The liver and muscle secreted HFE2-protein maintains central nervous system blood vessel integrity.

Liver failure causes breakdown of the Blood CNS Barrier (BCB) leading to damages of the Central-Nervous-System (CNS), however the mechanisms whereby the liver influences BCB-integrity remain elusive. One possibility is that the liver secretes an as-yet to be identified molecule(s) that circulate in the serum to directly promote BCB-integrity. To study BCB-integrity, we developed light-sheet imaging for three-dimensional analysis. We show that liver- or muscle-specific knockout of Hfe2/Rgmc induces BCB-breakdown, leading to accumulation of toxic-blood-derived fibrinogen in the brain, lower cortical neuron numbers, and behavioral deficits in mice. Soluble HFE2 competes with its homologue RGMa for binding to Neogenin, thereby blocking RGMa-induced downregulation of PDGF-B and Claudin-5 in endothelial cells, triggering BCB-disruption. HFE2 administration in female mice with experimental autoimmune encephalomyelitis, a model for multiple sclerosis, prevented paralysis and immune cell infiltration by inhibiting RGMa-mediated BCB alteration. This study has implications for the pathogenesis and potential treatment of diseases associated with BCB-dysfunction.

Spreading depolarization suppression from inter-astrocytic gap junction blockade assessed with multimodal imaging and a novel wavefront detection scheme.

Spreading depolarizations (SDs) are an enigmatic and ubiquitous co-morbidity of neural dysfunction. SDs are propagating waves of local field depolarization and increased extracellular potassium. They increase the metabolic demand on brain tissue, resulting in changes in tissue blood flow, and are associated with adverse neurological consequences including stroke, epilepsy, neurotrauma, and migraine. Their occurrence is associated with poor patient prognosis through mechanisms which are only partially understood. Here we show in vivo that two (structurally dissimilar) drugs, which suppress astroglial gap junctional communication, can acutely suppress SDs. We found that mefloquine hydrochloride (MQH), administered IP, slowed the propagation of the SD potassium waveform and intermittently led to its suppression. The hemodynamic response was similarly delayed and intermittently suppressed. Furthermore, in instances where SD led to transient tissue swelling, MQH reduced observable tissue displacement. Administration of meclofenamic acid (MFA) IP was found to reduce blood flow, both proximal and distal, to the site of SD induction, preceding a large reduction in the amplitude of the SD-associated potassium wave. We introduce a novel image processing scheme for SD wavefront localization under low-contrast imaging conditions permitting full-field wavefront velocity mapping and wavefront parametrization. We found that MQH administration delayed SD wavefront's optical correlates. These two clinically used drugs, both gap junctional blockers found to distinctly suppress SDs, may be of therapeutic benefit in the various brain disorders associated with recurrent SDs.

Effect of laser pulse shaping on photoacoustic dosimetry in retinal models.

Photoacoustic sensing can be a powerful technique to obtain real-time feedback of laser energy dose in treatments of biological tissue. However, when laser therapy uses pulses with microsecond duration, they are not optimal for photoacoustic pressure wave generation. This study examines a programmable fiber laser technique using pulse modulation in order to optimize the photoacoustic feedback signal to noise ratio (SNR) in a context where longer laser pulses are employed, such as in selective retinal therapy. We have demonstrated with a homogeneous tissue phantom that this method can yield a greater than seven-fold improvement in SNR over non-modulated square pulses of the same duration and pulse energy. This technique was further investigated for assessment of treatment outcomes in leporine retinal explants by photoacoustic mapping around the cavitation-induced frequency band.

Transitions between neocortical seizure and non-seizure-like states and their association with presynaptic glutamate release.

