ATP-dependent conformational dynamics in a photoactivated adenylate cyclase revealed by fluorescence spectroscopy and small-angle X-ray scattering.

Structural insights into the photoactivated adenylate cyclases can be used to develop new ways of controlling cellular cyclic adenosine monophosphate (cAMP) levels for optogenetic and other applications. In this work, we use an integrative approach that combines biophysical and structural biology methods to provide insight on the interaction of adenosine triphosphate (ATP) with the dark-adapted state of the photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata (OaPAC). A moderate affinity of the nucleotide for the enzyme was calculated and the thermodynamic parameters of the interaction have been obtained. Stopped-flow fluorescence spectroscopy and small-angle solution scattering have revealed significant conformational changes in the enzyme, presumably in the adenylate cyclase (AC) domain during the allosteric mechanism of ATP binding to OaPAC with small and large-scale movements observed to the best of our knowledge for the first time in the enzyme in solution upon ATP binding. These results are in line with previously reported drastic conformational changes taking place in several class III AC domains upon nucleotide binding.

Transparent neural interfaces: challenges and solutions of microengineered multimodal implants designed to measure intact neuronal populations using high-resolution electrophysiology and microscopy simultaneously.

The aim of this review is to present a comprehensive overview of the feasibility of using transparent neural interfaces in multimodal in vivo experiments on the central nervous system. Multimodal electrophysiological and neuroimaging approaches hold great potential for revealing the anatomical and functional connectivity of neuronal ensembles in the intact brain. Multimodal approaches are less time-consuming and require fewer experimental animals as researchers obtain denser, complex data during the combined experiments. Creating devices that provide high-resolution, artifact-free neural recordings while facilitating the interrogation or stimulation of underlying anatomical features is currently one of the greatest challenges in the field of neuroengineering. There are numerous articles highlighting the trade-offs between the design and development of transparent neural interfaces; however, a comprehensive overview of the efforts in material science and technology has not been reported. Our present work fills this gap in knowledge by introducing the latest micro- and nanoengineered solutions for fabricating substrate and conductive components. Here, the limitations and improvements in electrical, optical, and mechanical properties, the stability and longevity of the integrated features, and biocompatibility during in vivo use are discussed.

Histological and electrophysiological evidence on the safe operation of a sharp-tip multimodal optrode during infrared neuromodulation of the rat cortex.

Infrared neuromodulation is an emerging technology in neuroscience that exploits the inherent thermal sensitivity of neurons to excite or inhibit cellular activity. Since there is limited information on the physiological response of intracortical cell population in vivo including evidence on cell damage, we aimed to create and to validate the safe operation of a microscale sharp-tip implantable optrode that can be used to suppress the activity of neuronal population with low optical power continuous wave irradiation. Effective thermal cross-section and electric properties of the multimodal microdevice was characterized in bench-top tests. The evoked multi-unit activity was monitored in the rat somatosensory cortex, and using NeuN immunocytochemistry method, quantitative analysis of neuronal density changes due to the stimulation trials was evaluated. The sharp tip implant was effectively used to suppress the firing rate of neuronal populations. Histological staining showed that neither the probe insertion nor the heating protocols alone lead to significant changes in cell density in the close vicinity of the implant with respect to the intact control region. Our study shows that intracortical stimulation with continuous-wave infrared light at 1550 nm using a sharp tip implantable optical microdevice is a safe approach to modulate the firing rate of neurons.

A multimodal, implantable sensor array and measurement system to investigate the suppression of focal epileptic seizure using hypothermia.

