Myelin sheaths comprise compacted layers of membrane wrapped around axons. Each sheath, if 'unwrapped', has a cytoplasm-filled space at its perimeter. Transmission electron microscopy reveals that this space contains microtubules and organelles; however, whether these are developmental remnants or serve a specific function remains unknown. Live imaging of myelinating oligodendrocytes in mice showed microtubule-dependent organelle transport within myelin sheath cytoplasmic spaces. Further, movement of myelin-located peroxisomes was modulated by neuronal electrical activity, in vitro and in vivo. Loss of oligodendroglial KIF21B and/or CNP in vivo led to the apparent stasis of myelin organelles and secondary axon pathology. We, thus, propose the acronym TRAM (transport route across myelin), defining the continuous cytoplasmic network in oligodendrocytes and myelin. This system, comprising the inner and outer tongues and paranodal loops, spans compact myelin and enables transport of metabolites and organelles between the oligodendrocyte soma and the periaxonal space.

Myelin sheaths comprise compacted layers of oligodendroglial membrane wrapped spirally around axons. Each sheath, if imagined unwrapped, has a cytoplasm-filled space at its perimeter, linking it to the oligodendrocyte soma via a short process. By electron microscopy (EM), this space, which we term the ‘ myelinic channel system ’ contains microtubules and membranous organelles, but whether these are remnants of development or serve a function is unknown. Performing live imaging of myelinating oligodendrocytes expressing fluorescent reporters, we found that the myelinic channel system serves microtubule-dependent organelle transport. Further, the intra-myelinic movement of peroxisomes was modulated by neuronal electrical activity in these mixed neural cell cultures. Loss of oligodendroglial Kif21b or CNP in vivo led to apparent stasis of myelin organelles and secondary axon pathology. This suggests that oligodendrocytes require motor transport in the myelinic channel system to maintain axonal integrity. ### Competing Interest Statement The authors have declared no competing interest.

Astrocyte-derived cholesterol supports brain cells under physiological conditions. However, in demyelinating lesions, astrocytes downregulate cholesterol synthesis and the cholesterol that is essential for remyelination has to originate from other cellular sources. Here, we show that repair following acute versus chronic demyelination involves distinct processes. In particular, we found that in chronic myelin disease, when recycling of lipids is often defective, de novo neuronal cholesterol synthesis is critical for regeneration. By gene expression profiling, genetic loss of function experiments and comprehensive phenotyping, we provide evidence that neurons increase cholesterol synthesis in chronic myelin disease models and MS patients. In mouse models, neuronal cholesterol facilitated remyelination specifically by triggering OPC proliferation. Our data contribute to the understanding of disease progression and have implications for therapeutic strategies in MS patients.

The normal development and maintenance of CNS white matter, and its responses to disease and injury, are defined by synergies between axons, oligodendrocytes, astrocytes and microglia, and further influenced by peripheral components such as the gut microbiome and the endocrine and immune systems. Consequently, mechanistic insights, therapeutic approaches and safety tests rely ultimately on in vivo models and clinical trials. However, in vitro models that replicate the cellular complexity of the CNS can inform these approaches, reducing costs and minimising the use of human material or experimental animals; in line with the principles of the 3Rs. Using electrophysiology, pharmacology, time-lapse imaging, and immunological assays, we demonstrate that murine spinal cord-derived myelinating cell cultures recapitulate spinal-like electrical activity and innate CNS immune functions, including responses to disease-relevant myelin debris and pathogen associated molecular patterns (PAMPs). Further, we show they are (i) amenable to siRNA making them suitable for testing gene-silencing strategies; (ii) can be established on microelectrode arrays (MEAs) for electrophysiological studies; and (iii) are compatible with multi-well microplate formats for semi-high throughput screens, maximising information output whilst further reducing animal use. We provide protocols for each of these. Together, these advances increase the utility of this in vitro tool for studying normal and pathological development and function of white matter, and for screening therapeutic molecules or gene targets for diseases such as multiple sclerosis, motor neuron disease or spinal cord injury, whilst avoiding in vivo approaches on experimental animals.

