Resilience of compound action potential peaks to high-frequency firing in the mouse optic nerve.

Action potential conduction in axons triggers trans-membrane ion movements, where Na+ enters and K+ leaves axons, leading to disruptions in resting trans-membrane ion gradients that must be restored for optimal axon conduction, an energy dependent process. The higher the stimulus frequency, the greater the ion movements and the resulting energy demand. In the mouse optic nerve (MON), the stimulus evoked compound action potential (CAP) displays a triple peaked profile, consistent with subpopulations of axons classified by size producing the distinct peaks. The three CAP peaks show differential sensitivity to high-frequency firing, with the large axons, which contribute to the 1st peak, more resilient than the small axons, which produce the 3rd peak. Modeling studies predict frequency dependent intra-axonal Na+ accumulation at the nodes of Ranvier, sufficient to attenuate the triple peaked CAP. Short bursts of high-frequency stimulus evoke transient elevations in interstitial K+ ([K+ ]o ), which peak at about 50 Hz. However, powerful astrocytic buffering limits the [K+ ]o increase to levels insufficient to cause CAP attenuation. A post-stimulus [K+ ]o undershoot below baseline coincides with a transient increase in the amplitudes of all three CAP peaks. The volume specific scaling relating energy expenditure to increasing axon size dictates that large axons are more resilient to high-frequency firing than small axons.

Activity-Dependent Fluctuations in Interstitial [K+]: Investigations Using Ion-Sensitive Microelectrodes.

In the course of action potential firing, all axons and neurons release K+ from the intra- cellular compartment into the interstitial space to counteract the depolarizing effect of Na+ influx, which restores the resting membrane potential. This efflux of K+ from axons results in K+ accumulation in the interstitial space, causing depolarization of the K+ reversal potential (EK), which can prevent subsequent action potentials. To ensure optimal neuronal function, the K+ is buffered by astrocytes, an energy-dependent process, which acts as a sink for interstitial K+, absorbing it at regions of high concentration and distributing it through the syncytium for release in distant regions. Pathological processes in which energy production is compromised, such as anoxia, ischemia, epilepsy and spreading depression, can lead to excessive interstitial K+ accumulation, disrupting sensitive trans-membrane ion gradients and attenuating neuronal activity. The changes that occur in interstitial [K+] resulting from both physiological and pathological processes can be monitored accurately in real time using K+-sensitive microelectrodes, an invaluable tool in electrophysiological studies.

A color-coded graphical guide to the Hodgkin and Huxley papers.

The five papers published by Hodgkin and Huxley in 1952 are seminal works in the field of physiology, earning their authors the Nobel Prize in 1963 and ushering in the era of membrane biophysics. The papers present a considerable challenge to the novice student, but this has been partly allayed by recent publications that have updated the reporting of current and voltage to reflect the modern convention and two books that describe the contents of the papers in detail. A disadvantage is that these guides contain hundreds of pages, requiring considerable time and energy on behalf of the reader. We present a concise guide to the Hodgkin and Huxley papers that includes only essential content, with the data presented in a linear and logical manner. We have color-coded figures for ease of understanding and included boxes that summarize key information for easy reference. It is our expectation that this article will act as an accessible introduction for students to the work of Hodgkin and Huxley and hopefully foster an appreciation for a fascinating story that repays in-depth study.NEW & NOTEWORTHY The Hodgkin and Huxley papers continue to inspire and intimidate, 70 years after their publication. The diverse subjects they cover include advanced experimental procedures, complex data analysis, calculus, and modeling, all of which ensure the papers can present a challenging read. We present a concise guide to the papers that includes only essential content depicted in color-coded graphs, allowing tracking of data from recordings to analysis and incorporation into the model to ease understanding.

Energy Metabolism in Mouse Sciatic Nerve A Fibres during Increased Energy Demand.

The ability of sciatic nerve A fibres to conduct action potentials relies on an adequate supply of energy substrate, usually glucose, to maintain necessary ion gradients. Under our ex vivo experimental conditions, the absence of exogenously applied glucose triggers Schwann cell glycogen metabolism to lactate, which is transported to axons to fuel metabolism, with loss of the compound action potential (CAP) signalling glycogen exhaustion. The CAP failure is accelerated if tissue energy demand is increased by high-frequency stimulation (HFS) or by blocking lactate uptake into axons using cinnemate (CIN). Imposing HFS caused CAP failure in nerves perfused with 10 mM glucose, but increasing glucose to 30 mM fully supported the CAP and promoted glycogen storage. A combination of glucose and lactate supported the CAP more fully than either substrate alone, indicating the nerve is capable of simultaneously metabolising each substrate. CAP loss resulting from exposure to glucose-free artificial cerebrospinal fluid (aCSF) could be fully reversed in the absence of glycogen by addition of glucose or lactate when minimally stimulated, but imposing HFS resulted in only partial CAP recovery. The delayed onset of CAP recovery coincided with the release of lactate by Schwann cells, suggesting that functional Schwann cells are a prerequisite for CAP recovery.

