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The movement of ions in and out of neurons can exert significant effects on neighboring cells. Here we report several experimentally important consequences of activation of the optogenetic chloride pump, halorhodopsin. We recorded extracellular K+concentration ([K+]extra) in neocortical brain slices prepared from young adult mice (both sexes) which express halorhodopsin in pyramidal cells. Strong halorhodopsin activation induced a pronounced drop in [K+]extrathat persisted for the duration of illumination. Pharmacological blockade of K+channels reduced the amplitude of this drop, indicating that it represents K+redistribution into cells during the period of hyperpolarization. Halorhodopsin thus drives the inward movement of both Cl–directly, and K+secondarily. When the illumination period ended, a rebound surge in extracellular [K+] developed over tens of seconds, partly reflecting the previous inward redistribution of K+, but additionally driven by clearance of Cl–coupled to K+by the potassium-chloride cotransporter, KCC2. The drop in [K+]extraduring light activation leads to a small (2-3 mV) hyperpolarization also of other cells that do not express halorhodopsin. Its activation therefore has both direct and indirect inhibitory effects. Finally, we show that persistent strong activation of halorhodopsin causes cortical spreading depolarizations (CSDs), bothin vitroandin vivo. This novel means of triggering CSDs is unusual, in that the events can arise during the actual period of illumination, when neurons are being hyperpolarized and [K+]extrais low. We suggest that this fundamentally different experimental model of CSDs will open up new avenues of research to explain how they occur naturally.SIGNIFICANCE STATEMENTHalorhodopsin is a light-activated electrogenic chloride pump, which has been widely used to inhibit neurons optogenetically. Here, we demonstrate three previously unrecognized consequences of its use: (1) intense activation leads to secondary movement of K+ions into the cells; (2) the resultant drop in extracellular [K+] reduces excitability also in other, nonexpressing cells; and (3) intense persistent halorhodopsin activation can trigger cortical spreading depolarization (CSD). Halorhodopsin-induced CSDs can occur when neurons are hyperpolarized and extracellular [K+] is low. This contrasts with the most widely used experimental models that trigger CSDs with high [K+]. Both models, however, are consistent with the hypothesis that CSDs arise following net inward ionic movement into the principal neuron population.

Investigations into seizure initiation, in recent years, have focused almost entirely upon alterations of interneuronal function, chloride homeostasis, and extracellular potassium levels. In contrast, little attention has been directed toward a possible role of dendritic plateau potentials in the actual ictogenic transition, despite a substantial literature dating back 40 years regarding its importance generally in epilepsy. Here, we argue that an increase in dendritic excitability, coordinated across the population of pyramidal cells, is a key stage in ictogenesis.

We recently reported that strong activation of the optogenetic chloride pump, Halorhodopsin leads to a secondary redistribution of K+ions into the cell, through tonically open, “leak” K+channels. Here we show that this effect is not unique to halorhodopsin, but is also seen with activation of another electrogenic ion pump, archaerhodopsin. The two opsins differ however in the size of the rebound rise in extracellular potassium, [K+]o, after the end of activation, which is far larger with halorhodopsin than for archaerhodopsin activation. Multiple linear regression modelling indicates that most of the variance in the post-illumination surge in [K+]o was explained by the type of opsin, and almost nothing by the size of the preceding, illumination-induced drop in [K+]o. These data provide additional support for the hypothesis that intense chloride-loading of cells, as occurs naturally following intense bursts of GABAergic synaptic bombardment, or artificially following halorhodopsin activation, is followed by extrusion of both Cl and K+coupled together. We discuss this with respect to the pattern of [K+]o rise that occurs at the onset of seizure-like events.