Membrane-nanoparticle interactions are important in determining the effects of manufactured nanomaterials on cell physiology and pathology. Here, silica, titanium, zinc, and magnesium oxide nanoparticles are screened against human hERG (Kv 11.1) voltage-gated potassium channels under a whole-cell voltage clamp. 10 µg mL-1 ZnO uniquely increases the amplitude of the steady-state current, decreases the rate of hERG current inactivation during steady-state depolarization, accelerates channel deactivation during resurgent tail currents, and shows no significant alteration of current activation rate or voltage dependence. In contrast, ZnCl2 causes increased current suppression with increasing concentration and fails to replicate the nanoparticle effect on decreasing inactivation. The results show a novel class of nanoparticle-biomembrane interaction involving channel gating rather than channel block, and have implications for the use of nanoparticles in biomedicine, drug delivery applications, and nanotoxicology.

Large conductance Ca2+‐activated K+ (BK) channels are central in diverse cellular processes including neurotransmitter release or smooth muscle contraction. BK channel activation is driven by both intracellular Ca2+ and voltage, and they are essential for rapid membrane repolarisation. Moreover, BK channels are expressed in human β‐cells regulating insulin secretion. Indeed, in diabetes mellitus BK channels activity is disrupted.Previous electrophysiological studies suggested that N‐type (Cav2.2) calcium‐ and BK‐channel molecules must be in close proximity in the plasma membrane. Therefore, we developed novel methods to analyze the dynamics and distribution of BK channels at the level of single molecules in cell membranes using super‐resolution microscopy, molecular manipulation and electrophysiology. We expressed a cDNA construct that encodes the BK‐channel α‐subunit (BKα) fused to a photoactivatable fluorescent protein mutant of mCherry (PAmCherry) and localized single channel molecules. We also analyzed BK channel localization with gated stimulated emission depletion (g‐STED) microscopy using a BKα‐EGFP construct. Furthermore, we integrated both total internal reflection fluorescence (TIRF) microscopy and patch‐clamp for the study of BK channels function and localization.In order to study Ca2+ channel proximity with BK channels and organelles like secretory vesicles we transfected secretory cells with our BKα‐PAmCherry construct or use the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system to create genetically modified cells, labelling endogenous Cav2.2 channels with fluorescent‐Conotoxin (specific blocker for Cav2.2) that can be photoactivated. With this experiment we could analyse both channel membrane localization and proximity to secretory vesicles and exocytosis sites, using caged Ca2+ to trigger secretory vesicle fusion with the cell membrane. By co‐transfecting cells with both a GCaMP construct localized in the inner cell membrane layer and BKα‐PAmCherry constructs we can track the intracellular Ca2+ rise due to GCaMP fluorescence emission and how depolarization affects BK channels dynamics.These experiments revealed the dynamics, proximity and patterning of large cohorts of single ion channel protein molecules and their functional relationship with the secretory machinery.