Objective.Local cooling of the brain as a therapeutic intervention is a promising alternative for patients with epilepsy who do not respond to medication.In vitroandin vivostudies have demonstrated the seizure-suppressing effect of local cooling in various animal models. In our work, focal brain cooling in a bicuculline induced epilepsy model in rats is demonstrated and evaluated using a multimodal micro-electrocorticography (microECoG) device.Approach.We designed and experimentally tested a novel polyimide-based sensor array capable of recording microECoG and temperature signals concurrently from the cortical surface of rats. The effect of cortical cooling after seizure onset was evaluated using 32 electrophysiological sites and eight temperature sensing elements covering the brain hemisphere, where injection of the epileptic drug was performed. The focal cooling of the cortex right above the injection site was accomplished using a miniaturized Peltier chip combined with a heat pipe to transfer heat. Control of cooling and collection of sensor data was provided by a custom designed Arduino based electronic board. We tested the experimental setup using an agar gel modelin vitro, and thenin vivoin Wistar rats.Main results.Spatial variation of temperature during the Peltier controlled cooling was evaluated through calibrated, on-chip platinum temperature sensors. We found that frequency of epileptic discharges was not substantially reduced by cooling the cortical surface to 30 °C, but was suppressed efficiently at temperature values around 20 °C. The multimodal array revealed that seizure-like ictal events far from the focus and not exposed to high drop in temperature can be also inhibited at an extent like the directly cooled area.Significance.Our results imply that not only the absolute drop in temperature determines the efficacy of seizure suppression, and distant cortical areas not directly cooled can be influenced.

Application of a flexible polymer microECoG array to map functional coherence in schizophrenia model.

Anatomically, connections form the fundamental brain network, functionally the different types of oscillatory electric activities are creating a temporarily connected fraction of the anatomical connectome generating an output to the motor system. Schizophrenia can be considered as a connectome disease, in which the sensory input generates a schizophrenia specific temporary connectome and the signal processing becomes diseased showing hallucinations and adverse behavioral reactions. In this work, flexible, 32-channel polymer microelectrode arrays fabricated by the authors are used to map the functional coherence on large cortical areas during physiological activities in a schizophrenia model in rats.-Fabrication of a flexible microECoG array is shown.-Protocol to use a flexible microECoG is demonstrated to characterize connectome diseases in rats.-Customized method to analyze the functional coherence between different cortical areas during visually evoked potential is detailed.-R-based implementation of the analysis method is presented.

Infrared neuromodulation:a neuroengineering perspective.

Infrared neuromodulation (INM) is a branch of photobiomodulation that offers direct or indirect control of cellular activity through elevation of temperature in a spatially confined region of the target tissue. Research on INM started about 15 ago and is gradually attracting the attention of the neuroscience community, as numerous experimental studies have provided firm evidence on the safe and reproducible excitation and inhibition of neuronal firing in both in vitro and in vivo conditions. However, its biophysical mechanism is not fully understood and several engineered interfaces have been created to investigate infrared stimulation in both the peripheral and central nervous system. In this review, recent applications and present knowledge on the effects of INM on cellular activity are summarized, and an overview of the technical approaches to deliver infrared light to cells and to interrogate the optically evoked response is provided. The micro- and nanoengineered interfaces used to investigate the influence of INM are described in detail.

Electrophysiological and behavioral properties of 4-aminopyridine-induced epileptic activity in mice.

4-aminopyridine (4-AP) is a widely used drug that induces seizure activity in rodents, especially in rats, although there is no consensus in the literature on the dose to be used in mice. The aim of the present study was to investigate the effect of the intraperitoneal administration of 4-AP in two doses (4 and 10 mg/kg) in vivo. EEG, movement, and video recordings were made simultaneously in male B6 mice to specify the details of the seizures and to determine whether there is a suitable non-lethal dose for seizure induction and for further molecular studies. Seizure behavior in mice differs from that seen in rats, with no characteristic stages of epileptic seizures, but with spiking and seizure activity. Seizure activity, although produced at both doses without being lethal, induced different changes of the EEG pattern. Smaller dose induced a lower amplitude seizure activity, decreased spiking activity and later onset of seizures, while higher dose induced a much more intense brain seizure activity and severe trembling. It is concluded that the intraperitoneal administration of 4-AP at a dose of 10 mg/kg induces explicit seizure activity in mice which is repeatable and can be suitable for further molecular research.

Transparent, low-autofluorescence microECoG device for simultaneous Ca2+ imaging and cortical electrophysiology in vivo.

