Tool In Neuroscience Research Research Articles

Noninvasive optical activation of Flp recombinase for genetic manipulation in deep mouse brain regions

Spatiotemporal control of gene expression or labeling is a valuable strategy for identifying functions of genes within complex neural circuits. Here, we develop a highly light-sensitive and efficient photoactivatable Flp recombinase (PA-Flp) that is suitable for genetic manipulation in vivo. The highly light-sensitive property of PA-Flp is ideal for activation in deep mouse brain regions by illumination with a noninvasive light-emitting diode. In addition, PA-Flp can be extended to the Cre-lox system through a viral vector as Flp-dependent Cre expression platform, thereby activating both Flp and Cre. Finally, we demonstrate that PA-Flp–dependent, Cre-mediated Cav3.1 silencing in the medial septum increases object-exploration behavior in mice. Thus, PA-Flp is a noninvasive, highly efficient, and easy-to-use optogenetic module that offers a side-effect-free and expandable genetic manipulation tool for neuroscience research.

Фармакологическая характеристика пептидных лигандов калиевых каналов MeKTx13-2 и MeKTx13-3 из яда скорпиона Mesobuthus eupeus

Scorpion toxins that block potassium channels currently serve as indispensable molecular research tools in neuroscience. However, there is a grave shortage of compounds that selectively act on particular channel isoforms. Earlier, from the venom of the Central Asian scorpion Mesobuthus eupeus, we have isolated a number of toxins that act on voltage-gated potassium channels. Now, we have studied the activity of two similar M. eupeus toxins, named MeKTx13-2 and MeKTx13-3, on a panel of potassium channels and found that they are characterized by a selective effect on the Kv1.1 isoform, the expression of which is characteristic of the central nervous system of mammals. We believe that our results will allow to obtain derivatives of toxins with increased selectivity for Kv1.1, which will be in demand in studies of the nervous system.

Third Order Mathematical Model of the Brain Validated via Cerebral Evoked Potentials

This paper presents the vital role mathematical modeling plays in the analysis of evoked potentials obtained from auditory, somatosensory, visual, and cognitive stimuli. Analysis of this data was performed by using the concepts of Closed Loop Methodology (CLM) to create specific mathematical modes representative of the signals that were being analyzed. As a result of this effort, a Forensic Simulator was design and built to signal process the EEG's provided by patients of the Craig Hospital Neuroscience Laboratory located in Englewood, Colorado. The Forensic Simulator provides a modern system analysis tool for neuroscience research. This device provides a user friendly environmental from which the electrical activity of the brain can be quantified. By using an adaptive processor to null out the error generate by comparing measured evoked potentials to those produce by a mathematical model, the neurophysiology of the central nervous system may be evaluated. Nulling of this error is achieved by defining the neurological classification set. Members of the set are defined to be: the uncertainty of the neuron firing, the neuronal staee characteristics, the characteristic brain parameters, estimates of the neuronal states, variances associated with the neuronal state estimates, and the variances attributable toe measurement error. Members of the classification set are obtained by solving the modeling problem via solution to the subproblems of estimation, identification, and control. Algorithms solving the the estimation problem provide numerical time varying values for the states of the system. This ability is assured once the SID (System IDentification) has been solved. This solution yields the quantification of the uncertainties associated with the state estimates as well as the uncertainties associated with the evoked potential measurements.

Parawixin2 Protects Hippocampal Cells in Experimental Temporal Lobe Epilepsy.

