Riddled with holes: Understanding air space formation in plant leaves.

Plants use energy from sunlight to transform carbon dioxide from the air into complex organic molecules, ultimately producing much of the food we eat. To make this complex chemistry more efficient, plant leaves are intricately constructed in 3 dimensions: They are flat to maximise light capture and contain extensive internal air spaces to increase gas exchange for photosynthesis. Many years of work has built up an understanding of how leaves form flat blades, but the molecular mechanisms that control air space formation are poorly understood. Here, I review our current understanding of air space formation and outline how recent advances can be harnessed to answer key questions and take the field forward. Increasing our understanding of plant air spaces will not only allow us to understand a fundamental aspect of plant development, but also unlock the potential to engineer the internal structure of crops to make them more efficient at photosynthesis with lower water requirements and more resilient in the face of a changing environment.

Electrode pooling can boost the yield of extracellular recordings with switchable silicon probes.

State-of-the-art silicon probes for electrical recording from neurons have thousands of recording sites. However, due to volume limitations there are typically many fewer wires carrying signals off the probe, which restricts the number of channels that can be recorded simultaneously. To overcome this fundamental constraint, we propose a method called electrode pooling that uses a single wire to serve many recording sites through a set of controllable switches. Here we present the framework behind this method and an experimental strategy to support it. We then demonstrate its feasibility by implementing electrode pooling on the Neuropixels 1.0 electrode array and characterizing its effect on signal and noise. Finally we use simulations to explore the conditions under which electrode pooling saves wires without compromising the content of the recordings. We make recommendations on the design of future devices to take advantage of this strategy.

Extracellular detection of neuronal coupling.

We developed a method to non-invasively detect synaptic relationships among neurons from in vitro networks. Our method uses microelectrode arrays on which neurons are cultured and from which propagation of extracellular action potentials (eAPs) in single axons are recorded at multiple electrodes. Detecting eAP propagation bypasses ambiguity introduced by spike sorting. Our methods identify short latency spiking relationships between neurons with properties expected of synaptically coupled neurons, namely they were recapitulated by direct stimulation and were sensitive to changing the number of active synaptic sites. Our methods enabled us to assemble a functional subset of neuronal connectivity in our cultures.

Axonal injury alters the extracellular glial environment of the axon initial segment and allows substantial mitochondrial influx into axon initial segment.

The axon initial segment (AIS) is structurally and functionally distinct from other regions of the axon, yet alterations in the milieu of the AIS after brain injury have not been well characterized. In this study, we have examined extracellular and intracellular changes in the AIS after hypoglossal nerve injury. Microglial adhesions to the AIS were rarely observed in healthy controls, whereas microglial adhesions to the AIS became apparent in the axonal injury model. Regarding intra-AIS morphology, we focused on mitochondria because mitochondrial flow into the injured axon appears critical for axonal regeneration. To visualize mitochondria specifically in injured axons, we used Atf3:BAC transgenic mice whose mitochondria were labeled with GFP in response to nerve injury. These mice clearly showed mitochondrial localization in the AIS after nerve injury. To precisely confirm the light microscopic observations, we performed three-dimensional ultrastructural analysis using focused ion beam/scanning electron microscopy (FIB/SEM). Although the healthy AIS was not surrounded by microglia, tight microglial adhesions with thick processes adhering to the AIS were observed after injury. FIB/SEM simultaneously allowed the observation of mitochondrial localization in the AIS. In the AIS of non-injured neurons, few mitochondria were observed, whereas mitochondria were abundantly localized in the cell body, axon hillock, and axon. Intriguingly, in the injured AIS, numerous mitochondria were observed throughout the AIS. Taken together, axonal injury changes the extracellular glial environment surrounding the AIS and intracellular mitochondrial localization in the AIS. These changes would be crucial responses, perhaps for injured neurons to regenerate after axonal injury.

An electrodiffusive neuron-extracellular-glia model for exploring the genesis of slow potentials in the brain.

