Photo Control of Protein Function Using Photoactive Yellow Protein
Photoswitchable proteins are becoming increasingly common tools for manipulating cellular processes with high spatial and temporal precision. Photoactive yellow protein (PYP) is a small, water-soluble protein that undergoes a blue light induced change in conformation. It can serve as a scaffold for designing new tools to manipulate biological processes, but with respect to other protein scaffolds it presents some technical challenges. Here, we present practical information on how to overcome these, including how to synthesize the PYP chromophore, how to express and purify PYP, and how to screen for desired activity. (Sourc...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Controlling Protein Activity and Degradation Using Blue Light
Regulation of protein stability is a fundamental process in eukaryotic cells and pivotal to, e.g., cell cycle progression, faithful chromosome segregation, or protein quality control. Synthetic regulation of protein stability requires conditional degradation sequences (degrons) that induce a stability switch upon a specific signal. Fusion to a selected target protein permits to influence virtually every process in a cell. Light as signal is advantageous due to its precise applicability in time, space, quality, and quantity. Light control of protein stability was achieved by fusing the LOV2 photoreceptor domain of Arabidops...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Reversible Photoregulation of Gene Expression and Translation
Several methods for controlling gene expression by light illumination have been reported. Most of these methods control transcription by regulating the interaction between DNA and transcription factors. The use of a photolabile protecting compound (cage compound) is another promising approach for controlling gene expression, although typically in an irreversible manner. We here describe a new approach for reversibly controlling translation using a photoresponsive 8-styryl cap (8ST-cap) that can be reversibly isomerized by illumination with light of a specific wavelength. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Algal Photobiology: A Rich Source of Unusual Light Sensitive Proteins for Synthetic Biology and Optogenetics
The light absorption system in eukaryotic (micro)algae includes highly sensitive photoreceptors, which change their conformation in response to different light qualities on a subsecond time scale and induce physiological and behavioral responses. Some of the light sensitive modules are already in use to engineer and design photoswitchable tools for control of cellular and physiological activities in living organisms with various degrees of complexity. Thus, identification of new light sensitive modules will not only extend the source material for the generation of optogenetic tools but also foster the development of new li...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Natural Resources for Optogenetic Tools
Photoreceptors are found in all kingdoms of life and mediate crucial responses to environmental challenges. Nature has evolved various types of photoresponsive protein structures with different chromophores and signaling concepts for their given purpose. The abundance of these signaling proteins as found nowadays by (meta-)genomic screens enriched the palette of optogenetic tools significantly. In addition, molecular insights into signal transduction mechanisms and design principles from biophysical studies and from structural and mechanistic comparison of homologous proteins opened seemingly unlimited possibilities for cu...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Erratum to: Optogenetics in Plants: Red/Far-Red Light Control of Gene Expression
(Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Guidelines for Photoreceptor Engineering
Sensory photoreceptors underpin optogenetics by mediating the noninvasive and reversible perturbation of living cells by light with unprecedented temporal and spatial resolution. Spurred by seminal optogenetic applications of natural photoreceptors, the engineering of photoreceptors has recently garnered wide interest and has led to the construction of a broad palette of novel light-regulated actuators. Photoreceptors are modularly built of photosensors that receive light signals, and of effectors that carry out specific cellular functions. These modules have to be precisely connected to allow efficient communication, such...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Optogenetic Manipulation of Selective Neural Activity in Free-Moving Drosophila Adults
Activating selected neurons elicits specific behaviors in Drosophila adults. By combining optogenetics and laser-tracking techniques, we have recently developed an automated laser-tracking and optogenetic manipulation system (ALTOMS) for studying how brain circuits orchestrate complex behaviors. The established ALTOMS can independently target three lasers (473-nm blue laser, 593.5-nm yellow laser, and 1064-nm infrared laser) on any specified body part of two freely moving flies. Triggering light-sensitive proteins in real time, the blue laser and yellow laser can respectively activate and inhibit target neurons in artifici...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Protein Inactivation by Optogenetic Trapping in Living Cells
Optogenetic modules that use genetically encoded elements to control protein function in response to light allow for precise spatiotemporal modulation of signaling pathways. As one of optical approaches, LARIAT (Light-Activated Reversible Inhibition by Assembled Trap) is a unique light-inducible inhibition system that reversibly sequesters target proteins into clusters, generated by multimeric proteins and a blue light-induced heterodimerization module. Here we present a method based on LARIAT for optical inhibition of targets in living mammalian cells. In the protocol, we focus on the inhibition of proteins that modulate ...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Enhancing Channelrhodopsins: An Overview
