Squid Giant Axon Research Articles

Sir Andrew Fielding Huxley OM. 22 November 1917—30 May 2012

Andrew Huxley was a physiologist, possessing a combination of practical skill, invention and mathematical ability that has few parallels, and is famous for his contributions to the understanding of how nerve and muscle work. He came from an illustrious family: his paternal grandfather was Thomas Henry Huxley, and Julian Huxley and Aldous Huxley were his half-brothers. After completing his undergraduate degree in Cambridge in 1939, he joined Alan Hodgkin in his research on the squid giant axon and they made the first intracellular recording of a nerve action potential. Together, they used the voltage clamp technique to elucidate the ionic mechanism of the action potential; for this they were awarded a share of the Nobel Prize in Physiology or Medicine in 1963. Turning to research on muscle, he elucidated the sliding filament mechanism of contraction (in parallel with Hugh Huxley), and went on to formulate a quantitative account of the steady state properties of muscle based on the interaction between myosin crossbridges and actin sites. Moving to University College London (UCL) in 1960 as Jodrell Professor of Physiology, he provided quantitative evidence for the sliding filament theory through a study of the length–tension relation. His final studies were on the transient mechanical properties of muscle, resulting in a theory of force-generation by crossbridges. During the war, Huxley was engaged in operational research on anti-aircraft artillery and naval gunnery. He was President of the Royal Society in 1980–85, and Master of Trinity College Cambridge in 1984–90.

Colin Brownlee.

Colin Brownlee.

Modeling squid axon Na+ channel by a nucleation and growth kinetic mechanism

A kinetic model accounting for all salient features of the Na+ channel of the squid giant axon is provided. The model furnishes explanations for the Cole-Moore-like effect, the rising phase of the ON gating current and the slow ‘intermediate component’ of its decaying phase, as well as the gating charge immobilization. Experimental ON ionic currents are semi-quantitatively simulated by the use of only three free parameters, upon assuming that the Na+ channel opening proceeds along with the stepwise aggregation of its four domains, while they are moving their gating charge outward under depolarizing conditions. The inactivation phase of the ON ionic current is interpreted by a progressive electrostatic attraction between the positively charged ‘hinged lid’ containing the hydrophobic IFM triad and its receptor inside the channel pore, as the stepwise outward movement of the S4 segments of the Na+ channel progressively increases the negative charge attracting the triad to its receptor. The Na+ channel closing is assumed to proceed by repolarization-induced disaggregation of its domains, accompanied by inward movement of their gating charge. The phenomenon of ‘gating charge immobilization’ can be explained by assuming that gradual structural changes of the receptor over the time course of depolarization strengthen the interaction between the IFM triad and its receptor, causing a slow release of the gating charge during the subsequent repolarization.

Cellular function given parametric variation in the Hodgkin and Huxley model of excitability

How is reliable physiological function maintained in cells despite considerable variability in the values of key parameters of multiple interacting processes that govern that function? Here, we use the classic Hodgkin-Huxley formulation of the squid giant axon action potential to propose a possible approach to this problem. Although the full Hodgkin-Huxley model is very sensitive to fluctuations that independently occur in its many parameters, the outcome is in fact determined by simple combinations of these parameters along two physiological dimensions: structural and kinetic (denoted S and K, respectively). Structural parameters describe the properties of the cell, including its capacitance and the densities of its ion channels. Kinetic parameters are those that describe the opening and closing of the voltage-dependent conductances. The impacts of parametric fluctuations on the dynamics of the system-seemingly complex in the high-dimensional representation of the Hodgkin-Huxley model-are tractable when examined within the S-K plane. We demonstrate that slow inactivation, a ubiquitous activity-dependent feature of ionic channels, is a powerful local homeostatic control mechanism that stabilizes excitability amid changes in structural and kinetic parameters.