Within the computational neuroscience community, there has been a focus on simulating the electrical activity of neurons, while other components of brain tissue, such as glia cells and the extracellular space, are often neglected. Standard models of extracellular potentials are based on a combination of multicompartmental models describing neural electrodynamics and volume conductor theory. Such models cannot be used to simulate the slow components of extracellular potentials, which depend on ion concentration dynamics, and the effect that this has on extracellular diffusion potentials and glial buffering currents. We here present the electrodiffusive neuron-extracellular-glia (edNEG) model, which we believe is the first model to combine compartmental neuron modeling with an electrodiffusive framework for intra- and extracellular ion concentration dynamics in a local piece of neuro-glial brain tissue. The edNEG model (i) keeps track of all intraneuronal, intraglial, and extracellular ion concentrations and electrical potentials, (ii) accounts for action potentials and dendritic calcium spikes in neurons, (iii) contains a neuronal and glial homeostatic machinery that gives physiologically realistic ion concentration dynamics, (iv) accounts for electrodiffusive transmembrane, intracellular, and extracellular ionic movements, and (v) accounts for glial and neuronal swelling caused by osmotic transmembrane pressure gradients. The edNEG model accounts for the concentration-dependent effects on ECS potentials that the standard models neglect. Using the edNEG model, we analyze these effects by splitting the extracellular potential into three components: one due to neural sink/source configurations, one due to glial sink/source configurations, and one due to extracellular diffusive currents. Through a series of simulations, we analyze the roles played by the various components and how they interact in generating the total slow potential. We conclude that the three components are of comparable magnitude and that the stimulus conditions determine which of the components that dominate.

LONG-TERM COURSE AND VISUAL OUTCOMES OF PRECHOROIDAL CLEFT IN NEOVASCULAR AGE-RELATED MACULAR DEGENERATION AND POLYPOIDAL CHOROIDAL VASCULOPATHY.

PURPOSE: To evaluate the regression of prechoroidal cleft, its influence on visual outcomes, and differences in visual outcomes between neovascular age-related macular degeneration and polypoidal choroidal vasculopathy. METHODS: This retrospective study included 61 patients exhibiting prechoroidal cleft who were treated with antivascular endothelial growth factors. The patients were divided into two groups according to the following categories: 1) regression of prechoroidal cleft: regression group versus nonregression group and 2) type of neovascularization: neovascular age-related macular degeneration group versus polypoidal choroidal vasculopathy group. Changes in the visual acuity during the follow-up period were also compared between the two groups. RESULTS: During the 52.4 ± 17.4-month follow-up period, regression of prechoroidal cleft was noted in 17 patients (27.9%) at a mean of 25.7 ± 18.3 months after the first identification. The degree of the logarithm of the minimum angle of resolution of visual deterioration was greater in the nonregression group (0.59 ± 0.56, n = 17) than that in the regression group (0.25 ± 0.61, n = 44) (P = 0.007) and in the neovascular age-related macular degeneration group (0.56 ± 0.61, n = 51) than that in the polypoidal choroidal vasculopathy group (0.18 ± 0.33, n = 10) (P = 0.034). CONCLUSION: Approximately 27.9% of prechoroidal cleft cases eventually regressed, in conjunction with relatively favorable visual outcomes. Considering the poor visual prognosis in neovascular age-related macular degeneration accompanied by prechoroidal cleft, more caution is required for this condition.

What we can and what we cannot see with extracellular multielectrodes.

Extracellular recording is an accessible technique used in animals and humans to study the brain physiology and pathology. As the number of recording channels and their density grows it is natural to ask how much improvement the additional channels bring in and how we can optimally use the new capabilities for monitoring the brain. Here we show that for any given distribution of electrodes we can establish exactly what information about current sources in the brain can be recovered and what information is strictly unobservable. We demonstrate this in the general setting of previously proposed kernel Current Source Density method and illustrate it with simplified examples as well as using evoked potentials from the barrel cortex obtained with a Neuropixels probe and with compatible model data. We show that with conceptual separation of the estimation space from experimental setup one can recover sources not accessible to standard methods.

Extracellular water/total body water ratio as predictor of mortality in hemodialysis patients.

