in Drosophila melanogaster

A large number of different pathological L1CAM mutations have been identified that result in a broad spectrum of neurological and non-neurological phenotypes. While many of these mutations have been characterized for their effects on homophilic and heterophilic interactions, as well as expression levels in vitro, there are only few studies on their biological consequences in vivo. The single L1-type CAM gene in Drosophila, neuroglian (nrg), has distinct functions during axon guidance and synapse formation and the phenotypes of nrg mutants can be rescued by the expression of human L1CAM. We previously showed that the highly conserved intracellular FIGQY Ankyrin-binding motif is required for L1CAM-mediated synapse formation, but not for neurite outgrowth or axon guidance of the Drosophila giant fiber (GF) neuron. Here, we use the GF as a model neuron to characterize the pathogenic L120V, Y1070C, C264Y, H210Q, E309K and R184Q extracellular L1CAM missense mutations and a L1CAM protein with a disrupted ezrin–moesin–radixin (ERM) binding site to investigate the signaling requirements for neuronal development. We report that different L1CAM mutations have distinct effects on axon guidance and synapse formation. Furthermore, L1CAM homophilic binding and signaling via the ERM motif is essential for axon guidance in Drosophila. In addition, the human pathological H210Q, R184Q and Y1070C, but not the E309K and L120V L1CAM mutations affect outside-in signaling via the FIGQY Ankyrin binding domain which is required for synapse formation. Thus, the pathological phenotypes observed in humans are likely to be caused by the disruption of signaling required for both, guidance and synaptogenesis.     

Relationship between presynaptic calcium current and postsynaptic potential in squid giant synapse.

The relationship between calcium current and transmitter release was studied in squid giant synapse. It was found that the voltage-dependent calcium current triggers the release of synaptic transmitter in direct proportion to its magnitude and duration. Transmitter release occurs with a delay of approximately 200 mus after the influx of calcium. A model is presented which describes these relations formally.     

Predicting the synaptic information efficacy in cortical layer 5 pyramidal neurons using a minimal integrate-and-fire model

Synaptic information efficacy (SIE) is a statistical measure to quantify the efficacy of a synapse. It measures how much information is gained, on the average, about the output spike train of a postsynaptic neuron if the input spike train is known. It is a particularly appropriate measure for assessing the input–output relationship of neurons receiving dynamic stimuli. Here, we compare the SIE of simulated synaptic inputs measured experimentally in layer 5 cortical pyramidal neurons in vitro with the SIE computed from a minimal model constructed to fit the recorded data. We show that even with a simple model that is far from perfect in predicting the precise timing of the output spikes of the real neuron, the SIE can still be accurately predicted. This arises from the ability of the model to predict output spikes influenced by the input more accurately than those driven by the background current. This indicates that in this context, some spikes may be more important than others. Lastly we demonstrate another aspect where using mutual information could be beneficial in evaluating the quality of a model, by measuring the mutual information between the model’s output and the neuron’s output. The SIE, thus, could be a useful tool for assessing the quality of models of single neurons in preserving input–output relationship, a property that becomes crucial when we start connecting these reduced models to construct complex realistic neuronal networks.     

A Minimal Model of Signaling Network Elucidates Cell-to-Cell Stochastic Variability in Apoptosis

Background

Signaling networks are designed to sense an environmental stimulus and adapt to it. We propose and study a minimal model of signaling network that can sense and respond to external stimuli of varying strength in an adaptive manner. The structure of this minimal network is derived based on some simple assumptions on its differential response to external stimuli.

Methodology

We employ stochastic differential equations and probability distributions obtained from stochastic simulations to characterize differential signaling response in our minimal network model. Gillespie''s stochastic simulation algorithm (SSA) is used in this study.

Conclusions/Significance

We show that the proposed minimal signaling network displays two distinct types of response as the strength of the stimulus is decreased. The signaling network has a deterministic part that undergoes rapid activation by a strong stimulus in which case cell-to-cell fluctuations can be ignored. As the strength of the stimulus decreases, the stochastic part of the network begins dominating the signaling response where slow activation is observed with characteristic large cell-to-cell stochastic variability. Interestingly, this proposed stochastic signaling network can capture some of the essential signaling behaviors of a complex apoptotic cell death signaling network that has been studied through experiments and large-scale computer simulations. Thus we claim that the proposed signaling network is an appropriate minimal model of apoptosis signaling. Elucidating the fundamental design principles of complex cellular signaling pathways such as apoptosis signaling remains a challenging task. We demonstrate how our proposed minimal model can help elucidate the effect of a specific apoptotic inhibitor Bcl-2 on apoptotic signaling in a cell-type independent manner. We also discuss the implications of our study in elucidating the adaptive strategy of cell death signaling pathways.     

Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins

When isolated squid giant axons are incubated in radioactive amino acids, abundant newly synthesized proteins are found in the axoplasm. These proteins are translated in the adaxonal Schwann cells and subsequently transferred into the giant axon. The question as to whether any de novo protein synthesis occurs in the giant axon itself is difficult to resolve because the small contribution of the proteins possibly synthesized intra-axonally is not easily distinguished from the large amounts of the proteins being supplied from the Schwann cells. In this paper, we reexamine this issue by studying the synthesis of endogenous neurofilament (NF) proteins in the axon. Our laboratory previously showed that NF mRNA and protein are present in the squid giant axon, but not in the surrounding adaxonal glia. Therefore, if the isolated squid axon could be shown to contain newly synthesized NF protein de novo, it could not arise from the adaxonal glia. The results of experiments in this paper show that abundant 3H-labeled NF protein is synthesized in the squid giant fiber lobe containing the giant axon’s neuronal cell bodies, but despite the presence of NF mRNA in the giant axon no labeled NF protein is detected in the giant axon. This lends support to the glia–axon protein transfer hypothesis which posits that the squid giant axon obtains newly synthesized protein by Schwann cell transfer and not through intra-axonal protein synthesis, and further suggests that the NF mRNA in the axon is in a translationally repressed state.     