Calcium ions in the human body serve as second messengers and are responsible for cell homeostasis. Muscle contractions and neuronal communication are supported by electrical activity. Voltage‐activated calcium channels are crucial for many cellular events. They play an important role in transducing the initial stimulus to the effector systems that modulate insulin secretion in pancreatic cells or neurotransmitter at nerve terminals. Dysfunction of these processes may cause many conditions, including diabetes or Alzheimer's disease. In recent years the amount of research on Ca2+ channels has markedly increased, but there are limitations related to physical restrictions in the spatial resolution of fluorescence microscopy. This phenomenon results in a loss of information with regard to the true location of a point source that is emitting light. Newly developed methods such as Stimulated Emission Depletion Microscopy (STED) or Photoactivated localization microscopy (PALM) allow imaging close to the molecular scale.The general aims of this project so far were focused on understanding the mechanism of action and distribution of N‐type calcium channels by using novel tools I have helped to develop for calcium channel imaging.To find out if Cav2.2 Ca2+ (Neuronal type) channels are in a clustered conformation or randomly distributed in the cellular plasma membrane, gated‐ (g)STED was used. After measuring the size of the signals in gSTED image data we conclude that Cav2.2 are patterned in clusters at the plasma membrane. To support that conclusion and characterize our imaging capability, more experiments using known size, sub‐diffraction fluorescent beads were conducted.To quantify the mobility and location of N‐type calcium channels PALM was used. The principle of PALM is activating several photoactivable fluorescent molecules and imaging their signals until irreversible photo‐bleaching occurs. This process of activation and inactivation is then repeated over thousands of frames to ensure all molecules have been imaged. Using a photo‐uncageable fluorescent dye mono‐conjugated with a peptide toxin from a marine snail, allows for imaging single endogenous channel nano‐scale locations. This experiment is significantly better than the current state‐of‐the‐art and revealed the patterning of large cohorts of single endogenous ion channels. To verify mobility of N‐type calcium channels series of images were analysed using bespoke particle tracking scripts.Exocytosis is a process in which secretory vesicles are fused to the plasma membrane. Vesicles proximal to the plasma membrane are triggered to fuse by a rise in local free Ca2+ ions. The probability of the release of synaptic vesicles is hypothesized to increase with the number of proximal calcium channels. We combined our novel approaches to generate calcium ‘activity maps’ of the plasma membrane to investigate the relationship between calcium channels and exocytosis.

The nematode 'worm' >> fast forward for physiology 20 Drosophila -a model for all reasons 24 Are medicinal leeches still a useful model for studying neurophysiology?27 Why do physiologists work on snails?A personal perspective 31 Tunicates: not just little squirts?34 Some recent advances in spider sensory physiology

In many human cell types, the class I phosphoinositide 3-kinases play key roles in the control of diverse cellular processes including growth, proliferation, survival and polarity. This is achieved through their activation by many cell surface receptors, leading to the synthesis of the phosphoinositide lipid signal, PIP3, which in turn influences the function of numerous direct PIP3-binding proteins. Here we review PI3K pathway biology and analyse the evolutionary distribution of its components and their functions. The broad phylogenetic distribution of class I PI3Ks in metazoa, amoebozoa and choannoflagellates, implies that these enzymes evolved in single celled organisms and were later co-opted into metazoan intercellular communication. A similar distribution is evident for the AKT and Cytohesin groups of downstream PIP3-binding proteins, with other effectors and pathway components appearing to evolve later. The genomic and functional phylogeny of regulatory systems such as the PI3K pathway provides a framework to improve our understanding of the mechanisms by which key cellular processes are controlled in humans.

The effects of nanomaterials (NMs) on biological systems, especially their ability to stimulate inflammatory responses requires urgent investigation. We evaluated the response of the human differentiated HL60 neutrophil-like cell line to NMs. It was hypothesised that NM physico-chemical characteristics would influence cell responsiveness by altering intracellular Ca2+ concentration [Ca2+]i and reactive oxygen species production. Cells were exposed (1.95-125 μg/ml, 24 h) to silver (Ag), zinc oxide (ZnO), titanium dioxide (TiO2), multi-walled carbon nanotubes (MWCNTs) or ultrafine carbon black (ufCB) and cytotoxicity assessed (alamar blue assay). Relatively low (TiO2, MWCNTs, ufCB) or high (Ag, ZnO) cytotoxicity NMs were identified. Sub-lethal impacts of NMs on cell function were investigated for selected NMs only, namely TiO2, Ag and ufCB. Only Ag stimulated cell activation. Within minutes, Ag stimulated an increase in [Ca2+]i (in Fura-2 loaded cells), and a prominent inward ion current (assessed by electrophysiology). Within 2-4 h, Ag increased superoxide anion release and stimulated cytokine production (MCP-1, IL-8) that was diminished by Ca2+ inhibitors or trolox. Light microscopy demonstrated that cells had an activated phenotype. In conclusion NM toxicity was ranked; Ag>ufCB>TiO2, and the battery of tests used provided insight into the mechanism of action of NM toxicity to guide future testing strategies.