The relationship between clinically accessible epileptic biomarkers and neuronal activity underlying the seizure transition is complex, potentially leading to imprecise delineation of epileptogenic brain areas. In particular, the pattern of interneuronal firing at seizure onset remains under debate, with some studies demonstrating increased firing while others suggest reductions. Previous study of neocortical sites suggests that seizure recruitment occurs upon failure of inhibition, with intact feedforward inhibition in non-recruited territories. We investigated whether the same principles applied also in limbic structures.We analyzed simultaneous ECoG and neuronal recordings during 34 seizures in a cohort of 19 patients (10 male, 9 female) undergoing surgical evaluation for pharmacoresistant focal epilepsy. A clustering approach with five quantitative metrics computed from ECoG and multiunit data was used to distinguish three types of site-specific activity patterns during seizures, at times co-existing within seizures. 156 single-units were isolated, subclassified by cell-type, and tracked through the seizure using our previously published methods to account for impacts of increased noise and single-unit waveshape changes caused by seizures.One cluster was closely associated with clinically defined seizure onset or spread. Entrainment of high-gamma activity to low-frequency ictal rhythms was the only metric that reliably identified this cluster at the level of individual seizures (p< 0.001). A second cluster demonstrated multi-unit characteristics resembling those in the first cluster, without concomitant high-gamma entrainment, suggesting feedforward effects from the seizure. The last cluster captured regions apparently unaffected by the ongoing seizure. Across all territories, the majority of both excitatory and inhibitory neurons reduced (69.2%) or ceased firing (21.8%). Transient increases in interneuronal firing rates were rare (13.5%) but showed evidence of intact feedforward inhibition with maximal firing rate increases and waveshape deformations in territories not fully recruited but showing feedforward activity from the seizure, and a shift to burst-firing in seizure-recruited territories (p= 0.014).This study provides evidence for entrained high gamma activity as an accurate biomarker of ictal recruitment in limbic structures. However, our results of reduced neuronal firing suggest preserved inhibition in mesial temporal structures despite simultaneous indicators of seizure recruitment, in contrast to the inhibitory collapse scenario documented in neocortex. Further study is needed to determine if this activity is ubiquitous to hippocampal seizures or if it indicates a “seizure-responsive” state in which the hippocampus is not the primary driver. If the latter, distinguishing such cases may help refine surgical treatment of mesial temporal lobe epilepsy.

Brain-state transitions are readily apparent from changes in brain rhythms,1 but are difficult to predict, suggestive that the underlying cause is latent to passive recording methods. Among the most important transitions, clinically, are the starts of seizures. We here show that an ‘active probing’ approach may have several important benefits for epileptic management, including by helping predict these transitions. We used mice expressing the optogenetic actuator, channelrhodopsin, in pyramidal cells, allowing this population to be stimulated in isolation. Intermittent stimulation at frequencies as low as 0.033 Hz (period = 30 s) delayed the onset of seizure-like events in an acute brain slice model of ictogenesis, but the effect was lost if stimulation was delivered at even lower frequencies (1/min). Notably, active probing additionally provides advance indication of when seizure-like activity is imminent, revealed by monitoring the postsynaptic response to stimulation. The postsynaptic response, recorded extracellularly, showed an all-or-nothing change in both amplitude and duration, a few hundred seconds before seizure-like activity began—a sufficient length of time to provide a helpful warning of an impending seizure. The change in the postsynaptic response then persisted for the remainder of the recording, indicative of a state change from a pre-epileptic to a pro-epileptic network. This occurred in parallel with a large increase in the stimulation-triggered Ca2+ entry into pyramidal dendrites, and a step increase in the number of evoked postsynaptic action potentials, both consistent with a reduction in the threshold for dendritic action potentials. In 0 Mg2+ bathing media, the reduced threshold was not associated with changes in glutamatergic synaptic function, nor of GABAergic release from either parvalbumin or somatostatin interneurons, but simulations indicate that the step change in the optogenetic response can instead arise from incremental increases in intracellular [Cl−]. The change in the response to stimulation was replicated by artificially raising intracellular [Cl−], using the optogenetic chloride pump, halorhodopsin. By contrast, increases in extracellular [K+] cannot account for the firing patterns in the response to stimulation, although this, and other cellular changes, may contribute to ictal initiation in other circumstances. We describe how these various cellular changes form a synergistic network of positive feedback mechanisms, which may explain the precipitous nature of seizure onset. This model of seizure initiation draws together several major lines of epilepsy research as well as providing an important proof-of-principle regarding the utility of open-loop brain stimulation for clinical management of the condition.

Background: Intravenous ferric carboxymaltose has been shown to improve symptoms and quality of life in patients with chronic heart failure and iron deficiency. We aimed to evaluate the effect of ferric carboxymaltose, compared with placebo, on outcomes in patients who were stabilised after an episode of acute heart failure. Methods: AFFIRM-AHF was a multicentre, double-blind, randomised trial done at 121 sites in Europe, South America, and Singapore. Eligible patients were aged 18 years or older, were hospitalised for acute heart failure with concomitant iron deficiency (defined as ferritin