OBJECTIVE: Multimodal neuroimaging approaches are beneficial to discover brain functionalities at high spatial and temporal resolution. In our work, a novel material composition of a microECoG device relying on Parylene HT and indium-tin-oxide (ITO) is presented, which facilitates two-photon imaging of Ca2+ signals and concurrent recording of cortical EEG. APPROACH: Long-term stability of the interfaces of the transparent microdevice is confirmed in vitro by electrochemical and mechanical tests. The outstanding optical properties, like high transmittance and low auto-fluorescent are proven by fluorimetric measurements. Spatial resolution of fluorescent two-photon imaging through the microECoG device is presented in transgenic hippocampal slices, while concurrent recording of Ca2+ signals and cortical EEG is demonstrated in vivo. Photoartefacts and photodegradation of the materials are also investigated in detail to provide safety guidelines for further use in two-photon in vivo imaging schemes. MAIN RESULTS: Two-photon imaging of Ca signals can be safely performed through the proposed transparent ECoG device, without significant distortion in the dimensions of detected neuronal structures or in the temporal signaling. In chronic use, we demonstrated that fluorescent Ca signals of individual neurons can be clearly recorded even after 51 d. SIGNIFICANCE: Our results give a firm indication that highly transparent microECoG electrode arrays made of Parylene HT/ITO/Parylene HT multilayer are excellent candidates for synergetic recording of optical signals and EEG from intact brains with high resolution and are free of electrical and optical artefacts.

Optimization of an optrode microdevice for infrared neural stimulation.

Infrared light is a promising candidate for the treatment of neurodegenerative diseases. Optimizing the device parameters to achieve the best optical and mechanical performance is essential for reliable in vivo operation. In this work, mechanical strength simulations and coupled optical and thermal model were used to determine optimal design parameters for maximizing overall device efficiency. Our analysis reveals that minimizing the number of integrated optical elements and optimizing the optical path leads to a 33% relative in-coupling efficiency improvement at equal mechanical robustness. Using a symmetric optrode tip with an angle of 15°, the efficiency showed a further 17% relative improvement due to the enhancement of out-coupling at the tip. To investigate the temperature rise of the brain tissue during the infrared stimulation in the case of the optimized device, a thermal simulation with pulsed infrared excitation was developed. Our results show that the optimized device provides a temperature rise of 4.42°C as opposed to 3°C for the original setup.

A softening laminar electrode for recording single unit activity from the rat hippocampus.

Softening neural implants that change their elastic modulus under physiological conditions are promising candidates to mitigate neuroinflammatory response due to the reduced mechanical mismatch between the artificial interface and the brain tissue. Intracortical neural probes have been used to demonstrate the viability of this material engineering approach. In our paper, we present a robust technology of softening neural microelectrode and demonstrate its recording performance in the hippocampus of rat subjects. The 5 mm long, single shank, multi-channel probes are composed of a custom thiol-ene/acrylate thermoset polymer substrate, and were micromachined by standard MEMS processes. A special packaging technique is also developed, which guarantees the stable functionality and longevity of the device, which were tested under in vitro conditions prior to animal studies. The 60 micron thick device was successfully implanted to 4.5 mm deep in the hippocampus without the aid of any insertion shuttle. Spike amplitudes of 84 µV peak-to-peak and signal-to-noise ratio of 6.24 were achieved in acute experiments. Our study demonstrates that softening neural probes may be used to investigate deep layers of the rat brain.

Effect of nanostructures on anchoring stem cell-derived neural tissue to artificial surfaces.

OBJECTIVE: Chronic application of brain implants monitoring or modulating neuronal activity are hindered by the foreign body response of the tissue. Topographical modification of implant surfaces may reduce negative tissue response by imitating the structure of the extracellular matrix and therefore affecting the attachment and behavior of neural cells. APPROACH: In our in vitro study, the effect of nanostructuring was investigated on two commercially used neural implant materials: silicon and platinum. The adhesion, survival and arrangement of neural stem cells (NE4C) and microglial cells (BV2) were investigated and compared to nanostructured and flat Si and Pt surfaces using cell viability studies and fluorescent microscopy image analysis. MAIN RESULTS: Our data indicated that neural cells established strong adhesive couplings with each other, instead of binding to the artificial surfaces. SIGNIFICANCE: The phenomena resemble some features of in vivo separation of living tissue from the implanted artificial material, providing an in vitro model for studying immune response.