Epilepsy is considered as one of the major disabling neuropathologies. Almost one third of adult patients with temporal lobe epilepsy (TLE) do not respond to current antiepileptic drugs (AEDs). Additionally, most AEDs do not have neuroprotective effects against the inherent neurodegenerative process underlying the hippocampal sclerosis on TLE. Dysfunctions in the GABAergic neurotransmission may contribute not only to the onset of epileptic activity but also constitute an important system for therapeutic approaches. Therefore, molecules that enhance GABA inhibitory effects could open novel avenues for the understanding of epileptic plasticity and for drug development. Parawixin2, a compound isolated from Parawixia bistriata spider venom, inhibits both GABA and glycine uptake and has an anticonvulsant effect against a wide range of chemoconvulsants. The neuroprotective potential of Parawixin2 was analyzed in a model of TLE induced by a long-lasting Status Epilepticus (SE), and its efficiency was compared to well-known neuroprotective drugs, such as riluzole and nipecotic acid. Neuroprotection was assessed through histological markers for cell density (Nissl), astrocytic reactivity (GFAP) and cell death labeling (TUNEL), which were performed 24 h and 72 h after SE. Parawixin2 treatment resulted in neuroprotective effects in a dose dependent manner at 24 h and 72 h after SE, as well as reduced reactive astrocytes and apoptotic cell death. Based on these findings, Parawixin2 has a great potential to be used as a tool for neuroscience research and as a probe to the development of novel GABAergic neuroprotective agents.

LED Optrode with Integrated Temperature Sensing for Optogenetics.

In optogenetic studies, the brain is exposed to high-power light sources and inadequate power density or exposure time can cause cell damage from overheating (typically temperature increasing of 2 C). In order to overcome overheating issues in optogenetics, this paper presents a neural tool capable of assessing tissue temperature over time, combined with the capability of electrical recording and optical stimulation. A silicon-based 8 mm long probe was manufactured to reach deep neural structures. The final proof-of-concept device comprises a double-sided function: on one side, an optrode with LED-based stimulation and platinum (Pt) recording points; and, on the opposite side, a Pt-based thin-film thermoresistance (RTD) for temperature assessing in the photostimulation site surroundings. Pt thin-films for tissue interface were chosen due to its biocompatibility and thermal linearity. A single-shaft probe is demonstrated for integration in a 3D probe array. A 3D probe array will reduce the distance between the thermal sensor and the heating source. Results show good recording and optical features, with average impedance magnitude of 371 k, at 1 kHz, and optical power of 1.2 mW·mm (at 470 nm), respectively. The manufactured RTD showed resolution of 0.2 C at 37 C (normal body temperature). Overall, the results show a device capable of meeting the requirements of a neural interface for recording/stimulating of neural activity and monitoring temperature profile of the photostimulation site surroundings, which suggests a promising tool for neuroscience research filed.

Functional Neuroimaging: Single Photon Emission Computed Tomography (SPECT) In Neurological Disorders

A single photon emission computed tomography (SPECT) scan is a functional nuclear imaging technique performed to evaluate regional cerebral perfusion. Because cerebral blood flow is closely linked to neuronal activity, the activity distribution is presumed to reflect neuronal activity levels in several areas of the brain. Although structural magnetic resonance imaging (MRI) and computed tomography (CT) provide exquisite anatomical detail, SPECT provide complementary functional information. Frequently, brain pathology will manifest as functional changes before anatomical changes are detectable. SPECT has clinical value in the diagnosis, therapeutic management, and follow-up of patients. A general consideration of the clinical value of this technique is followed by relevant information on cerebral physiology and pathology for proper understanding of brain SPECT images. The diversity of central nervous system diseases and therefore the still incomplete knowledge of the mechanisms that underlie them have contributed to the success of brain perfusion SPECT as a research tool in neurosciences. Finally, stepby- step recommendations for interpreting and reporting brain perfusion SPECT images are provided to get the utmost clinical beneût from this technique. Bangladesh Journal of Neuroscience 2018; Vol. 34 (2): 96-105

Ultrasonic Neuromodulation Causes Widespread Cortical Activation via an Indirect Auditory Mechanism