BACKGROUND: Overhydration is a predictor of mortality in hemodialysis (HD) patients. Bioimpedance spectroscopy (BIS) is used to determine the body composition. Extracellular Water/Total Body Water (ECW/TBW) ratio has been proposed to predict mortality. METHODS: Multicenter, prospective, observational, proof-of-concept study to estimate the impact of ECW/TBW in global and cardiovascular mortality and the relationship with cardiovascular biomarkers. The study included 60 patients (mean age, 71.8 ± 11.4 years; mean time on HD, 52.3 ± 30.8 months) with a median follow-up of 30.5 months (IQ range, 17.2-34 months). RESULTS: Post-dialysis ECW/TBW was directly associated with NT-proBNP and cTnT. During the study 28 patients died, most of them (43%) due to cardiovascular events. Compared to the survivors, these subjects had a higher post-dialysis ECW/TBW ratio (p = 0.006), while for cardiovascular mortality the only significant difference was a higher pre-dialysis ECW/TBW. The ability of post-dialysis ECW/TBW ratio to predict all-cause mortality had an area under the ROC curve (AUC) of 0.71 (CI 95%, 0.57-0.81; p = 0.002), with a cutoff point of 0.5023. For cardiovascular mortality the AUC was 0.66 (CI 95%, 0.52-0.77; p = 0.045), with a cutoff point of 0.4713. CONCLUSIONS: The post-dialysis ECW/TBW ratio measured by BIS can be a predictor of all-cause and cardiovascular mortality.

Examining the evidence for extracellular RNA function in mammals.

The presence of RNAs in the extracellular milieu has sparked the hypothesis that RNA may play a role in mammalian cell-cell communication. As functional nucleic acids transfer from cell to cell in plants and nematodes, the idea that mammalian cells also transfer functional extracellular RNA (exRNA) is enticing. However, untangling the role of mammalian exRNAs poses considerable experimental challenges. This Review discusses the evidence for and against functional exRNAs in mammals and their proposed roles in health and disease, such as cancer and cardiovascular disease. We conclude with a discussion of the forward-looking prospects for studying the potential of mammalian exRNAs as mediators of cell-cell communication.

Microscale Physiological Events on the Human Cortical Surface.

Despite ongoing advances in our understanding of local single-cellular and network-level activity of neuronal populations in the human brain, extraordinarily little is known about their "intermediate" microscale local circuit dynamics. Here, we utilized ultra-high-density microelectrode arrays and a rare opportunity to perform intracranial recordings across multiple cortical areas in human participants to discover three distinct classes of cortical activity that are not locked to ongoing natural brain rhythmic activity. The first included fast waveforms similar to extracellular single-unit activity. The other two types were discrete events with slower waveform dynamics and were found preferentially in upper cortical layers. These second and third types were also observed in rodents, nonhuman primates, and semi-chronic recordings from humans via laminar and Utah array microelectrodes. The rates of all three events were selectively modulated by auditory and electrical stimuli, pharmacological manipulation, and cold saline application and had small causal co-occurrences. These results suggest that the proper combination of high-resolution microelectrodes and analytic techniques can capture neuronal dynamics that lay between somatic action potentials and aggregate population activity. Understanding intermediate microscale dynamics in relation to single-cell and network dynamics may reveal important details about activity in the full cortical circuit.

Small Extracellular Vesicles Control Dendritic Spine Development through Regulation of HDAC2 Signaling.

The release of small extracellular vesicles (sEVs) has recently been reported, but knowledge of their function in neuron development remains limited. Using LC-MS/MS, we found that sEVs released from developing cortical neurons in vitro obtained from mice of both sexes were enriched in cytoplasm, exosome, and protein-binding and DNA/RNA-binding pathways. The latter included HDAC2, which was of particular interest, because HDAC2 regulates spine development, and populations of neurons expressing different levels of HDAC2 co-exist in vivo during the period of spine growth. Here, we found that HDAC2 levels decrease in neurons as they acquire synapses and that sEVs from HDAC2-rich neurons regulate HDAC2 signaling in HDAC2-low neurons possibly through HDAC2 transfer. This regulation led to a transcriptional decrease in HDAC2 synaptic targets and the density of excitatory synapses. These data suggest that sEVs provide inductive cell-cell signaling that coordinates the development of dendritic spines via the activation of HDAC2-dependent transcriptional programs.SIGNIFICANCE STATEMENT A role of small extracellular vesicles (sEVs; also called exosomes) in neuronal development is of particular interest, because sEVs could provide a major signaling modality between developing neurons when synapses are not fully functional or immature. However, knowledge of sEVs on neuron, and more precisely spine development, is limited. We provide several lines of evidence that sEVs released from developing cortical neurons regulate the development of dendritic spines via the regulation of HDAC2 signaling. This paracrine communication is temporally restricted during development because of the age-dependent decrease in sEV release as neurons mature and acquire spines.