The RING-between-RING (RBR) E3 ubiquitin ligase family in humans comprises 14 members and is defined by a two-step catalytic mechanism in which ubiquitin is first transferred from an E2 to the RBR active site and then to the substrate. To define the core features of this catalytic mechanism, we structurally and biochemically characterise the two RBRs HOIL-1 and RNF216. Crystal structures of both enzymes in their RBR/E2-Ub/Ub transthiolation complexes capturing the first catalytic step, in combination with complementary functional experiments, reveal the defining features of the RBR catalytic mechanism. RBRs catalyse ubiquitination via a conserved transthiolation complex structure that enables efficient E2-to-RBR ubiquitin transfer. Our data also highlight a conserved RBR allosteric mechanism by distinct ubiquitin linkages that suggests that RBRs employ a feed-forward mechanism accelerating their signalling pathways. We finally identify that the HOIL-1 RING2 domain contains an unusual Zn2/Cys6 bivalent zinc cluster that may represent a specific adaptation enabling HOIL-1’s atypical substrate specificity.

Early infantile developmental and epileptic encephalopathies are devastating conditions, generally of genetic origin, but the pathological mechanisms often remain obscure. A major obstacle in this field of research is the difficulty of studying cortical brain development in humans, at the relevant time period in utero. To address this, we established an in vitro assay to study the impact of gene variants on the developing human brain by using living organotypic cultures of the human subplate and neighbouring cortical regions, prepared from ethically sourced, 14–17 post-conception week brain tissue (www.hdbr.org). We were able to maintain cultures for several months, during which time the gross anatomical structures of the cortical plate, subplate and marginal zone persisted, while neurons continued to develop morphologically and form new synaptic networks. This preparation thus permits the study of genetic manipulations and their downstream effects on an intact developing human cortical network. We focused on STXBP1 haploinsufficiency, which is among the most common genetic causes of developmental and epileptic encephalopathy. This was induced using shRNA interference, leading to impaired synaptic function and a reduced density of glutamatergic synapses. We thereby provide a critical proof-of-principle for how to study the impact of any gene of interest on the development of the human cortex.

Clinical analyses of neuronal activity during seizures, invariably using extracellular recordings, is greatly hindered by various phenomena that are well established in animal studies: changes in local ionic concentration, changes in ionic conductance, and intense, hypersynchronous firing. The first two alter the action potential waveform, whereas the third increases the “noise”; all three factors confound attempts to detect and classify single neurons (units). To address these analytical difficulties, we developed a novel template-matching based spike sorting method, which enabled identification of 1,239 single units in 27 patients with intractable focal epilepsy, that were tracked throughout multiple seizures. These new analyses showed continued neuronal firing through the ictal transition, which was defined as a transient period of intense tonic firing consistent with previous descriptions of the ictal wavefront. After the ictal transition, neurons displayed increased spike duration (p < 0.001) and reduced spike amplitude (p < 0.001), in keeping with prior animal studies; units in non-recruited territories, by contrast, showed more stable waveforms. All units returned to their pre-ictal waveforms after seizure termination. Waveshape changes were stereotyped across seizures within patients. Our analyses of single neuron firing patterns, at the ictal wavefront, showed widespread intense activation, and commonly involving marked waveshape alteration. We conclude that the distinction between tissue that has been recruited to the seizure versus non-recruited territories is evident at the level of single neurons, and that increased waveform duration and decreased waveform amplitude are hallmarks of seizure invasion that could be used as defining characteristics of local recruitment.Significance StatementAnimal studies consistently show marked changes in action potential waveform during epileptic discharges, but acquiring similar evidence in humans has proved difficult. Assessing neuronal involvement in ictal events is pivotal to understanding seizure dynamics and in defining clinical localization of epileptic pathology. Using a novel method to track neuronal firing, we analyzed microelectrode array recordings of spontaneously occurring human seizures, and here report two dichotomous activity patterns. In cortex that is recruited to the seizure, neuronal firing rates increase and waveforms become longer in duration and shorter in amplitude, while penumbral tissue shows stable action potentials, in keeping with the “dual territory” model of seizure dynamics.

Nearly all life forms require iron to survive and function. Microorganisms utilize a number of mechanisms to acquire iron including the production of siderophores, which are organic compounds that combine with ferric iron into forms that are easily absorbed by the microorganism. There has been significant experimental investigation into the role, distribution and function of siderophores in fungi but until now no predictive tools have been developed to qualify or quantify fungi-initiated siderophore–iron interactions. In this investigation, we construct the first mathematical models of siderophore function related to fungi. Initially, a set of partial differential equations are calibrated and integrated numerically to generate quantitative predictions on the spatio-temporal distributions of siderophores and related populations. This model is then reduced to a simpler set of equations that are solved algebraically giving rise to solutions that predict the distributions of siderophores and resultant compounds. These algebraic results require the calculation of zeros of cross products of Bessel functions and thus new algebraic expansions are derived for a variety of different cases that are in agreement with numerically computed values. The results of the modelling are consistent with experimental data while the analysis provides new quantitative predictions on the time scales involved between siderophore production and iron uptake along with how the total amount of iron acquired by the fungus depends on its environment. The implications to bio-technological applications are briefly discussed.