Model of Impedance Changes in Unmyelinated Nerve Fibers

Currently, there is no imaging method that is able to distinguish the functional activity inside nerves. Such a method would be essential for understanding peripheral nerve physiology and would allow precise neuromodulation of organs these nerves supply. Electrical impedance tomography (EIT) is a method that produces images of electrical impedance change (dZ) of an object by injecting alternating current and recording surface voltages. It has been shown to be able to image fast activity in the brain and large peripheral nerves. To image inside small autonomic nerves, mostly containing unmyelinated fibers, it is necessary to maximize SNR and optimize the EIT parameters. An accurate model of the nerve is required to identify these optimal parameters as well as to validate data obtained in the experiments. In this study, we developed two three-dimensional models of unmyelinated fibers: Hodgkin-Huxley (HH) squid giant axon (single and multiple) and mammalian C-nociceptor. A coupling feedback system was incorporated into the models to simulate direct and alternating current application and simultaneously record external field during action potential propagation. Parameters of the developed models were varied to study their influence on the recorded impedance changes; the optimal parameters were identified. The negative dZ was found to monotonically decrease with frequency for both HH and C fiber models, in accordance with the experimental data. The accurate realistic model of unmyelinated nerve allows the optimization of EIT parameters and matches literature and experimental results.

Saddle Slow Manifolds and Canard Orbits in mathbb{R}^{4} and Application to the Full Hodgkin\u2013Huxley Model

Many physiological phenomena have the property that some variables evolve much faster than others. For example, neuron models typically involve observable differences in time scales. The Hodgkin–Huxley model is well known for explaining the ionic mechanism that generates the action potential in the squid giant axon. Rubin and Wechselberger (Biol. Cybern. 97:5–32, 2007) nondimensionalized this model and obtained a singularly perturbed system with two fast, two slow variables, and an explicit time-scale ratio ε. The dynamics of this system are complex and feature periodic orbits with a series of action potentials separated by small-amplitude oscillations (SAOs); also referred to as mixed-mode oscillations (MMOs). The slow dynamics of this system are organized by two-dimensional locally invariant manifolds called slow manifolds which can be either attracting or of saddle type.In this paper, we introduce a general approach for computing two-dimensional saddle slow manifolds and their stable and unstable fast manifolds. We also develop a technique for detecting and continuing associated canard orbits, which arise from the interaction between attracting and saddle slow manifolds, and provide a mechanism for the organization of SAOs in mathbb{R}^{4}. We first test our approach with an extended four-dimensional normal form of a folded node. Our results demonstrate that our computations give reliable approximations of slow manifolds and canard orbits of this model. Our computational approach is then utilized to investigate the role of saddle slow manifolds and associated canard orbits of the full Hodgkin–Huxley model in organizing MMOs and determining the firing rates of action potentials. For ε sufficiently large, canard orbits are arranged in pairs of twin canard orbits with the same number of SAOs. We illustrate how twin canard orbits partition the attracting slow manifold into a number of ribbons that play the role of sectors of rotations. The upshot is that we are able to unravel the geometry of slow manifolds and associated canard orbits without the need to reduce the model.

Reflections on the Design of Clinical Trials: A Conversation With Raymond J. Lipicky, MD.

Raymond Lipicky was born in Cleveland, OH. He received an MD from the University of Cincinnati in 1960 and completed an internship and residency at Barnes Hospital at the Washington University School of Medicine in St. Louis, MO. He trained in cardiology at Strong Memorial Hospital in Rochester, NY, and in 1966 joined the faculty of the University of Cincinnati, where he became Director, Division of Pharmacology, and Professor of Medicine and Pharmacology. From 1979 until 2002, he served at the FDA, initially as Medical Officer, and later as Director, Division of Cardiovascular and Renal Products. He maintained an appointment as Visiting Scientist at the Marine Biological Laboratory, Woods Hole, MA. He received the FDA Award of Merit in 1988 and 1997. ### Dr Rutherford asks: How did your career lead you to become the Director of the FDA’s Cardiovascular and Renal Products Division? Dr Lipicky replies: For 13 years I was at the University of Cincinnati, and I was a tenured Professor of Medicine and Pharmacology and Director of the Division of Clinical Pharmacology. I decided that academic medicine was morphing into a business whose goals I could no longer support. The practice of medicine was not something I wished to pursue, and decisions in the pharmaceutical industry were driven by the need to make a profit (also something I could not support). My primary research interests were in basic science with funded National Institutes of Health and Muscular Dystrophy grants. One of the projects was Drug Effects on Electrically Excitable Membranes, with the primary assay being the squid giant axon. I spent summers at the Marine Biological Laboratory in Woods Hole, MA, which had the only daily source of live squid in the USA. During those summers I collaborated a lot with the Biophysics Laboratory at the National Institutes of Health. The FDA had a basic science laboratory located in Washington, DC, that had a job vacancy and I went …

Demonstration of ion channel synthesis by isolated squid giant axon provides functional evidence for localized axonal membrane protein translation