A fit to nerve membrane rectification curves with a double-dipole-layer membrane model

Following Wei's suggestion that nerve stimulation and conduction properties are due to dipole layers at the two membrane surfaces (Wei, 1969), we have done steady-state electro-diffusion calculations in the constant field approximation for a simple double-dipole-layer model. We are thereby able to quantitatively fit the recent potassium iso-osmotic rectification curves of Gilbert and Ehrenstein for the squid giant axon membrane. For the squid axon membrane in a natural ion environment, only the outside dipole layer is present in the fit to the data.     

The nicotinic acetylcholine receptor Dalpha7 is required for an escape behavior in Drosophila

Acetylcholine is the major excitatory neurotransmitter in the central nervous system of insects. Mutant analysis of the Dalpha7 nicotinic acetylcholine receptor (nAChR) of Drosophila shows that it is required for the giant fiber-mediated escape behavior. The Dalpha7 protein is enriched in the dendrites of the giant fiber, and electrophysiological analysis of the giant fiber circuit showed that sensory input to the giant fiber is disrupted, as is transmission at an identified cholinergic synapse between the peripherally synapsing interneuron and the dorsal lateral muscle motor neuron. Moreover, we found that gfA1, a mutation identified in a screen for giant fiber defects more than twenty years ago, is an allele of Dalpha7. Therefore, a combination of behavioral, electrophysiological, anatomical, and genetic data indicate an essential role for the Dalpha7 nAChR in giant fiber-mediated escape in Drosophila.     

Interactions between neurons and their targets during in vivo synaptogenesis.

In vivo synaptogenesis is described in a simple vertebrate system, the chick ciliary ganglion, a parasympathetic autonomic ganglion. An attempt is made to integrate anatomical, physiological and biochemical observations during synapse formation in the ganglion and in the peripheral target structures; the iris, ciliary muscle, and smooth muscle of the choroid coat. The relationship between synaptogenesis and neuron survival is explored, and it is shown that a critically timed interaction between the neuron and target organ is necessary for full neuronal maturation and survival. The existence of an active competition between neurons for survival is documented, and the possible relationship between neuronal cell death and specificity of connections is discussed.     

Giant Glial Cell: New Insight Through Mechanism-Based Modeling

The paper describes a detailed mechanism-based model of a tripartite synapse consisting of P- and R-neurons together with a giant glial cell in the ganglia of the medical leech (Hirudo medicinalis), which is a useful object for experimental studies in situ. We describe the two main pathways of the glial cell activation: (1) via IP₃ production and Ca^2?+? release from the endoplasmic reticulum and (2) via increase of the extracellular potassium concentration, glia depolarization, and opening of voltage-dependent Ca^2?+? channels. We suggest that the second pathway is the more significant for establishing the positive feedback in glutamate release that is critical for the self-sustained activity of the postsynaptic neuron. This mechanism differs from the mechanisms of the astrocyte-neuron signaling previously reported.     

Astrocytic Ephrin-B1 Regulates Synapse Remodeling Following Traumatic Brain Injury

Traumatic brain injury (TBI) can result in tissue alterations distant from the site of the initial injury, which can trigger pathological changes within hippocampal circuits and are thought to contribute to long-term cognitive and neuropsychological impairments. However, our understanding of secondary injury mechanisms is limited. Astrocytes play an important role in brain repair after injury and astrocyte-mediated mechanisms that are implicated in synapse development are likely important in injury-induced synapse remodeling. Our studies suggest a new role of ephrin-B1, which is known to regulate synapse development in neurons, in astrocyte-mediated synapse remodeling following TBI. Indeed, we observed a transient upregulation of ephrin-B1 immunoreactivity in hippocampal astrocytes following moderate controlled cortical impact model of TBI. The upregulation of ephrin-B1 levels in hippocampal astrocytes coincided with a decline in the number of vGlut1-positive glutamatergic input to CA1 neurons at 3 days post injury even in the absence of hippocampal neuron loss. In contrast, tamoxifen-induced ablation of ephrin-B1 from adult astrocytes in ephrin-B1^loxP/yERT2-Cre^GFAP mice accelerated the recovery of vGlut1-positive glutamatergic input to CA1 neurons after TBI. Finally, our studies suggest that astrocytic ephrin-B1 may play an active role in injury-induced synapse remodeling through the activation of STAT3-mediated signaling in astrocytes. TBI-induced upregulation of STAT3 phosphorylation within the hippocampus was suppressed by astrocyte-specific ablation of ephrin-B1 in vivo, whereas the activation of ephrin-B1 in astrocytes triggered an increase in STAT3 phosphorylation in vitro. Thus, regulation of ephrin-B1 signaling in astrocytes may provide new therapeutic opportunities to aid functional recovery after TBI.