Optical and thermal modeling of an optrode microdevice for infrared neural stimulation.

Infrared neural stimulation is a promising medical technique using pulsed infrared light for generating temperature-controlled firing of neurons. A combined optical and thermal model of a stimulating microtool-or so-called optrode-has been developed to investigate the amount, the spatial distribution, and the temporal behavior of the thermal excitation. Ray tracing and Fourier optics were used to describe the propagation and scattering of light in the optrode, and the finite element method was applied to model heat transfer. The scattered intensity distribution profiles were calculated based on measured surface roughness of the device and were integrated into the ray optics model. As a validation of the optical model, the simulated and measured values of the light efficiency of the microoptical system are compared. The temperature rise of the brain tissue during the infrared stimulation was estimated using the combined model. Using 30 mW total power and a single 100 ms pulse, the excitation resulted in a temperature rise of 3°C of the brain tissue. The spatial and temporal distributions of the tissue temperature are discussed in the paper. The proposed combined model is an efficient tool for the investigation and optimization of the stimulation process and for further development of the optrode configuration.

In vitro and in vivo stability of black-platinum coatings on flexible, polymer microECoG arrays.

OBJECTIVE: Intracranial EEG (iEEG) or micro-electrocorticography (µECoG) microelectrodes offer high spatial resolution in recordings of neuronal activity from the exposed brain surface. Reliability of dielectric substrates and conductive materials of these devices are under intensive research in terms of functional stability in biological environments. APPROACH: The aim of our study is to investigate the stability of electroplated platinum recording sites on 16-channel, 8 micron thick, polyimide based, flexible µECoG arrays implanted underneath the skull of rats. Scanning electron microscopy and electrochemical impedance spectroscopy was used to reveal changes in either surface morphology or interfacial characteristics. The effect of improved surface area (roughness factor = 23 ± 0.12) on in vivo recording capability was characterized in both acute and chronic experiments. MAIN RESULTS: Besides the expected reduction in thermal noise and enhancement in signal-to-noise ratio (up to 39.8), a slight increase in the electrical impedance of individual sites was observed, as a result of changes in the measured interfacial capacitance. In this paper, we also present technology processes and protocols in detail to use such implants without crack formation of the porous platinum surfaces. SIGNIFICANCE: Our findings imply that black-platinum coating deposited on the recording sites of flexible microelectrodes (20 microns in diameter) provides a stable interface between tissue and device.

Editor's Choice - Very Urgent Carotid Endarterectomy is Associated with an Increased Procedural Risk: The Carotid Alarm Study.

OBJECTIVE/BACKGROUND: The aim of the Carotid Alarm Study was to compare the procedural risk of carotid endarterectomy (CEA) performed within 48 hours with that after 48 hours to 14 days following an ipsilateral cerebrovascular ischaemic event. METHODS: Consecutive patients with symptomatic carotid stenosis undergoing CEA were prospectively recruited. Time to surgery was calculated as time from the most recent ischaemic event preceding surgery. A neurologist examined patients before and, after CEA. The primary endpoint was the composite endpoint of death and/or any stroke within 30 days of the surgical procedure. The study was designed to include 600 patients, with 150 operated on within 48 hours. RESULTS: From October 2010 to December 2015, 418 patients were included, of whom 75 were operated within 48 hours of an ischaemic event. The study was prematurely terminated owing to the slow recruitment rate in the group operated on within 48 hours. Patients undergoing CEA within 48 hours had a higher risk of reaching the primary endpoint than those operated on later (8.0% vs. 2.9%). Multivariate logistic regression analyses showed that CEA performed within 48 h (odds ratio [OR] 3.07; 95% confidence interval [CI] 1.04-9.09), CEA performed out of office hours (OR 3.65; 95% CI 1.14-11.67), and use of shunt (OR 4.02; 95% CI 1.36-11.93) were all independently associated with an increased risk of reaching the primary endpoint. CONCLUSION: CEA performed within 48 hours was associated with a higher risk of complications compared with surgery performed 48 hours-14 days after the most recent ischaemic event.