Local translation of membrane proteins in neuronal subcellular domains like soma, dendrites and axon termini is well-documented. In this study, we isolated the electrical signaling unit of an axon by dissecting giant axons from mature squids (Dosidicus gigas). Axoplasm extracted from these axons was found to contain ribosomal RNAs, ~8000 messenger RNA species, many encoding the translation machinery, membrane proteins, translocon and signal recognition particle (SRP) subunits, endomembrane-associated proteins, and unprecedented proportions of SRP RNA (~68% identical to human homolog). While these components support endoplasmic reticulum-dependent protein synthesis, functional assessment of a newly synthesized membrane protein in axolemma of an isolated axon is technically challenging. Ion channels are ideal proteins for this purpose because their functional dynamics can be directly evaluated by applying voltage clamp across the axon membrane. We delivered in vitro transcribed RNA encoding native or Drosophila voltage-activated Shaker KV channel into excised squid giant axons. We found that total K+ currents increased in both cases; with added inactivation kinetics on those axons injected with RNA encoding the Shaker channel. These results provide unambiguous evidence that isolated axons can exhibit de novo synthesis, assembly and membrane incorporation of fully functional oligomeric membrane proteins.

Effects of channel blocking on information transmission and energy efficiency in squid giant axons.

Action potentials are the information carriers of neural systems. The generation of action potentials involves the cooperative opening and closing of sodium and potassium channels. This process is metabolically expensive because the ions flowing through open channels need to be restored to maintain concentration gradients of these ions. Toxins like tetraethylammonium can block working ion channels, thus affecting the function and energy cost of neurons. In this paper, by computer simulation of the Hodgkin-Huxley neuron model, we studied the effects of channel blocking with toxins on the information transmission and energy efficiency in squid giant axons. We found that gradually blocking sodium channels will sequentially maximize the information transmission and energy efficiency of the axons, whereas moderate blocking of potassium channels will have little impact on the information transmission and will decrease the energy efficiency. Heavy blocking of potassium channels will cause self-sustained oscillation of membrane potentials. Simultaneously blocking sodium and potassium channels with the same ratio increases both information transmission and energy efficiency. Our results are in line with previous studies suggesting that information processing capacity and energy efficiency can be maximized by regulating the number of active ion channels, and this indicates a viable avenue for future experimentation.

Modeling squid axon K+ channel by a nucleation and growth kinetic mechanism

A kinetic model accounting for all salient features of the K+ channel of the squid giant axon, including the rising phase of the ON gating charge and the Cole-Moore effect, is provided. Upon accounting for a significant feature distinguishing K+, Na+ and Ca2+ channels from channel-forming peptides modeled in our previous 2016 BBA paper, the nucleation-and-growth kinetic model developed therein is extended to simulate ON ionic and gating currents of the K+ channel of the squid giant axon at different depolarization potentials by the use of only two free parameters. K+ channel opening is considered to proceed by progressive aggregation of single subunits, while they are moving their gating charge outward under depolarizing conditions within their tetrameric structure; K+ channel closing proceeds in the opposite direction, by repolarization-induced disaggregation of subunits, accompanied by inward movement of their gating charge.

Electrothermal Equivalent Three-Dimensional Finite-Element Model of a Single Neuron.

We propose a novel approach for modelling the interdependence of electrical and mechanical phenomena in nervous cells, by using electrothermal equivalences in finite element (FE) analysis so that existing thermomechanical tools can be applied. First, the equivalence between electrical and thermal properties of the nerve materials is established, and results of a pure heat conduction analysis performed in Abaqus CAE Software 6.13-3 are validated with analytical solutions for a range of steady and transient conditions. This validation includes the definition of equivalent active membrane properties that enable prediction of the action potential. Then, as a step toward fully coupled models, electromechanical coupling is implemented through the definition of equivalent piezoelectric properties of the nerve membrane using the thermal expansion coefficient, enabling prediction of the mechanical response of the nerve to the action potential. Results of the coupled electromechanical model are validated with previously published experimental results of deformation for squid giant axon, crab nerve fibre, and garfish olfactory nerve fibre. A simplified coupled electromechanical modelling approach is established through an electrothermal equivalent FE model of a nervous cell for biomedical applications. One of the key findings is the mechanical characterization of the neural activity in a coupled electromechanical domain, which provides insights into the electromechanical behaviour of nervous cells, such as thinning of the membrane. This is a first step toward modelling three-dimensional electromechanical alteration induced by trauma at nerve bundle, tissue, and organ levels.