Neuronal synapses are among the most scrutinized of cellular systems, serving as a model for all membrane trafficking studies. Despite this, synaptic biology has proven difficult to interrogate directly in situ due to the small size and dynamic nature of central synapses and the molecules within them. Here we determine the spatial and temporal interaction status of presynaptic proteins, imaging large cohorts of single molecules inside active synapses. Measuring rapid interaction dynamics during synaptic depolarization identified the small number of syntaxin1a and munc18-1 protein molecules required to support synaptic vesicle exocytosis. After vesicle fusion and subsequent SNARE complex disassembly, a prompt switch in syntaxin1a and munc18-1-binding mode, regulated by charge alteration on the syntaxin1a N-terminal, sequesters monomeric syntaxin1a from other disassembled fusion complex components, preventing ectopic SNARE complex formation, readying the synapse for subsequent rounds of neurotransmission.

Co-option of cis-regulatory modules has been suggested as a mechanism for the evolution of expression sites during development. However, the extent and mechanisms involved in mobilization of cis-regulatory modules remains elusive. To trace the history of non-coding elements, which may represent candidate ancestral cis-regulatory modules affirmed during chordate evolution, we have searched for conserved elements in tunicate and vertebrate (Olfactores) genomes. We identified, for the first time, 183 non-coding sequences that are highly conserved between the two groups. Our results show that all but one element are conserved in non-syntenic regions between vertebrate and tunicate genomes, while being syntenic among vertebrates. Nevertheless, in all the groups, they are significantly associated with transcription factors showing specific functions fundamental to animal development, such as multicellular organism development and sequence-specific DNA binding. The majority of these regions map onto ultraconserved elements and we demonstrate that they can act as functional enhancers within the organism of origin, as well as in cross-transgenesis experiments, and that they are transcribed in extant species of Olfactores. We refer to the elements as 'Olfactores conserved non-coding elements'.

As calcium is the most important signaling molecule in neurons and secretory cells, amongst many other cell types, it follows that an understanding of calcium channels and their regulation of exocytosis is of vital importance. Calcium imaging using calcium dyes such as Fluo3, or FRET-based dyes that have been used widely has provided invaluable information, which combined with modeling has estimated the subtypes of channels responsible for triggering the exocytotic machinery as well as inferences about the relative distances away from vesicle fusion sites these molecules adopt. Importantly, new super-resolution microscopy techniques, combined with novel Ca(2+) indicators and imaginative imaging approaches can now define directly the nano-scale locations of very large cohorts of single channel molecules in relation to single vesicles. With combinations of these techniques the activity of individual channels can be visualized and quantified using novel Ca(2+) indicators. Fluorescently labeled specific channel toxins can also be used to localize endogenous assembled channel tetramers. Fluorescence lifetime imaging microscopy and other single-photon-resolution spectroscopic approaches offer the possibility to quantify protein-protein interactions between populations of channels and the SNARE protein machinery for the first time. Together with simultaneous electrophysiology, this battery of quantitative imaging techniques has the potential to provide unprecedented detail describing the locations, dynamic behaviors, interactions, and conductance activities of many thousands of channel molecules and vesicles in living cells.

The outstanding behavioural capacity of cephalopods is underpinned by a highly sophisticated nervous system anatomy and neural mechanisms that often differ significantly from similarly complex systems in vertebrates and insects. Cephalopods exhibit considerable behavioural flexibility and adaptability, and it might be expected that this should be supported by evident cellular and synaptic plasticity. Here, we review what little is known of the cellular mechanisms that underlie plasticity in cephalopods, particularly from the point of view of synaptic function. We conclude that cephalopods utilise short-, medium-, and long-term plasticity mechanisms that are superficially similar to those so far described in vertebrate and insect synapses. These mechanisms, however, often differ significantly from those in other animals at the biophysical level and are deployed not just in the central nervous system, but also to a limited extent in the peripheral nervous system and neuromuscular junctions.