Ultrasound has received widespread attention as anemerging technology for targeted, non-invasive neuromodulation based on its ability to evoke electrophysiological and motor responses in animals. However, little is known about the spatiotemporal pattern of ultrasound-induced brain activity that could drive these responses. Here, we address this question by combining focused ultrasound with wide-field optical imaging of calcium signals in transgenic mice. Surprisingly, we find cortical activity patterns consistent with indirect activation of auditory pathways rather than direct neuromodulation at the ultrasound focus. Ultrasound-induced activity is similar to that evoked by audible sound. Furthermore, both ultrasound and audible sound elicit motor responses consistent with a startle reflex, with both responses reduced by chemical deafening. These findings reveal an indirect auditory mechanism for ultrasound-induced cortical activity and movement requiring careful consideration in future development of ultrasonic neuromodulation as a tool in neuroscience research.

Consumer neuroscience for marketing researchers

AbstractPurposeFor many consumer neuromarketing researchers, the use of functional magnetic resonance imaging has been the most preferred neuroscience technique. However, electroencephalography, eye tracking, and implicit measurements are becoming increasingly popular market research methods due to rapid technological improvements and reduced costs.DesignThis article is an overview of the most commonly used consumer neuroscience techniques in marketing research.FindingsMany peer‐reviewed journal articles in the consumer neuroscience literature usually provide only a brief summary of the actual functions of each neuroscience technique used in their research. Throughout the consumer neuroscience and marketing research literature, there is a lack of comprehensive evaluation of the relative merits of all neuroscience research tools. There is no rigorous analysis of the relative appropriateness of all the neuroscience, physiological, and biometric research tools currently used in consumer neuroscience market research.Originality/valueThis is the first paper that provides a comprehensive review of all relevant neuroscience techniques with a proper explanation of their functions and utility for different types of research. Each individual neuromarketing research tools' strengths and weaknesses are analysed for particular types of marketing research questions and highlights some of the key research papers and authors for interested researchers to follow up.

Beyond Fluorescent Proteins: Hybrid and Bioluminescent Indicators for Imaging Neural Activities

Optical biosensors have been invaluable tools in neuroscience research, as they provide the ability to directly visualize neural activity in real time, with high specificity, and with exceptional spatial and temporal resolution. Notably, a majority of these sensors are based on fluorescent protein scaffolds, which offer the ability to target specific cell types or even subcellular compartments. However, fluorescent proteins are intrinsically bulky tags, often insensitive to the environment, and always require excitation light illumination. To address these limitations, there has been a proliferation of alternative sensor scaffolds developed in recent years, including hybrid sensors that combine the advantages of synthetic fluorophores and genetically encoded protein tags, as well as bioluminescent probes. While still in their early stage of development as compared with fluorescent protein-based sensors, these novel probes have offered complementary solutions to interrogate various aspects of neuronal communication, including transmitter release, changes in membrane potential, and the production of second messengers. In this Review, we discuss these important new developments with a particular focus on design strategies.

Comparative neurotoxicity study of mercury-based inorganic compounds including Ayurvedic medicines Rasasindura and Kajjali in zebrafish model

Zebrafish behavioral model is a powerful tool for neuroscience research. Behavioral changes in the zebrafish are studied by administering drugs. With the aid of automated and open-source MATLAB program, high-accuracy tracking of zebrafish can be achieved, and the important behavioral parameters can be calculated. Although mercury is accepted as a potent neurotoxin, used as a key element for preparing certain Ayurvedic medicines. In this work, mercury-based inorganic compounds, including HgCl2, HgS, and Ayurvedic medicines (Rasasindura and Kajjali) were administrated in zebrafish, and the effects on various behavioral parameters and cortisol levels were studied. A significant change in the basic locomotor parameters of fish was observed including speed (43% reduction), meander (150% increment), and a number of freeze points (125% increment), during 5-day treatment of HgCl2 along with a 3-fold increase in cortisol level against the control groups. Abnormal behavior was also recorded in color preference test, and novel tank diving behavior of HgCl2-treated groups, which can be attributed to the neurotoxicity induced by the HgCl2 administration. Contrary to this, the Rasasindura-treated group showed a significant increase in speed by 33%, decrease in meander by 20%, decrease in freeze points by 30%, and insignificant alteration in cortisol levels, which can be related to the rejuvenating nature of the Ayurvedic medicine Rasasindura. Additionally, Kajjali treated group did not show any substantial changes in zebrafish cortisol level and behavioral parameters except one in the diving test that indicates lowering stress. Similarly, HgS group showed normal behaviors except two irregular motor behaviors identical with the HgCl2 group. From these results, it can be concluded that the mercury-based Ayurvedic Rasasindura and Kajjali did not show any adverse effect or toxicity on zebrafish behavior model.