Conformational changes, from β-strand to α-helix, of the fatty acid-binding protein ReP1-NCXSQ in anionic lipid membranes: dependence with the vesicle curvature.

We studied the conformational changes of the fatty acid-binding protein ReP1-NCXSQ in the interface of anionic lipid membranes. ReP1-NCXSQ is an acidic protein that regulates the activity of the Na+/Ca2+ exchanger in squid axon. The structure is a flattened barrel composed of two orthogonal β-sheets delimiting an inner cavity and a domain of two α-helix segments arranged as a hairpin. FTIR and CD spectroscopy showed that the interactions with several anionic lipids in the form of small unilamellar vesicles (SUVs) induced an increase in the proportion of helix secondary structure. Lower amount or no increase in α-helix was observed upon the interaction with anionic lipids in the form of large unilamellar vesicles (LUVs). The exception was 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) that was equally efficien to to induce the conformational change both in SUVs and in LUVs. In solution, the infrared spectra of ReP1-NCXSQ at temperatures above the unfolding displayed a band at 1617cm-1 characteristic of aggregated strands. This band was not observed when the protein interacted with DMPG, indicating inhibition of aggregation in the interface. Similarly to the observed in L-BABP, another member of the fatty acid binding proteins, a conformational change in ReP1-NCXSQ was coupled to the gel to liquid-crystalline lipid phase transition.

Invisible motion: Paratactical curation of bio art and performative political imaginaries

Abstract Bio artworks tend towards social reflection, conveying political and societal criticism through the combination of artistic and scientific processes. Some bio artworks also challenge to subvert hegemonic political imaginaries. In this article, focusing on the curation of a bio artwork and making an ethnographic research based on my participatory observation, I suggest a new conception of contemporary curatorial practice, ‘paratactical curation’, which aims to create performative political imaginaries and alternative futures. The article focuses on The Invisible Life, a series of a bio artwork of Joseph A. DeGiorgis and Ayse Gul Suter. This series was exhibited in Contemporary Istanbul Art Fair – Plugin New Media Section that I have curated in 2015. The main discussion is developed by paying attention to the giant squid axon work, in which the article responds to the emergence of a new political realm that reflects the communication problems of post-control societies: connection failures, memory loss and noise.

Squid Giant Axons Synthesize NF Proteins.

Squid giant axon has been an excellent model system for studying fundamental topics in neurobiology such as neuronal signaling. It has been also useful in addressing the questions of local protein synthesis in the axons. Incubation of isolated squid giant axons with [35S]methionine followed by immunoprecipitation with a rabbit antibody against all squid neurofilament (NF) proteins demonstrates the local synthesis of a major 180kDa NF protein and of several NF proteins of lower molecular weights. Their identification as NF proteins is based on their absence in the preimmune precipitates. Immunoprecipitates washed with more stringent buffers confirmed these results. Our data are at variance with a recent study based on the same experimental procedure that failed to visualize the local synthesis of NF proteins by the giant axon and thereby suggested their exclusive derivation from nerve cell bodies (as reported by Gainer et al. in Cell Mol Neurobiol 37:475-486, 2017). By reviewing the pertinent literature, we confute the claims that mRNA translation is absent in mature axons because of a putative translation block and that most proteins of mature axons are synthesized in the surrounding glial cells. Given the intrinsic axonal capacity to synthesize proteins, we stress the glial derivation of axonal and presynaptic RNAs and the related proposal that these neuronal domains are endowed with largely independent gene expression systems (as reported by Giuditta et al. in Physiol Rev 88:515-555, 2008).

Elucidation of pyrethroid and DDT receptor sites in the voltage-gated sodium channel

DDT and pyrethroid insecticides were among the earliest neurotoxins identified to act on voltage-gated sodium channels. In the 1960s, equipped with, at the time, new voltage-clamp techniques, Professor Narahashi and associates provided the initial evidence that DDT and allethrin (the first commercial pyrethroid insecticide) caused prolonged flow of sodium currents in lobster and squid giant axons. Over the next several decades, continued efforts by Prof. Narahashi’s group as well as other laboratories led to a comprehensive understanding of the mechanism of action of DDT and pyrethroids on sodium channels. Fast forward to the 1990s, genetic, pharmacological and toxicological data all further confirmed voltage-gated sodium channels as the primary targets of DDT and pyrethroid insecticides. Modifications of the gating kinetics of sodium channels by these insecticides result in repetitive firing and/or membrane depolarization in the nervous system. This mini-review focuses on studies from Prof. Narahashi’s pioneer work and more recent mutational and computational modeling analyses which collectively elucidated the elusive pyrethroid receptor sites as well as the molecular basis of differential sensitivities of insect and mammalian sodium channels to pyrethroids.