Ephaptic coupling in white matter fibre bundles modulates axonal transmission delays.

Axonal connections are widely regarded as faithful transmitters of neuronal signals with fixed delays. The reasoning behind this is that extracellular potentials caused by spikes travelling along axons are too small to have an effect on other axons. Here we devise a computational framework that allows us to study the effect of extracellular potentials generated by spike volleys in axonal fibre bundles on axonal transmission delays. We demonstrate that, although the extracellular potentials generated by single spikes are of the order of microvolts, the collective extracellular potential generated by spike volleys can reach several millivolts. As a consequence, the resulting depolarisation of the axonal membranes increases the velocity of spikes, and therefore reduces axonal delays between brain areas. Driving a neural mass model with such spike volleys, we further demonstrate that only ephaptic coupling can explain the reduction of stimulus latencies with increased stimulus intensities, as observed in many psychological experiments.

Three-dimensional structure analysis of melanocytes and keratinocytes in senile lentigo.

Senile lentigo or age spots are hyperpigmented macules of skin that commonly develop following long-term exposure to ultraviolet radiation. This condition is caused by accumulation of large numbers of melanosomes (melanin granules) produced by melanocytes within neighboring keratinocytes. However, there is still no consensus regarding the melanosome transfer mechanism in senile lentigo. To date, most pathohistological studies of skin have been two-dimensional and do not provide detailed data on the complex interactions of the melanocyte-keratinocyte network involved in melanosome transfer. We performed a three-dimensional reconstruction of the epidermal microstructure in senile lentigo using three different microscopic modalities to visualize the topological melanocyte-keratinocyte relationship and melanosome distribution. Confocal laser microscopy images showed that melanocyte dendritic processes are more frequently branched and elongated in senile lentigo skin than in normal skin. Serial transmission electron micrographs showed that dendritic processes extend into intercellular spaces between keratinocytes. Focused ion beam-scanning electron micrographs showed that dendritic processes in senile lentigo encircle adjacent keratinocytes and accumulate large numbers of melanosomes. Moreover, melanosomes transferred to keratinocytes are present not only in the supranuclear area but throughout the perinuclear area except on the basal side. The use of these different microscopic methods helped to elucidate the three-dimensional morphology and topology of melanocytes and keratinocytes in senile lentigo. We show that the localization of melanosomes in dendritic processes to the region encircling recipient keratinocytes contributes to efficient melanosome transfer in senile lentigo.

Extracellular Multi-Unit Recording from the Olfactory Nerve of Teleosts.

Recent studies have shown that ocean acidification affects olfactory-driven behavior in fish. This may be due in part to a reduction in olfactory sensitivity in high PCO2/low pH water. To assess the effects of ocean acidification, or olfactory sensitivity in marine fish in general, we propose that extracellular multi-unit recording from the olfactory nerve is the method of choice. Although invasive, it is sensitive, robust, reproducible and independent of external salinity (unlike the electro-olfactogram [EOG], for example). Furthermore, it records a primary sensory input into the CNS, prior to any central processing. We show that this method can show a reduction in olfactory sensitivity that is both temporary and odorant-dependent, using a range of amino acids to construct concentration-response curves and calculate the thresholds of detection.

TARPs Modulate Receptor-Mediated Paired-Pulse Depression and Recovery from Desensitization.