The Nernst equation: using physico-chemical laws to steer novel experimental design.

The application of physico-chemical principles has been routinely used to explain various physiological concepts. The Nernst equation is one example of this, used to predict the potential difference created by the transmembrane ion gradient resulting from uneven ion distribution within cellular compartments and the interstitial space. This relationship remains of fundamental importance to the understanding of electrical signaling in the brain, which relies on current flow across cell membranes. We describe four distinct occasions when the Nernst equation was ingeniously applied in experimental design to illuminate diverse cellular functions, from the dependence of the action potential on Na+ influx to K+ buffering in astrocytes. These examples are discussed with the aim of inspiring students to appreciate how the application of seemingly textbook-bound concepts can dictate novel experimental design across physiological disciplines.

A classic experiment revisited: membrane permeability changes during the action potential.

The ability to understand the relationship between the reversal potential and the membrane potential is a fundamental skill that must be mastered by students studying membrane excitability. To clarify this relationship, we have reframed a classic experiment carried out by Hodgkin and Katz, where we compare graphically the membrane potential at three phases of the action potential (resting potential, action potential peak, and afterhyperpolarization) to reversal potential for K+ (EK), reversal potential for Na (ENa), and membrane potential (Em) (calculated by the Goldman Hodgkin Katz equation) to illustrate that the membrane potential approaches the reversal potential of the ion to which it is most permeable at that instant.

Universal Glia to Neurone Lactate Transfer in the Nervous System: Physiological Functions and Pathological Consequences.

Whilst it is universally accepted that the energy support of the brain is glucose, the form in which the glucose is taken up by neurones is the topic of intense debate. In the last few decades, the concept of lactate shuttling between glial elements and neural elements has emerged in which the glial cells glycolytically metabolise glucose/glycogen to lactate, which is shuttled to the neural elements via the extracellular fluid. The process occurs during periods of compromised glucose availability where glycogen stored in astrocytes provides lactate to the neurones, and is an integral part of the formation of learning and memory where the energy intensive process of learning requires neuronal lactate uptake provided by astrocytes. More recently sleep, myelination and motor end plate integrity have been shown to involve lactate shuttling. The sequential aspect of lactate production in the astrocyte followed by transport to the neurones is vulnerable to interruption and it is reported that such disparate pathological conditions as Alzheimer's disease, amyotrophic lateral sclerosis, depression and schizophrenia show disrupted lactate signalling between glial cells and neurones.

A primer on tissue pH and local anesthetic potency.

The relationship between pH, pKa, and degree of local anesthetic ionization is quantified by the Henderson-Hasselbalch equation. As presented in standard textbooks, the effect of pH on the degree of ionization of any particular local anesthetic is not immediately clear due to the x-axis displaying pH - pKa, which requires conversion to pH, based on the pKa for each local anesthetic, a complex process. We present a graphical solution that clarifies the interrelationships between pH, pKa, and degree of ionization by plotting pKa on the x-axis versus the percentage of unionized local anesthetic on the y-axis. The vertical intercept from the x-axis to the pH curves allows rapid and accurate estimation of the degree of ionization of any local anesthetic of known pKa.

A method for reducing animal use whilst maintaining statistical power in electrophysiological recordings from rodent nerves.

The stimulus evoked compound action potential, recorded from ex vivo nerve trunks such as the rodent optic and sciatic nerve, is a popular model system used to study aspects of nervous system metabolism. This includes (1) the role of glycogen in supporting axon conduction, (2) the injury mechanisms resulting from metabolic insults, and (3) to test putative benefits of clinically relevant neuroprotective strategies. We demonstrate the benefit of simultaneously recording from pairs of nerves in the same superfusion chamber compared with conventional recordings from single nerves. Experiments carried out on mouse optic and sciatic nerves demonstrate that our new recording configuration decreased the relative standard deviation from samples when compared with recordings from an equivalent number of individually recorded nerves. The new method reduces the number of animals required to produce equivalent Power compared with the existing method, where single nerves are used. Adopting this method leads to increased experimental efficiency and productivity. We demonstrate that reduced animal use and increased Power can be achieved by recording from pairs of rodent nerve trunks simultaneously.

Calbindin Deficits May Underlie Dissociable Effects of 5-HT6 and mGlu7 Antagonists on Glutamate and Cognition in a Dual-Hit Neurodevelopmental Model for Schizophrenia.