The transition between seizure and non-seizure states in neocortical epileptic networks is governed by distinct underlying dynamical processes. Based on the gamma distribution of seizure and inter-seizure durations, over time, seizures are highly likely to self-terminate; whereas, inter-seizure durations have a low chance of transitioning back into a seizure state. Yet, the chance of a state transition could be formed by multiple overlapping, unknown synaptic mechanisms. To identify the relationship between the underlying synaptic mechanisms and the chance of seizure-state transitions, we analyzed the skewed histograms of seizure durations in human intracranial EEG and seizure-like events (SLEs) in local field potential activity from mouse neocortical slices, using an objective method for seizure state classification. While seizures and SLE durations were demonstrated to have a unimodal distribution (gamma distribution shape parameter >1), suggesting a high likelihood of terminating, inter-SLE intervals were shown to have an asymptotic exponential distribution (gamma distribution shape parameter <1), suggesting lower probability of cessation. Then, to test cellular mechanisms for these distributions, we studied the modulation of synaptic neurotransmission during, and between, the in vitro SLEs. Using simultaneous local field potential and whole-cell voltage clamp recordings, we found a suppression of presynaptic glutamate release at SLE termination, as demonstrated by electrically- and optogenetically-evoked excitatory postsynaptic currents (EPSCs), and focal hypertonic sucrose application. Adenosine A1 receptor blockade interfered with the suppression of this release, changing the inter-SLE shape parameter from asymptotic exponential to unimodal, altering the chance of state transition occurrence with time. These findings reveal a critical role for presynaptic glutamate release in determining the chance of neocortical seizure state transitions.

Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue.

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics and investigate its underlying mechanisms. Acute seizure models are convenient, offer the ability to perform electrophysiological recordings, and can generate a large volume of electrographic seizure-like (ictal) events. The promising findings from acute seizure models can then be advanced to chronic epilepsy models and clinical trials. Thus, studying seizures in acute models that faithfully replicate the electrographic and dynamical signatures of a clinical seizure will be essential for making clinically relevant findings. Studying ictal events in acute seizure models prepared from human tissue is also important for making findings that are clinically relevant. The key focus in this paper is on the cortical 4-AP model due to its versatility in generating ictal events in both in vivo and in vitro studies, as well as in both mouse and human tissue. The methods in this paper will also describe an alternative method of seizure induction using the Zero-Mg2+ model and provide a detailed overview of the advantages and limitations of the epileptiform-like activity generated in the different acute seizure models. Moreover, by taking advantage of commercially available optogenetic mouse strains, a brief (30 ms) light pulse can be used to trigger an ictal event identical to those occurring spontaneously. Similarly, 30 - 100 ms puffs of neurotransmitters (Gamma-Amino Butyric Acid or glutamate) can be applied to the human tissue to trigger ictal events that are identical to those occurring spontaneously. The ability to trigger ictal events on-demand in acute seizure models offers the newfound ability to observe the exact sequence of events that underlie seizure initiation dynamics and efficiently evaluate potential anti-seizure therapies.

Brief activation of GABAergic interneurons initiates the transition to ictal events through post-inhibitory rebound excitation.

Activation of γ-aminobutyric acid (GABAA) receptors have been associated with the onset of epileptiform events. To investigate if a causal relationship exists between GABAA receptor activation and ictal event onset, we activated inhibitory GABAergic networks in the superficial layer (2/3) of the somatosensory cortex during hyperexcitable conditions using optogenetic techniques in mice expressing channelrhodopsin-2 in all GABAergic interneurons. We found that a brief 30ms light pulse reliably triggered either an interictal-like event (IIE) or ictal-like ("ictal") event in the in vitro cortical 4-Aminopyridine (4-AP) slice model. The link between light pulse and epileptiform event onset was lost following blockade of GABAA receptors with bicuculline methiodide. Additionally, recording the chronological sequence of events following a light pulse in a variety of configurations (whole-cell, gramicidin-perforated patch, and multi-electrode array) demonstrated an initial hyperpolarization followed by post-inhibitory rebound spiking and a subsequent slow depolarization at the transition to epileptiform activity. Furthermore, the light-triggered ictal events were independent of the duration or intensity of the initiating light pulse, suggesting an underlying regenerative mechanism. Moreover, we demonstrated that brief GABAA receptor activation can initiate ictal events in the in vivo 4-AP mouse model, in another common in vitro model of epileptiform activity, and in neocortical tissue resected from epilepsy patients. Our findings reveal that the synchronous activation of GABAergic interneurons is a robust trigger for ictal event onset in hyperexcitable cortical networks.

Continuous multi-modality brain imaging reveals modified neurovascular seizure response after intervention.