Significance Epilepsy is an episodic disease characterized by brief periods of abnormal brain activity, known as seizures, that often have clinical correlates. In many patients, seizures preferentially happen during certain stages of daily and multiday cycles. However, it is unclear whether and how seizures themselves change over time, even though such variability may have clinical implications. To address this knowledge gap, we quantitatively analyze the nature of within-patient variability in seizure network evolutions. Contrary to common expectations, we find seizure variability throughout our cohort. Moreover, we demonstrate that seizures do not change randomly; instead, they also appear to fluctuate over daily and slower timescales. Ultimately, we may improve treatments by tailoring interventions to the full repertoire of seizures in each patient., Personalized medicine requires that treatments adapt to not only the patient but also changing factors within each individual. Although epilepsy is a dynamic disorder characterized by pathological fluctuations in brain state, surprisingly little is known about whether and how seizures vary in the same patient. We quantitatively compared within-patient seizure network evolutions using intracranial electroencephalographic (iEEG) recordings of over 500 seizures from 31 patients with focal epilepsy (mean 16.5 seizures per patient). In all patients, we found variability in seizure paths through the space of possible network dynamics. Seizures with similar pathways tended to occur closer together in time, and a simple model suggested that seizure pathways change on circadian and/or slower timescales in the majority of patients. These temporal relationships occurred independent of whether the patient underwent antiepileptic medication reduction. Our results suggest that various modulatory processes, operating at different timescales, shape within-patient seizure evolutions, leading to variable seizure pathways that may require tailored treatment approaches.

The stability of some simple density profiles in a vertically orientated two-dimensional porous medium is considered. The quasi-steady-state approximation is made so that the stability of the system can be approximated. As the profiles diffuse in time, the instantaneous growth rates evolve in time. For an initial step function density profile, the instantaneous growth rate was numerically found to decay like $$T^{-1/2}$$ for large times T, and the corresponding eigenfunctions scale with $$\mathrm{e}^{\omega \sqrt{T}}$$ where $$\omega $$ is a constant. For density profiles initially corresponding to a finite layer, the instantaneous growth rate eventually decayed like $$T^{-1}$$. This corresponds to an instability with algebraic growth, and the eigenfunctions scale with $$T^p$$ (where p is a constant) for large time. For a species initially linearly distributed in a finite layer, when the concentration has an increasing gradient in the downwards direction, the stability of the system was similar to that found for a uniformly distributed finite layer. However, when the concentration had a decreasing gradient in the downwards direction, the growth rates remained constant for a long period time, but eventually decayed in the same way as found in a uniformly distributed finite layer, for very large times. Numerical simulations were performed to validate the predictions made by the linear stability analysis.

Neocortical circuits exhibit a rich dynamic repertoire, and their ability to achieve entrainment (adjustment of their frequency to match the input frequency) is thought to support many cognitive functions and indicate functional flexibility. Although previous studies have explored the influence of various circuit properties on this phenomenon, the role of divisive gain modulation (or divisive inhibition) is unknown. This gain control mechanism is thought to be delivered mainly by the soma-targeting interneurons in neocortical microcircuits. In this study, we use a neural mass model of the neocortical microcircuit (extended Wilson–Cowan model) featuring both soma-targeting and dendrite-targeting interneuronal subpopulations to investigate the role of divisive gain modulation in entrainment. Our results demonstrate that the presence of divisive inhibition in the microcircuit, as delivered by the soma-targeting interneurons, enables its entrainment to a wider range of input frequencies. Divisive inhibition also promotes a faster entrainment, with the microcircuit needing less time to converge to the fully entrained state. We suggest that divisive inhibition, working alongside subtractive inhibition, allows for more adaptive oscillatory responses in neocortical circuits and, thus, supports healthy brain functioning. NEW & NOTEWORTHY We introduce a computational neocortical microcircuit model that features two inhibitory neural populations, with one providing subtractive and the other divisive inhibition to the excitatory population. We demonstrate that divisive inhibition widens the range of input frequencies to which the microcircuit can become entrained and diminishes the time needed to reach full entrainment. We suggest that divisive inhibition enables more adaptive oscillatory activity, with important implications for both normal and pathological brain function.