Assessing denoising strategies to increase signal to noise ratio in spinal cord and in brain cortical and subcortical regions

Functional Magnetic Resonance Imaging (fMRI) based on Blood Oxygenation Level Dependent (BOLD) contrast has become one of the most powerful tools in neuroscience research. On the other hand, fMRI approaches have seen limited use in the study of spinal cord and subcortical brain regions (such as the brainstem and portions of the diencephalon). Indeed obtaining good BOLD signal in these areas still represents a technical and scientific challenge, due to poor control of physiological noise and to a limited overall quality of the functional series. A solution can be found in the combination of optimized experimental procedures at acquisition stage, and well-adapted artifact mitigation procedures in the data processing. In this framework, we studied two different data processing strategies to reduce physiological noise in cortical and subcortical brain regions and in the spinal cord, based on the aCompCor and RETROICOR denoising tools respectively. The study, performed in healthy subjects, was carried out using an ad hoc isometric motor task. We observed an increased signal to noise ratio in the denoised functional time series in the spinal cord and in the subcortical brain region.

Survey of electrically evoked responses in the retina - stimulus preferences and oscillation among neurons

Electrical stimulation is an important tool in neuroscience research and clinically. In the retina, extensive work has revealed how the retinal ganglion cells respond to extracellular electrical stimulation. But little is known about the responses of other neuronal types, and more generally, how the network responds to stimulation. We conducted a survey of electrically evoked responses, over a range of pulse amplitudes and pulse widths, for 21 cell types spanning the inner two layers of the rabbit retina. It revealed: (i) the evoked responses of some neurons were charge insensitive; (ii) pulse-width sensitivity varied between cell types, allowing preferential recruitment of cell types; and (iii) 10–20 Hz damped oscillations across retinal layers. These oscillations were generated by reciprocal excitatory / inhibitory synapses, at locations as early as the cone-horizontal-cell synapses. These results illustrate at cellular resolution how a network responds to extracellular stimulation, and could inform the development of bioelectronic implants for treating blindness.

High resolution modulation of human brain circuits using transcranial focused ultrasound

Current non-invasive electric and electromagnetic non-invasive neuromodulatory approaches have proven effective for inducing transient plastic changes in human cortex. However, these technologies have poor spatial resolution and suffer from a depth-focality tradeoff. Transcranial focused ultrasound (tFUS) is an emerging non-surgical low-energy technique for inducing transient plasticity in sub-cortical and cortical areas with high spatial resolution and adjustable focus. tFUS has been successfully employed in small and large animal preparations and our work has also demonstrated tFUS to be effective for human cortical and subcortical neuromodulation. Here, we will discuss tFUS current approaches as well as challenges and future directions for this technology as a tool for neuroscience research and human brain mapping and potential clinical applications.

Magnetic eye tracking in mice.

Eye movements provide insights about a wide range of brain functions, from sensorimotor integration to cognition; hence, the measurement of eye movements is an important tool in neuroscience research. We describe a method, based on magnetic sensing, for measuring eye movements in head-fixed and freely moving mice. A small magnet was surgically implanted on the eye, and changes in the magnet angle as the eye rotated were detected by a magnetic field sensor. Systematic testing demonstrated high resolution measurements of eye position of <0.1°. Magnetic eye tracking offers several advantages over the well-established eye coil and video-oculography methods. Most notably, it provides the first method for reliable, high-resolution measurement of eye movements in freely moving mice, revealing increased eye movements and altered binocular coordination compared to head-fixed mice. Overall, magnetic eye tracking provides a lightweight, inexpensive, easily implemented, and high-resolution method suitable for a wide range of applications.