We developed a multi-modal brain imaging system to investigate the relationship between blood flow, blood oxygenation/volume, intracellular calcium and electrographic activity during acute seizure-like events (SLEs), both before and after pharmacological intervention. Rising blood volume was highly specific to SLE-onset whereas blood flow was more correlated with all eletrographic activity. Intracellular calcium spiked between SLEs and at SLE-onset with oscillation during SLEs. Modified neurovascular and ionic SLE responses were observed after intervention and the interval between SLEs became shorter and more inconsistent. Comparison of artery and vein pulsatile flow suggest proximal interference and greater vascular leakage prior to intervention.

Biofouling-Resistant Impedimetric Sensor for Array High-Resolution Extracellular Potassium Monitoring in the Brain.

Extracellular potassium concentration, [K⁺]o, plays a fundamental role in the physiological functions of the brain. Studies investigating changes in [K⁺]o have predominantly relied upon glass capillary electrodes with K⁺-sensitive solution gradients for their measurements. However, such electrodes are unsuitable for taking spatio-temporal measurements and are limited by the surface area of their tips. We illustrate seizures invoked chemically and in optogenetically modified mice using blue light exposure while impedimetrically measuring the response. A sharp decrease of 1-2 mM in [K⁺]o before each spike has shown new physiological events not witnessed previously when measuring extracellular potassium concentrations during seizures in mice. We propose a novel approach that uses multichannel monolayer coated gold microelectrodes for in vivo spatio-temporal measurements of [K⁺]o in a mouse brain as an improvement to the conventional glass capillary electrode.

Extracellular Potassium and Seizures: Excitation, Inhibition and the Role of Ih.

Seizure activity leads to increases in extracellular potassium concentration ([K[Formula: see text]]o), which can result in changes in neuronal passive and active membrane properties as well as in population activities. In this study, we examined how extracellular potassium modulates seizure activities using an acute 4-AP induced seizure model in the neocortex, both in vivo and in vitro. Moderately elevated [K[Formula: see text]]o up to 9[Formula: see text]mM prolonged seizure durations and shortened interictal intervals as well as depolarized the neuronal resting membrane potential (RMP). However, when [K[Formula: see text]]o reached higher than 9[Formula: see text]mM, seizure like events (SLEs) were blocked and neurons went into a depolarization-blocked state. Spreading depression was never observed as the blockade of ictal events could be reversed within 1-2[Formula: see text]min after the raised [K[Formula: see text]]o was changed back to control levels. This concentration-dependent dual effect of [K[Formula: see text]]o was observed using in vivo and in vitro mouse brain preparations as well as in human neocortical tissue resected during epilepsy surgery. Blocking the Ih current, mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, modulated the elevated [K[Formula: see text]]o influence on SLEs by promoting the high [K[Formula: see text]]o inhibitory actions. These results demonstrate biphasic actions of raised [K[Formula: see text]]o on neuronal excitability and seizure activity.

Optrodes for combined optogenetics and electrophysiology in live animals.

Optical tissue properties limit visible light depth penetration in tissue. Because of this, the recent development of optogenetic tools was quickly followed by the development of light delivery devices for in vivo optogenetics applications. We summarize the efforts made in the last decade to design neural probes that combine conventional electrophysiological recordings and optical channel(s) for optogenetic activation, often referred to as optodes or optrodes. Several aspects including challenges for light delivery in living brain tissue, the combination of light delivery with electrophysiological recordings, probe designs, multimodality, wireless implantable system, and practical considerations guiding the choice of configuration depending on the questions one seeks to address are presented.

Wide field fluorescent imaging of extracellular spatiotemporal potassium dynamics in vivo.

Potassium homeostasis is fundamental for the physiological functioning of the brain. Increased [K(+)] in the extracellular fluid has a major impact on neuronal physiology and can lead to ictal events. Compromised regulation of extracellular [K(+)] is involved in generation of seizures in animal models and potentially also in humans. For this reason, the investigation of K(+) spatio-temporal dynamics is of fundamental importance for neuroscientists in the field of epilepsy and other related pathologies. To date, the majority of studies investigating changes in extracellular K(+) have been conducted using a micropipette filled with a K(+) sensitive solution. However, this approach presents a major limitation: the area of the measurement is circumscribed to the tip of the pipette and it is not possible to know the spatiotemporal distribution or origin of the focally measured K(+) signal. Here we propose a novel approach, based on wide field fluorescence, to measure extracellular K(+) dynamics in neural tissue. Recording the local field potential from the somatosensory cortex of the mouse, we compared responses obtained from a K(+)-sensitive microelectrode to the spatiotemporal increases in fluorescence of the fluorophore, Asante Potassium Green-2, in physiological conditions and during 4-AP induced ictal activity. We conclude that wide field imaging is a valuable and versatile tool to measure K(+) dynamics over a large area of the cerebral cortex and is capable of capturing fast dynamics such as during ictal events. Moreover, the present technique is potentially adaptable to address questions regarding spatiotemporal dynamics of other ionic species.