Transmembrane AMPA receptor regulatory proteins (TARPs) are auxiliary AMPA receptor subunits that play a key role in receptor trafficking and in modulating receptor gating. The ability of TARPs to slow both deactivation and desensitization is isoform specific. However, TARP isoform-specific modulation of receptor properties remains uncharacterized. Here, we compare the isoform-specific effects of γ-2, γ-3, γ-4, and γ-8 TARPs on recovery from desensitization and responses to pairs of brief applications of glutamate. All four isoforms were able to reduce receptor-mediated paired-pulse depression and significantly speed recovery from desensitization in an isoform-specific manner. In the presence of TARPs, recovery time courses were observed to contain two components, fast and slow. The proportion of fast and slow components was determined by the TARP isoform. The time constant of recovery was also altered by the duration of glutamate application. When studies with TARP chimeras were performed, TARP extracellular loops were found to play a vital role in TARP modulation of recovery. Thus, isoform-specific differences in TARP modulation of recovery from desensitization influence receptor responses to repeated brief applications of glutamate, and these differences may impact frequency-dependent synaptic signaling in the mammalian central nervous system.SIGNIFICANCE STATEMENT AMPA receptors are major determinants of excitatory synaptic strength. The channel kinetics of AMPA receptors contribute to the kinetics of synaptic transmission. Transmembrane AMPA receptor regulatory proteins (TARPs) auxiliary subunits can modulate the decay kinetics of AMPA receptors. However, whether TARP isoforms specifically modulate receptor recovery is unclear. Here, we investigated the recovery kinetics of AMPA receptors by expressing various TARP isoforms and chimeras. We observed that the TARP isoforms and duration of glutamate application uniquely modulate time constants and the proportion of fast and slow components through a previously unidentified TARP domain. Given the impact of recovery kinetics on receptor responses to repetitive stimulation such as synaptic transmission, this work will be of great interest in the field of excitatory synaptic transmission research.

Spatial Organization and Dynamics of the Extracellular Space in the Mouse Retina.

The extracellular space (ECS) plays an important role in the physiology of neural circuits. Despite our detailed understanding of the cellular architecture of the mammalian retina, little is known about the organization and dynamics of the retinal ECS. We developed an optical technique based on two-photon imaging of fluorescently labeled extracellular fluid to measure the ECS volume fraction (α) in the ex vivo retina of male and female mice. This method has high spatial resolution and can detect rapid changes in α evoked by osmotic challenge and neuronal activity. The measured ECS α varied dramatically in different layers of the adult mouse retina, with α equaling ∼0.050 in the ganglion cell layer, ∼0.122 in the inner plexiform layer (IPL), ∼0.025 in the inner nuclear layer (INL), ∼0.087 in the outer plexiform layer, and ∼0.026 in the outer nuclear layer (ONL). ECS α was significantly larger early in retinal development; α was 67% larger in the IPL and 100% larger in the INL in neonatal mice compared with adults. In adult retinas, light stimulation evoked rapid decreases in ECS α. Light-driven reductions in ECS α were largest in the IPL, where visual stimuli decreased α values ∼10%. These light-evoked decreases demonstrate that a physiological stimulus can lead to rapid changes in ECS α and indicate that activity-dependent regulation of extracellular space may contribute to visual processing in the retina.SIGNIFICANCE STATEMENT The volume fraction of the extracellular space (ECS α), that portion of CNS tissue occupied by interstitial space, influences the diffusion of neurotransmitters from the synaptic cleft and the volume transmission of transmitters. However, ECS α has never been measured in live retina, and little is known about how ECS α varies following physiological stimulation. Here we show that ECS α values vary dramatically between different retinal layers and decrease by 10% following light stimulation. ECS α differences within the retina will influence volume transmission and light-evoked α variations may modulate synaptic transmission and visual processing in the retina. Activity-dependent ECS α variations may represent a mechanism of synaptic modulation throughout the CNS.

Stochastic intracellular regulation can remove oscillations in a model of tissue growth.

The work is devoted to the analysis of cell population dynamics where cells make a choice between differentiation and apoptosis. This choice is based on the values of intracellular proteins whose concentrations are described by a system of ordinary differential equations with bistable dynamics. Intracellular regulation and cell fate are controlled by the extracellular regulation through the number of differentiated cells. It is shown that the total cell number necessarily oscillates if the initial condition in the intracellular regulation is fixed. These oscillations can be suppressed if the initial condition is a random variable with a sufficiently large variation. Thus, the result of the work suggests a possible answer to the question about the role of stochasticity in the intracellular regulation.

Emerging roles of extracellular vesicles in the intercellular communication for exercise-induced adaptations.