The β-site Amyloid precursor protein Cleaving Enzyme 1 (BACE1) is an extensively studied therapeutic target for Alzheimer’s disease (AD), owing to its role in the production of neurotoxic amyloid beta (Aβ) peptides. However, despite numerous BACE1 inhibitors entering clinical trials, none have successfully improved AD pathogenesis, despite effectively lowering Aβ concentrations. This can, in part, be attributed to an incomplete understanding of BACE1, including its physiological functions and substrate specificity. We propose that BACE1 has additional important physiological functions, mediated through substrates still to be identified. Thus, to address this, we computationally analysed a list of 533 BACE1 dependent proteins, identified from the literature, for potential BACE1 substrates, and compared them against proteins differentially expressed in AD. We identified 15 novel BACE1 substrates that were specifically altered in AD. To confirm our analysis, we validated Protein tyrosine phosphatase receptor type D (PTPRD) and Netrin receptor DCC (DCC) using Western blotting. These findings shed light on the BACE1 inhibitor failings and could enable the design of substrate-specific inhibitors to target alternative BACE1 substrates. Furthermore, it gives us a greater understanding of the roles of BACE1 and its dysfunction in AD.

An isogeometric boundary element method (IGABEM) is developed for the analysis of two-dimensional linear and isotropic elastic bodies governed by the couple stress theory. This theory is the simplest generalised continuum theory that can eectively model size eects in solids. The couple stress fundamental solutions are explicitly derived and used to construct the boundary integral equations. A new boundary integral equation arises to obtain the moments and rotations introduced by the couple stress formulation. A new analytical solution is also derived in the present work for an elliptical opening in an innite sheet under uniaxial far-eld stress. Several stress concentration problems are examined to illustrate and validate the application of the IGABEM in couple stress elasticity. It is shown that the IGABEM scheme exhibits advantageous convergence properties in comparison with the conventional BEM for boundary value problems within the framework of couple stress elasticity.

Coagulation factor (F) Xa induces proinflammatory responses through activation of protease-activated receptors (PARs). However, the effect of FXa on cardiac fibroblasts (CFs) and the contribution of PARs in FXa-induced cellular signalling in CF has not been fully characterised. To answer these questions, human and rat CFs were incubated with FXa (or TRAP-14, PAR-1 agonist). Gene expression of pro-fibrotic and proinflammatory markers was determined by qRT-PCR after 4 and 24 h. Gene silencing of F2R (PAR-1) and F2RL1 (PAR-2) was achieved using siRNA. MCP-1 protein levels were measured by ELISA of FXa-conditioned media at 24 h. Cell proliferation was assessed after 24 h of incubation with FXa ± SCH79797 (PAR-1 antagonist). In rat CFs, FXa induced upregulation of Ccl2 (MCP-1, &gt, 30-fold at 4 h in atrial and ventricular CF) and Il6 (IL-6, ±7-fold at 4 h in ventricular CF). Increased MCP-1 protein levels were detected in FXa-conditioned media at 24 h. In human CF, FXa upregulated the gene expression of CCL2 (&gt, 3-fold) and IL6 (&gt, 4-fold) at 4 h. Silencing of F2R (PAR-1 gene), but not F2RL1 (PAR-2 gene), downregulated this effect. Selective activation of PAR-1 by TRAP-14 increased CCL2 and IL6 gene expression, this was prevented by F2R (PAR-1 gene) knockdown. Moreover, SCH79797 decreased FXa-induced proliferation after 24 h. In conclusion, our study shows that FXa induces overexpression of proinflammatory genes in human CFs via PAR-1, which was found to be the most abundant PARs isoform in this cell type.

Early infantile developmental and epileptic encephalopathies are devastating conditions, generally of genetic origin, but the pathological mechanisms often remain obscure. A major obstacle in this field of research is the difficulty of studying cortical brain development in humans,in utero. To address this, we established anin vitroassay to study the impact of gene variants on the developing human brain, using living organotypic cultures of the human subplate and neighbouring cortical regions, prepared from ethically sourced, 14-17 post conception week brain tissue (www.hdbr.org). We were able to maintain cultures for several months, during which time, the gross anatomical structures of the cortical plate, subplate and marginal zone persisted, while neurons continued to develop morphologically, and form new synaptic networks. This preparation thus permits the study of genetic manipulations, and their downstream effects upon an intact developing human cortical network. We focused upon STXBP1 haploinsufficiency, which is among the most common genetic causes of developmental and epileptic encephalopathy. This was induced using shRNA interference, leading to impaired synaptic function and a drop in the number of glutamatergic synapses. We thereby provide a critical proof-of-principle for how to study the impact of any gene of interest on the development of the human cortex.