Simultaneous in vivo recording of local brain temperature and electrophysiological signals with a novel neural probe.

OBJECTIVE: Temperature is an important factor for neural function both in normal and pathological states, nevertheless, simultaneous monitoring of local brain temperature and neuronal activity has not yet been undertaken. APPROACH: In our work, we propose an implantable, calibrated multimodal biosensor that facilitates the complex investigation of thermal changes in both cortical and deep brain regions, which records multiunit activity of neuronal populations in mice. The fabricated neural probe contains four electrical recording sites and a platinum temperature sensor filament integrated on the same probe shaft within a distance of 30 µm from the closest recording site. The feasibility of the simultaneous functionality is presented in in vivo studies. The probe was tested in the thalamus of anesthetized mice while manipulating the core temperature of the animals. MAIN RESULTS: We obtained multiunit and local field recordings along with measurement of local brain temperature with accuracy of 0.14 °C. Brain temperature generally followed core body temperature, but also showed superimposed fluctuations corresponding to epochs of increased local neural activity. With the application of higher currents, we increased the local temperature by several degrees without observable tissue damage between 34-39 °C. SIGNIFICANCE: The proposed multifunctional tool is envisioned to broaden our knowledge on the role of the thermal modulation of neuronal activity in both cortical and deeper brain regions.

Knowledge of Emergency Contraceptive Pills among Hungarian Women Presenting for Induced Abortion or Seeking Emergency Contraception.

Aim: To compare the differences in contraceptive characteristics and the knowledge of emergency contraception (ECP) among women who used ECP after unprotected intercourse and those who sought an abortion. Methods: A questionnaire survey was conducted in a Hungarian university hospital among women for whom ECP was prescribed after unprotected intercourse (n = 940) as well as women who presented for the termination of pregnancy (n = 1592) between January 1, 2005 and November 20, 2006. Their knowledge of ECP and their experience with and attitudes toward ECP use were targeted. Results: The availability of ECP was well known (87.9 %), but it was still greatly underutilized: applied by only 13 of the 1592 women who resorted to abortion. Primarily, the ECP group consisted of those who experienced a condom failure significantly more often (odds ratio [OR] = 4.1), followed by those cases where ECP applications was a consequence of not using any kind of contraception (OR = 3.8). Fewer than one third (32 %) of the abortion seekers had previously used ECP, and only one fifth knew how to obtain it. Appropriate awareness of ECP was influenced by information obtained from health-care providers (adjusted odds ratio [AOR] = 3.93) or school education (AOR = 1.82). Conclusions: More thorough education is needed to provide a deeper knowledge of ECP use during contraceptive counseling for women seeking abortion, including those contraceptive mishaps where unintended pregnancy can be prevented by ECP.

A silicon-based microelectrode array with a microdrive for monitoring brainstem regions of freely moving rats.

OBJECTIVE: Exploring neural activity behind synchronization and time locking in brain circuits is one of the most important tasks in neuroscience. Our goal was to design and characterize a microelectrode array (MEA) system specifically for obtaining in vivo extracellular recordings from three deep-brain areas of freely moving rats, simultaneously. The target areas, the deep mesencephalic reticular-, pedunculopontine tegmental-and pontine reticular nuclei are related to the regulation of sleep-wake cycles. APPROACH: The three targeted nuclei are collinear, therefore a single-shank MEA was designed in order to contact them. The silicon-based device was equipped with 3 × 4 recording sites, located according to the geometry of the brain regions. Furthermore, a microdrive was developed to allow fine actuation and post-implantation relocation of the probe. The probe was attached to a rigid printed circuit board, which was fastened to the microdrive. A flexible cable was designed in order to provide not only electronic connection between the probe and the amplifier system, but sufficient freedom for the movements of the probe as well. MAIN RESULTS: The microdrive was stable enough to allow precise electrode targeting into the tissue via a single track. The microelectrodes on the probe were suitable for recording neural activity from the three targeted brainstem areas. SIGNIFICANCE: The system offers a robust solution to provide long-term interface between an array of precisely defined microelectrodes and deep-brain areas of a behaving rodent. The microdrive allowed us to fine-tune the probe location and easily scan through the regions of interest.