Despite several compounds entering clinical trials for the negative and cognitive symptoms of schizophrenia, few have progressed beyond phase III. This is partly attributed to a need for improved preclinical models, to understand disease and enable predictive evaluation of novel therapeutics. To this end, one recent approach incorporates "dual-hit" neurodevelopmental insults like neonatal phencyclidine plus isolation rearing (PCP-Iso). Glutamatergic dysfunction contributes to schizophrenia pathophysiology and may represent a treatment target, so we used enzyme-based microsensors to evaluate basal- and drug-evoked glutamate release in hippocampal slices from rats that received neonatal PCP and/or isolation rearing. 5-HT6 antagonist-evoked glutamate release (thought to be mediated indirectly via GABAergic disinhibition) was reduced in PCP-Iso, as were cognitive effects of a 5-HT6 antagonist in a hippocampal glutamate-dependent novel object discrimination task. Yet mGlu7 antagonist-evoked glutamatergic and cognitive responses were spared. Immunohistochemical analyses suggest these findings (which mirror the apparent lack of clinical response to 5-HT6 antagonists in schizophrenia) are not due to reduced hippocampal 5-HT input in PCP-Iso, but may be explained by reduced calbindin expression. This calcium-binding protein is present in a subset of GABAergic interneurons receiving preferential 5-HT innervation and expressing 5-HT6 receptors. Its loss (in schizophrenia and PCP-Iso) would be expected to reduce interneuron firing and potentially prevent further 5-HT6 antagonist-mediated disinhibition, without impacting on responses of VIP-expressing interneurons to mGlu7 antagonism. This research highlights the importance of improved understanding for selection of appropriate preclinical models, especially where disease neurobiology impacts on cells mediating the effects of potential therapeutics.

Metabolism of Glycogen in Brain White Matter.

Brain glycogen is a specialized energy buffer, rather than a conventional reserve. In the rodent optic nerve, a central white matter tract, it is located in astrocytes, where it is converted to lactate, which is then shuttled intercellularly from the astrocyte to the axon. This basic pathway was elucidated from non-physiological experiments in which the nerve was deprived of exogenous glucose. However, this shuttling also occurs under physiological conditions, when tissue energy demand is increased above baseline levels in the presence of normoglycemic concentrations of glucose. The signaling mechanism by which axons alert astrocytes to their increased energy requirement is likely to be elevated interstitial K+, the inevitable consequence of increased neuronal activity.

The Role of Brain Glycogen in Supporting Physiological Function.

Glycogen is present in the mammalian brain but occurs at concentrations so low it is unlikely to act as a conventional energy reserve. Glycogen has the intriguing feature of being located exclusively in astrocytes, but its presence benefits neurones, suggesting that glycogen is metabolized to a conduit that is transported between the glia and neural elements. In the rodent optic nerve model glycogen supports axon conduction in the form of lactate to supplement axonal metabolism during aglycemia, hypoglycemia and during periods of increased energy demand under normoglycemic conditions. In the hippocampus glycogen plays a vital role in supplying the neurones with lactate during memory formation. The physiological processes that glycogen supports, such as learning and memory, imply an inclusive and vital role in supporting physiological brain functions.

Hypothermic neuroprotection during reperfusion following exposure to aglycemia in central white matter is mediated by acidification.

Hypoglycemia is a common iatrogenic consequence of type 1 diabetes therapy that can lead to central nervous system injury and even death if untreated. In the absence of clinically effective neuroprotective drugs we sought to quantify the putative neuroprotective effects of imposing hypothermia during the reperfusion phase following aglycemic exposure to central white matter. Mouse optic nerves (MONs), central white matter tracts, were superfused with oxygenated artificial cerebrospinal fluid (aCSF) containing 10 mmol/L glucose at 37°C. The supramaximal compound action potential (CAP) was evoked and axon conduction was assessed as the CAP area. Extracellular lactate was measured using an enzyme biosensor. Exposure to aglycemia, simulated by omitting glucose from the aCSF, resulted in axon injury, quantified by electrophysiological recordings, electron microscopic analysis confirming axon damage, the extent of which was determined by the duration of aglycemia exposure. Hypothermia attenuated injury. Exposing MONs to hypothermia during reperfusion resulted in improved CAP recovery compared with control recovery measured at 37°C, an effect attenuated in alkaline aCSF. Hypothermia decreases pH implying that the hypothermic neuroprotection derives from interstitial acidification. These results have important clinical implications demonstrating that hypothermic intervention during reperfusion can improve recovery in central white matter following aglycemia.

Fibre sub-type specific conduction reveals metabolic function in mouse sciatic nerve.