The transcriptional coactivator, PGC-1α (peroxisome proliferator activated receptor gamma coactivator 1α), plays a key role coordinating energy requirement within cells. Its importance is reflected in the growing number of psychiatric and neurological conditions that have been associated with reduced PGC-1α levels. In cortical networks, PGC-1α is required for the induction of parvalbumin (PV) expression in interneurons, and PGC-1α deficiency affects synchronous GABAergic release. It is unknown, however, how this affects cortical excitability. We show here that knocking down PGC-1α specifically in the PV-expressing cells (PGC-1αPV-/-), blocks the activity-dependent regulation of the synaptic proteins, SYT2 and CPLX1. More surprisingly, this cell-class specific knock-out of PGC-1α appears to have a novel anti-epileptic effect, as assayed in brain slices bathed in 0 Mg2+ media. The rate of pre-ictal discharges developed approximately equivalently in wild-type and PGC-1αPV-/- brain slices, but the intensity of these discharges was lower in PGC-1αPV-/- slices, as evident from the reduced power in the gamma range and reduced firing rates in both PV interneurons and pyramidal cells during these discharges. Reflecting this reduced intensity in the pre-ictal discharges, the PGC-1αPV-/- brain slices experienced many more discharges before transitioning into a seizure-like event. Consequently, there was a large increase in the latency to the first seizure-like event in brain slices lacking PGC-1α in PV interneurons. We conclude that knocking down PGC-1α limits the range of PV interneuron firing, and this slows the pathophysiological escalation during ictogenesis.

Insights into chloride regulation in neurons have come slowly, but they are likely to be critical for our understanding of how the brain works. The reason is that the intracellular Cl- level ([Cl-]i) is the key determinant of synaptic inhibitory function, and this in turn dictates all manner of neuronal network function. The true impact on the network will only be apparent, however, if Cl- is measured at many locations at once (multiple neurons, and also across the subcellular compartments of single neurons), which realistically, can only be achieved using imaging. The development of genetically-encoded anion biosensors (GABs) brings the additional benefit that Cl- imaging may be done in identified cell-classes and hopefully in subcellular compartments. Here, we describe the historical background and motivation behind the development of these sensors and how they have been used so far. There are, however, still major limitations for their use, the most important being the fact that all GABs are sensitive to both pH and Cl-. Disambiguating the two signals has proved a major challenge, but there are potential solutions; notable among these is ClopHensor, which has now been developed for in vivo measurements of both ion species. We also speculate on how these biosensors may yet be improved, and how this could advance our understanding of Cl- regulation and its impact on brain function.

SummaryThe main inhibitory synaptic currents, gated by gamma-aminobutyric acid (GABA), are mediated by Cl--conducting channels1–3, and are therefore sensitive to changes in the chloride electrochemical gradient. GABAergic activity dictates the neuronal firing range4,5 and timing6–9, which in turn influences the rhythms of the brain, synaptic plasticity, and flow of information in neuronal networks7,10–12. The intracellular chloride concentration [Cl-]i is, therefore, ideally placed to be a regulator of neuronal activity. Chloride levels have been thought to be stable in adult cortical networks, except when associated with pathological activation13–16. Here, we used 2-photon LSSmClopHensor imaging, in anaesthetized young adult mice13, to show that [Cl-] inside pyramidal cells shows a physiological diurnal rhythm, with an approximately 1.8-fold range, equating to an ~15mV positive shift in ECl at times when mice are typically awake (midnight), relative to when they are usually asleep (midday). This change of [Cl-]i alters the stability of cortical networks, as demonstrated by a greater than 3-fold longer latency to seizures induced by 4-aminopyridine at midday, compared to midnight. Importantly, both [Cl-]i and latency to seizure, in night-time experiments, were shifted in line with day-time measures, by inhibition of NKCC1. The redistribution of [Cl-]i reflects diurnal changes in surface expression and phosphorylation states of the cation-chloride-co-transporters, KCC2 and NKCC1, leading to a greatly reduced chloride-extrusion capacity at night (awake period). Our data demonstrate a means by which changes in the biochemical state of neurons are transduced into altered brain states.