WONOEP appraisal: optogenetic tools to suppress seizures and explore the mechanisms of epileptogenesis.

Optogenetics is a novel technology that combines optics and genetics by optical control of microbial opsins, targeted to living cell membranes. The versatility and the electrophysiologic characteristics of the light-sensitive ion-channels channelrhodopsin-2 (ChR2), halorhodopsin (NpHR), and the light-sensitive proton pump archaerhodopsin-3 (Arch) make these optogenetic tools potent candidates in controlling neuronal firing in models of epilepsy and in providing insights into the physiology and pathology of neuronal network organization and synchronization. Opsins allow selective activation of excitatory neurons and inhibitory interneurons, or subclasses of interneurons, to study their activity patterns in distinct brain-states in vivo and to dissect their role in generation of synchrony and seizures. The influence of gliotransmission on epileptic network function is another topic of great interest that can be further explored by using light-activated Gq protein-coupled opsins for selective activation of astrocytes. The ever-growing optogenetic toolbox can also be combined with emerging techniques that have greatly expanded our ability to record specific subtypes of cortical and hippocampal neurons in awake behaving animals such as juxtacellular recording and two-photon guided whole-cell recording, to identify the specific subtypes of neurons that are altered in epileptic networks. Finally, optogenetic tools allow rapid and reversible suppression of epileptic electroencephalography (EEG) activity upon photoactivation. This review outlines the most recent advances achieved with optogenetic techniques in the field of epilepsy by summarizing the presentations contributed to the 13th ILAE WONOEP meeting held in the Laurentian Mountains, Quebec, in June 2013.

Evaluation of laser speckle contrast imaging as an intrinsic method to monitor blood brain barrier integrity.

The integrity of the blood brain barrier (BBB) can contribute to the development of many brain disorders. We evaluate laser speckle contrast imaging (LSCI) as an intrinsic modality for monitoring BBB disruptions through simultaneous fluorescence and LSCI with vertical cavity surface emitting lasers (VCSELs). We demonstrated that drug-induced BBB opening was associated with a relative change of the arterial and venous blood velocities. Cross-sectional flow velocity ratio (veins/arteries) decreased significantly in rats treated with BBB-opening drugs, ≤0.81 of initial values.

Rapid multiexposure in vivo brain imaging system using vertical cavity surface emitting lasers as a light source.

We demonstrate an imaging technique implementing vertical cavity lasers with extremely low transient times for a greatly simplified realization of a multiexposure laser speckle contrast imaging system. Data from multiexposure laser speckle imaging was observed to more closely agree with absolute velocity measurements using time of flight technique, when compared to long-exposure laser speckle imaging. Furthermore, additional depth information of the vasculature morphology was inferred by accounting for the change in the static scattering from tissue above vessels with respect to the total scattering from blood flow and tissue.

A multimodal micro-optrode combining field and single unit recording, multispectral detection and photolabeling capabilities.

Microelectrodes have been very instrumental and minimally invasive for in vivo functional studies from deep brain structures. However they are limited in the amount of information they provide. Here, we describe a, aluminum-coated, fibre optic-based glass microprobe with multiple electrical and optical detection capabilities while retaining tip dimensions that enable single cell measurements (diameter ≤10 µm). The probe enables optical separation from individual cells in transgenic mice expressing multiple fluorescent proteins in distinct populations of neurons within the same deep brain nucleus. It also enables color conversion of photoswitchable fluorescent proteins, which can be used for post-hoc identification of the recorded cells. While metal coating did not significantly improve the optical separation capabilities of the microprobe, the combination of metal on the outside of the probe and of a hollow core within the fiber yields a microelectrode enabling simultaneous single unit and population field potential recordings. The extended range of functionalities provided by the same microprobe thus opens several avenues for multidimensional structural and functional interrogation of single cells and their surrounding deep within the intact nervous system.