KEY POINTS: We have developed an improved method that enables simultaneous recording of stimulus evoked compound action potentials from large myelinated A fibres and small unmyelinated C fibres in mouse sciatic nerves. Investigations into the ability of fructose to support conduction in sciatic nerve revealed a novel glia-to-axon metabolic pathway in which fructose is converted in Schwann cells to lactate for subsequent shuttling to A fibres. The C fibres most likely directly take up and metabolise fructose. These differences are indicative of fibre sub-type specific metabolic profiles. These results demonstrate that the physiological insights provided by the method can be applied to investigations of peripheral nerve, with a view to understanding the metabolic disruptions that underlie diabetic neuropathy. ABSTRACT: The stimulus evoked compound action potential (CAP), recorded using suction electrodes, provides an index of the relative number of conducting axons within a nerve trunk. As such the CAP has been used to elucidate the diverse mechanisms of injury resulting from a variety of metabolic insults to central nervous white matter, whilst also providing a model with which to assess the benefits of clinically relevant neuroprotective strategies. In addition the technique lends itself to the study of metabolic cell-to-cell signalling that occurs between glial cells and neurones, and to exploring the ability of non-glucose substrates to support axon conduction. Although peripheral nerves are sensitive to metabolic insult and are susceptible to diabetic neuropathy, there is a lack of fundamental information regarding peripheral nerve metabolism. A confounding factor in such studies is the extended duration demanded by the experimental protocol, requiring stable recording for periods of many hours. We describe a method that allows us to record simultaneously the stimulus evoked CAPs from A and C fibres from mouse sciatic nerve, and demonstrate its utility as applied to investigations into fibre sub-type substrate use. Our results suggest that C fibres directly take up and metabolise fructose, whereas A fibre conduction is supported by fructose-derived lactate, implying there exist unique metabolic profiles in neighbouring fibre sub-types present within the same nerve trunk.

Acute Effect of Pore-Forming Clostridium perfringens ε-Toxin on Compound Action Potentials of Optic Nerve of Mouse.

ε-Toxin is a pore forming toxin produced by Clostridium perfringens types B and D. It is synthesized as a less active prototoxin form that becomes fully active upon proteolytic activation. The toxin produces highly lethal enterotoxaemia in ruminants, has the ability to cross the blood-brain barrier (BBB) and specifically binds to myelinated fibers. We discovered that the toxin induced a release of ATP from isolated mice optic nerves, which are composed of myelinated fibers that are extended from the central nervous system. We also investigated the effect of the toxin on compound action potentials (CAPs) in isolated mice optic nerves. When nerves were stimulated at 100 Hz during 200 ms, the decrease of the amplitude and the area of the CAPs was attenuated in the presence of ε-toxin. The computational modelling of myelinated fibers of mouse optic nerve revealed that the experimental results can be mimicked by an increase of the conductance of myelin and agrees with the pore forming activity of the toxin which binds to myelin and could drill it by making pores. The intimate ultrastructure of myelin was not modified during the periods of time investigated. In summary, the acute action of the toxin produces a subtle functional impact on the propagation of the nerve action potential in myelinated fibers of the central nervous system with an eventual desynchronization of the information. These results may agree with the hypothesis that the toxin could be an environmental trigger of multiple sclerosis (MS).

A Lifetime's Adventure in Extracellular K+ Regulation: The Scottish Connection.

In a career that has spanned 45 years and shows no signs of slowing down, Dr Bruce Ransom has devoted considerable time and energy to studying regulation of interstitial K+. When Bruce commenced his studies in 1969 virtually nothing was known of the functions of glial cells, but Bruce's research contributed to the physiological assignation of function to mammalian astrocytes, namely interstitial K+ buffering. The experiments that I describe in this review concern the response of the membrane potential (Em) of in vivo cat cortical astrocytes to changes in [K+]o, an experimental manoeuvre that was achieved in two different ways. The first involved recording the Em of an astrocyte while the initial aCSF was switched to one with different K+, whereas in the second series of experiments the cortex was stimulated and the response of the astrocyte Em to the K+ released from neighbouring neurons was recorded. The astrocytes responded in a qualitatively predictable manner, but quantitatively the changes were not as predicted by the Nernst equation. Elevations in interstitial K+ are not sustained and K+ returns to baseline rapidly due to the buffering capacity of astrocytes, a phenomenon studied by Bruce, and his son Chris, published 27 years after Bruce's initial publications. Thus, a lifetime spent investigating K+ buffering has seen enormous advances in glial research, from the time cells were identified as 'presumed' glial cells or 'silent cells', to the present day, where glial cells are recognised as contributing to every important physiological brain function.