Polymer Thin Films with Tunable Acetylcholine-like Functionality Enable Long-Term Culture of Primary Hippocampal Neurons

In vitro culture systems for primary neurons have served as useful tools for neuroscience research. However, conventional in vitro culture methods are still plagued by challenging problems with respect to applications to neurodegenerative disease models or neuron-based biosensors and neural chips, which commonly require long-term culture of neural cells. These impediments highlight the necessity of developing a platform capable of sustaining neural activity over months. Here, we designed a series of polymeric bilayers composed of poly(glycidyl methacrylate) (pGMA) and poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA), designated pGMA:pDMAEMA, using initiated chemical vapor deposition (iCVD). Harnessing the surface-growing characteristics of iCVD polymer films, we were able to precisely engraft acetylcholine-like functionalities (tertiary amine and quaternary ammonium) onto cell culture plates. Notably, pGD3, a pGMA:pDMAEMA preparation with the highest surface composition of quaternary ammonium, fostered the most rapid outgrowth of neural cells. Clear contrasts in neural growth and survival between pGD3 and poly-l-lysine (PLL)-coated surfaces became apparent after 30 days in vitro (DIV). Moreover, brain-derived neurotrophic factor level continuously accumulated in pGD3-cultured neurons, reaching a 3-fold increase at 50 DIV. Electrophysiological measurements at 30 DIV revealed that the pGD3 surface not only promoted healthy maturation of hippocampal neurons but also enhanced the function of hippocampal ionotropic glutamate receptors in response to synaptic glutamate release. Neurons cultured long-term on pGD3 also maintained their characteristic depolarization-induced Ca2+ influx functions. Furthermore, primary hippocampal neurons cultured on pGD3 showed long-term survival in a stable state up to 90 days-far longer than neurons on conventional PLL-coated surfaces. Taken together, our findings indicate that a polymer thin film with optimal acetylcholine-like functionality enables a long-term culture and survival of primary neurons.

Of mice and people

N2 often includes articles on mouse models and therefore the title of this Corner would be applicable to most N2 issues. Animal models represent the quintessence of translational research and are invaluable tools in neuroscience research. Modeling starts with a basic understanding of a human disease translated into a model that, if successful, will lead researchers back to the clinics armed with new theories and therapeutic possibilities. In the current issue of N2, there are several studies that exemplify the power of animal modeling to link the laboratory to the clinics.

Shining a light on optogenetics: Advances in electronics and protein engineering advance the field of optogenetics to study the activity of neuronal networks.

Optogenetics is relatively new and has attracted much interest since the first publications from the field in the early 2000s. Both researchers and the media are fascinated by the notions it conjures of bionic implants. Notwithstanding potential applications, the technology has quickly become an important research tool in neuroscience with potential for developing a range of novel therapeutic approaches. Scientifically, optogenetics combines advances in optics, small‐scale power generation, electronics, the manipulation of cellular activity and gene therapy, to enable the study of complete systems, including neural networks in animals and humans. Moreover, some recent studies have highlighted potential applications in plants as an alternative to genetic modification (GM) for targeted alteration of phenotypes. Since the first experiments [1], optogenetics has consistently expanded in scope and definition. It can now be defined as the use of light to stimulate cells into which exogenous genes coding for light‐sensitive proteins have been introduced, and to measure the response of these transformed cells to stimulation. Typically, the light is generated by an artificial source powered by a battery or external electric field. The definition also embraces treatments for some forms of blindness based on implanting light‐sensitive proteins in the retina. However, much of the fields' focus revolves around the optical components, their implantation in the body, and the interface with the target cells to stimulate gene expression with high spatial and temporal resolution. In practice, an implanted LED or other light source illuminates a specific target area. Within this area, the target cells—genetically modified to express specific light‐sensitive proteins—react to this light by changing their membrane potential, which in turn starts signal cascades that activate or shut down specific target genes. The key biological challenges of optogenetics are the development of appropriate light‐sensitive proteins, mechanisms for delivering their genes to specific cells, …

Light-evoked hyperpolarization and silencing of neurons by conjugated polymers.