A microprobe for parallel optical and electrical recordings from single neurons in vivo.

Recording electrical activity from identified neurons in intact tissue is key to understanding their role in information processing. Recent fluorescence labeling techniques have opened new possibilities to combine electrophysiological recording with optical detection of individual neurons deep in brain tissue. For this purpose we developed dual-core fiberoptics-based microprobes, with an optical core to locally excite and collect fluorescence, and an electrolyte-filled hollow core for extracellular single unit electrophysiology. This design provides microprobes with tips < 10 µm, enabling analyses with single-cell optical resolution. We demonstrate combined electrical and optical detection of single fluorescent neurons in rats and mice. We combined electrical recordings and optical Ca²(+) measurements from single thalamic relay neurons in rats, and achieved detection and activation of single channelrhodopsin-expressing neurons in Thy1::ChR2-YFP transgenic mice. The microprobe expands possibilities for in vivo electrophysiological recording, providing parallel access to single-cell optical monitoring and control.

In vivo simultaneous intra- and extracellular potassium recordings using a micro-optrode.

This technique proposes a new approach to correlate intra- and extracellular variations of the ionic concentrations in vivo by means of tapered optical waveguides coupled to standard electrophysiological electrodes to monitor in vivo simultaneously the intracellular and extracellular K(+) concentration as well as the neighboring field potential. The optical fibers were tapered to a final diameter of approximately 10 µm and were used to guide the excitation light deep into the tissue and to collect the fluorescence emanating from the intracellular milieu. This fiber was coupled to a double barrel ion-sensitive electrode forming a micro-optrode with a final diameter around 15 µm. The method was successfully used to record the intracellular K(+) evolution with the fluorescent indicator PBFI during three states: normal sleep-like patterns, paroxysmal seizures, and coma. While we could not disclose any phasic fluctuations of the intracellular K(+) during normal sleep patterns, they were clearly present during seizures and coma. In the majority of cases (58%), paroxysmal discharges were associated with positive variations of the intracellular fluorescence of 62±5% corresponding to extracellular K(+) increases of 2.04±0.4 mM. In the remaining cases (42%) intracellular K(+) dropped by 44.4±12% for an extracellular K(+) increase of 2.62±0.47 mM. We suggest that this differential behavior might reflect different cellular populations (glia vs. neurons, respectively). Comatose states were accompanied by an extracellular drop of K(+) of 1.31±0.13 mM, which was reflected, in all cases, by an intracellular K(+) increase of 39±4%.

[Two-photon laser scanning fluorescence microscopy for functional cellular imaging: Advantages and challenges or One photon is good... but two is better!]. / Microscopie à deux photons pour l'imagerie cellulaire fonctionnelle: avantages et enjeux ou un photon c'est bien...mais deux c'est mieux!

One of the main challenges of modern biochemistry and cell biology is to be able to observe molecular dynamics in their functional context, i.e. in live cells in situ. Thus, being able to track ongoing molecular events with maximal spatial and temporal resolution (within subcellular compartments), while minimizing interference with tissue biology, is key to future developments for in situ imaging. The recent use of non-linear optics approaches in tissue microscopy, made possible in large part by the availability of femtosecond pulse lasers, has allowed major advances on this front that would not have been possible with conventional linear microscopy techniques. Of these approaches, the one that has generated most advances to date is two-photon laser scanning fluorescence microscopy. While this approach does not really provide improved resolution over linear microscopy in non absorbing media, it allows us to exploit a window of low absorbance in live tissue in the near infrared range. The end result is much improved tissue penetration, minimizing unwanted excitation outside the focal area, which yields an effective improvement in resolution and sensitivity. The optical system is also simplified and, more importantly, phototoxicity is reduced. These advantages are at the source of the success of two-photon microscopy for functional cellular imaging in situ. Yet, we still face further challenges, reaching the limits of resolution that conventional optics can offer. Here we review some recent advances in optics/photonics approaches that hold promises to improve our ability to probe the tissue in finer areas, at faster speed, and deeper into the tissue. These include super-resolution techniques, introduction of non paraxial optics in microscopy and use of amplified femtosecond lasers, yielding enhanced spatial and temporal resolution as well as tissue penetration.