Experimental study on the mechanical interaction between silicon neural microprobes and rat dura mater during insertion.

In vivo insertion experiments are essential to optimize novel neural implants. Our work focuses on the interaction between intact dura mater of rats and as-fabricated single-shaft silicon microprobes realized by deep reactive ion etching. Implantation parameters like penetration force and dimpling through intact dura mater were studied as a function of insertion speed, microprobe cross-section, tip angle and animal age. To reduce tissue resistance, we proposed a unique tip sharpening technique, which was also evaluated in in vivo insertion tests. By doubling the insertion speed (between 1.2 and 10.5 mm/min), an increase of 10-35% in penetration forces was measured. When decreasing the cross-section of the microprobes, penetration forces and dimpling was reduced by as much as 30-50% at constant insertion speeds. Force was noticed to gradually decrease by decreasing tip angles. Measured penetration forces through dura mater were reduced even down to 11±3 mN compared to unsharpened (49±13 mN) probes by utilizing our unique tip sharpening technique, which is very close to exerted penetration force in the case of retracted dura (5±1.5 mN). Our findings imply that age remarkably alters the elasticity of intact dura mater. The decreasing stiffness of dura mater results in a significant rise in penetration force and decrease in dimpling. Our work is the first in vivo comparative study on microelectrode penetration through intact and retracted dura mater.

Gas chromatographic-mass spectrometric analysis and subsequent quality improvement of plastic infusion packaging materials.

Although the opalescence of sterile transparent plastic materials utilized for the packaging of parenteral infusion drugs is a serious quality problem, most suppliers do not report the exact compositions of such polymers, and no literature data are available. Similarly, no information is available as concerns the potential incompatibility of the inner bag and the overpouch. Our gas chromatographic-mass spectrometric study revealed that the cause of the opalescence is the presence of a low-molecular-weight slip additive, 13-docosenamide (erucamide), which is transferred into the primary infusion bag from the overpouch during the heat-sterilization process. Autoclaving trials confirmed the analytical results. In view of these findings, a new slip additive-free overpouch has been produced as secondary packaging material, which does not give rise to opalescence.

Non-thromboembolic risk in systemic lupus erythematosus associated with antiphospholipid syndrome.

OBJECTIVES: We investigated the impact of secondary antiphospholipid syndrome (APS) and antiphospholipid antibody (aPL) positivity on the non-thromboembolic clinical manifestations of systemic lupus erythematosus (SLE). METHODS: In total, 224 patients with SLE were studied, of whom 105 were aPL-positive; 52 fulfilled the criteria for APS. SLE- and APS-related clinical and laboratory features were assesed: SLE patients with aPL or APS were compared with those without these features. RESULTS: Not only thromboembolic events, but also Coombs-positive haemolytic anaemia, thrombocytopenia and endocarditis occurred significantly more frequently in the aPL-positive than in the aPL-negative patients. In the APS + SLE subgroup, several non-thromboembolic symptoms occurred more often than in the absence of APS: pleuritis, interstitial lung disease, myocarditis, nephritis and organic brain syndrome. The mean number of major organ manifestations (1.2 vs. 0.5) and the overall number of organ manifestations (8.1 vs. 6.9) were higher in the APS + SLE patients than in those without APS (p < 0.05). The APS + SLE subgroup more frequently required intensive immunosuppressive treatment than did the APS-negative patients (p < 0.05). CONCLUSIONS: SLE patients with aPL positivity or secondary APS also have a higher risk to develop non-thromboembolic disease manifestations in addition to the aPL-related symptoms, and are predisposed to more severe SLE manifestations.