The right tool and the right place for the job: the importance of the field in experimental neurophysiology, 1880-1945.

This paper seeks to contribute to understandings of practice and place in the history of early American neurophysiology by exploring research with jellyfish at marine stations. Jellyfish became a particularly important research tool to experimental physiologists studying neurological subjects at the turn of the twentieth century. But their enthusiasm for the potential of this organism was constrained by its delicacy in captivity. The discovery of hardier species made experimentation at the shore possible and resulted in two epicenters of neurophysiological research on the American East Coast: the Marine Biological Laboratory and the Carnegie Institution's Dry Tortugas Laboratory. Work done in these locations had impacts on a wide range of physiological questions. These centers were short lived-researchers at the MBL eventually focused on the squid giant axon and the Tortugas lab closed after the death of Mayer-but the development of basic requirements and best practices to sustain these organisms paints an important picture of early experimental neurophysiology. Marine organisms and locations have played an integral role in the development of experimental life sciences in America. By understanding the earliest experimental research done at these locations, and the organisms that lured researchers from the campus to the coastline, we can begin to integrate marine stations into the larger historical narrative of American physiology.

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.

Thermodynamic analysis of the squid mantle muscles and giant axon during slow swimming and jet escape propulsion

Squids have two substantially different types of muscle fibers: superficial mitochondria rich fibers, which perform aerobic respiration during slow swimming, and central mitochondria poor fibers, which perform anaerobic respiration during jet escape. A detailed thermodynamic analysis shows that during slow swimming, 3.82 J/(kg s) of chemical exergy is consumed, and a total muscle work of 0.28 J/(kg s) is produced. 0.27 J/(kg s) of this is produced by the fin to generate lift, and the rest by the mantle volume contraction. During the jet escape at a speed of 3 mantle length/s, squid consumes an exergy of 9.97 J/(kg s) and produces a muscle work of 0.16 J/(kg s). Exergy destruction rates during slow swimming and jet escape modes are 3.54 and 9.81 J/(kg s), respectively. Exergy destroyed because of the action potential propagation in the squid giant axon is calculated as 0.03 and 0.10 J/(kg s) for the slow and fast swimming modes, respectively.

Mathematical Modeling in Neuroscience: Neuronal Activity and Its Modulation by Astrocytes.

Mathematical Modeling in Neuroscience: Neuronal Activity and Its Modulation by Astrocytes.

The Amyloid Precursor Protein of Alzheimer's Disease Clusters at the Organelle/Microtubule Interface on Organelles that Bind Microtubules in an ATP Dependent Manner.

The amyloid precursor protein (APP) is a causal agent in the pathogenesis of Alzheimer’s disease and is a transmembrane protein that associates with membrane-limited organelles. APP has been shown to co-purify through immunoprecipitation with a kinesin light chain suggesting that APP may act as a trailer hitch linking kinesin to its intercellular cargo, however this hypothesis has been challenged. Previously, we identified an mRNA transcript that encodes a squid homolog of human APP770. The human and squid isoforms share 60% sequence identity and 76% sequence similarity within the cytoplasmic domain and share 15 of the final 19 amino acids at the C-terminus establishing this highly conserved domain as a functionally import segment of the APP molecule. Here, we study the distribution of squid APP in extruded axoplasm as well as in a well-characterized reconstituted organelle/microtubule preparation from the squid giant axon in which organelles bind microtubules and move towards the microtubule plus-ends. We find that APP associates with microtubules by confocal microscopy and co-purifies with KI-washed axoplasmic organelles by sucrose density gradient fractionation. By electron microscopy, APP clusters at a single focal point on the surfaces of organelles and localizes to the organelle/microtubule interface. In addition, the association of APP-organelles with microtubules is an ATP dependent process suggesting that the APP-organelles contain a microtubule-based motor protein. Although a direct kinesin/APP association remains controversial, the distribution of APP at the organelle/microtubule interface strongly suggests that APP-organelles have an orientation and that APP like the Alzheimer’s protein tau has a microtubule-based function.