The ability to control and modulate the action potential firing in neurons represents a powerful tool for neuroscience research and clinical applications. While neuronal excitation has been achieved with many tools, including electrical and optical stimulation, hyperpolarization and neuronal inhibition are typically obtained through patch-clamp or optogenetic manipulations. Here we report the use of conjugated polymer films interfaced with neurons for inducing a light-mediated inhibition of their electrical activity. We show that prolonged illumination of the interface triggers a sustained hyperpolarization of the neuronal membrane that significantly reduces both spontaneous and evoked action potential firing. We demonstrate that the polymeric interface can be activated by either visible or infrared light and is capable of modulating neuronal activity in brain slices and explanted retinas. These findings prove the ability of conjugated polymers to tune neuronal firing and suggest their potential application for the in-vivo modulation of neuronal activity.

Exosome-associated AAV vector as a robust and convenient neuroscience tool.

Adeno-associated virus (AAV) vectors are showing promise in gene therapy trials and have proven to be extremely efficient biological tools in basic neuroscience research. One major limitation to their widespread use in the neuroscience laboratory is the cost, labor, skill, and time intense purification process of AAV. We have recently shown that AAV can associate with exosomes (exo-AAV) when vector is isolated from conditioned media of producer cells, and the exo-AAV is more resistant to neutralizing anti-AAV antibodies compared to standard AAV. Here we demonstrate that simple pelleting of exo-AAV from media via ultracentrifugation, results in high-titer vector preparations capable of efficient transduction of central nervous system (CNS) cells after systemic injection in mice. We observed that exo-AAV is more efficient at gene delivery to the brain at low vector doses relative to conventional AAV, even when derived from a serotype that does not normally efficiently cross the blood brain barrier. Similar cell types were transduced by exo-AAV and conventionally purified vector. Importantly, no cellular toxicity was noted in exo-AAV transduced cells. We demonstrated the utility and robustness of exo-AAV-mediated gene delivery by detecting direct GFP fluorescence after systemic injection, allowing 3-dimensional reconstruction of transduced Purkinje cells in the cerebellum using ex-vivo serial 2-photon tomography. The ease of isolation combined with the high efficiency of transgene expression in the CNS, may enable widespread use of exo-AAV as a neuroscience research tool. Furthermore, the ability of exo-AAV to evade neutralizing antibodies while still transducing CNS after peripheral delivery is clinically relevant.

Realistic Numerical and Analytical Modeling of Light Scattering in Brain Tissue for Optogenetic Applications(1,2,3).

In recent years, optogenetics has become a central tool in neuroscience research. Estimating the transmission of visible light through brain tissue is of crucial importance for controlling the activation levels of neurons in different depths, designing optical systems, and avoiding lesions from excessive power density. The Kubelka–Munk model and Monte Carlo simulations have previously been used to model light propagation through rodents' brain tissue, however, these prior attempts suffer from fundamental shortcomings. Here, we introduce and study two modified approaches for modeling the distributions of light emanating from a multimode fiber and scattering through tissue, using both realistic numerical Monte Carlo simulations and an analytical approach based on the beam-spread function approach. We demonstrate a good agreement of the new methods' predictions both with recently published data, and with new measurements in mouse brain cortical slices, where our results yield a new cortical scattering length estimate of ∼47 µm at λ = 473 nm, significantly shorter than ordinarily assumed in optogenetic applications.