Complex organisms rely heavily on intercellular communication. The rapidly expanding field of extracellular vesicle biology has made it clear that the necessary intercellular communication occurs partly through their paracrine and endocrine actions. Extracellular vesicles are nanoscale lipid membranes (30-2,000 nm in diameter) that shuttle functional biological material between cells. They are released from numerous tissues and are isolated from nearly all biofluids and cell cultures. Although their biogenesis, cell targeting, and functional roles are incompletely understood, they appear to have crucial roles in physiological and disease processes. Their enormous potential to serve as sensitive biomarkers of disease and also new therapeutic interventions for diseases have gained them considerable attention in recent years. Regular physical exercise training confers systemic health benefits and consequently prevents many age-related degenerative diseases. Many of the molecular mechanisms responsible for the salubrious effects of exercise are known, yet a common underlying mechanism potentially responsible for the holistic health benefits of exercise has only recently been explored (i.e., via extracellular vesicle transport of biological material). Here, we provide an overview of extracellular vesicle biology before outlining the current evidence on the capacity for a single bout and chronic exercise to elicit changes in extracellular vesicle content and modulate their molecular cargo (e.g., small RNAs). We highlight areas for future research and emphasize their potential utility as biomarkers and therapeutic strategies of disease and its prevention.

Dynamics of a neuron-glia system: the occurrence of seizures and the influence of electroconvulsive stimuli : A mathematical and numerical study.

In this paper, we investigate the dynamics of a neuron-glia cell system and the underlying mechanism for the occurrence of seizures. For our mathematical and numerical investigation of the cell model we will use bifurcation analysis and some computational methods. It turns out that an increase of the potassium concentration in the reservoir is one trigger for seizures and is related to a torus bifurcation. In addition, we will study potassium dynamics of the model by considering a reduced version and we will show how both mechanisms are linked to each other. Moreover, the reduction of the potassium leak current will also induce seizures. Our study will show that an enhancement of the extracellular potassium concentration, which influences the Nernst potential of the potassium current, may lead to seizures. Furthermore, we will show that an external forcing term (e.g. electroshocks as unidirectional rectangular pulses also known as electroconvulsive therapy) will establish seizures similar to the unforced system with the increased extracellular potassium concentration. To this end, we describe the unidirectional rectangular pulses as an autonomous system of ordinary differential equations. These approaches will explain the appearance of seizures in the cellular model. Moreover, seizures, as they are measured by electroencephalography (EEG), spread on the macro-scale (cm). Therefore, we extend the cell model with a suitable homogenised monodomain model, propose a set of (numerical) experiment to complement the bifurcation analysis performed on the single-cell model. Based on these experiments, we introduce a bidomain model for a more realistic modelling of white and grey matter of the brain. Performing similar (numerical) experiment as for the monodomain model leads to a suitable comparison of both models. The individual cell model, with its seizures explained in terms of a torus bifurcation, extends directly to corresponding results in both the monodomain and bidomain models where the neural firing spreads almost synchronous through the domain as fast traveling waves, for physiologically relevant paramenters.

Division of labour in a matrix, rather than phagocytosis or endosymbiosis, as a route for the origin of eukaryotic cells.

Two apparently irreconcilable models dominate research into the origin of eukaryotes. In one model, amitochondrial proto-eukaryotes emerged autogenously from the last universal common ancestor of all cells. Proto-eukaryotes subsequently acquired mitochondrial progenitors by the phagocytic capture of bacteria. In the second model, two prokaryotes, probably an archaeon and a bacterial cell, engaged in prokaryotic endosymbiosis, with the species resident within the host becoming the mitochondrial progenitor. Both models have limitations. A search was therefore undertaken for alternative routes towards the origin of eukaryotic cells. The question was addressed by considering classes of potential pathways from prokaryotic to eukaryotic cells based on considerations of cellular topology. Among the solutions identified, one, called here the "third-space model", has not been widely explored. A version is presented in which an extracellular space (the third-space), serves as a proxy cytoplasm for mixed populations of archaea and bacteria to "merge" as a transitionary complex without obligatory endosymbiosis or phagocytosis and to form a precursor cell. Incipient nuclei and mitochondria diverge by division of labour. The third-space model can accommodate the reorganization of prokaryote-like genomes to a more eukaryote-like genome structure. Nuclei with multiple chromosomes and mitosis emerge as a natural feature of the model. The model is compatible with the loss of archaeal lipid biochemistry while retaining archaeal genes and provides a route for the development of membranous organelles such as the Golgi apparatus and endoplasmic reticulum. Advantages, limitations and variations of the "third-space" models are discussed. REVIEWERS: This article was reviewed by Damien Devos, Buzz Baum and Michael Gray.