After the discovery of Channelrhodopsin, a light-gated ion channel, only a few people saw the diverse range of applications for such a protein. Now, more than 10 years later Channelrhodopsins have become widely accepted as the ultimate tool to control the membrane potential of excitable cells via illumination. The demand for more application-specific Channelrhodopsin variants started a race between protein engineers to design improved variants. Even though many engineered variants have undisputable advantages compared to wild-type variants, many users are alienated by the tremendous amount of new variants and their perplex...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Combined Optogenetic and Chemogenetic Control of Neurons
We describe applications of this approach in cultured neurons in vitro, in brain slices ex vivo, and in awake and anesthetized animals in vivo. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Optogenetic Modulation of Locomotor Activity on Free-Behaving Rats
The technology of optogenetics provides a new method to modulate neural activity with spatial specificity and millisecond-temporal scale. This nonelectrical modulation method also gives chance for simultaneous electrophysiological recording during stimulations. Here, we describe our locomotor activity modulation on free-behaving rats using optogenetic techniques. The target sites of the rat brain were dorsal periaqueductal gray (dPAG) and ventral tegmental area (VTA) for the modulation of defensive and reward behaviors, respectively. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Optogenetic Control of Mammalian Ion Channels with Chemical Photoswitches
In neurons, ligand-gated ion channels decode the chemical signal of neurotransmitters into an electric response, resulting in a transient excitation or inhibition. Neurotransmitters act on multiple receptor types and subtypes, with spatially and temporally precise patterns. Hence, understanding the neural function of a given receptor requires methods for its targeted, rapid activation/inactivation in defined brain regions. To address this, we have developed a versatile optochemical genetic strategy, which allows the reversible control of defined receptor subtypes in designated cell types, with millisecond and micrometer pr...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Optogenetics in Drosophila Neuroscience
Optogenetic techniques enable one to target specific neurons with light-sensitive proteins, e.g., ion channels, ion pumps, or enzymes, and to manipulate their physiological state through illumination. Such artificial interference with selected elements of complex neuronal circuits can help to determine causal relationships between neuronal activity and the effect on the functioning of neuronal circuits controlling animal behavior. The advantages of optogenetics can best be exploited in genetically tractable animals whose nervous systems are, on the one hand, small enough in terms of cell numbers and to a certain degree ste...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

An Optimized Calcium-Phosphate Transfection Method for Characterizing Genetically Encoded Tools in Primary Neurons
In order to characterize genetically encoded tools under the most relevant conditions, the constructs need to be expressed in the cell type in which they will be used. This is a major hurdle in developing optogenetic tools for neuronal cells, due to the difficulty of gene transfer to these cells. Several protocols have been developed for transfecting neurons, focusing on improved transfection efficiency. However, obtaining healthy cells is as important. We monitored transfected cell health by measuring electrophysiological parameters, and used them as a guideline to optimize transfection. Here we describe an optimized tran...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Intracranial Injection of an Optogenetics Viral Vector Followed by Optical Cannula Implantation for Neural Stimulation in Rat Brain Cortex
Optogenetics is rapidly gaining acceptance as a preferred method to study specific neuronal cell types using light. Optogenetic neuromodulation requires the introduction of a cell-specific viral vector encoding for a light activating ion channel or ion pump and the utilization of a system to deliver light stimulation to brain. Here, we describe a two-part methodology starting with a procedure to inject an optogenetic AAV virus into rat cortex followed by a second procedure to surgically implant an optical cannula for light delivery to the deeper cortical layers. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Optogenetic Approaches for Mesoscopic Brain Mapping
Recent advances in identifying genetically unique neuronal proteins has revolutionized the study of brain circuitry. Researchers are now able to insert specific light-sensitive proteins (opsins) into a wide range of specific cell types via viral injections or by breeding transgenic mice. These opsins enable the activation, inhibition, or modulation of neuronal activity with millisecond control within distinct brain regions defined by genetic markers. Here we present a useful guide to implement this technique into any lab. We first review the materials needed and practical considerations and provide in-depth instructions fo...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Optogenetic Tools for Confined Stimulation in Deep Brain Structures
Optogenetics has emerged in the past decade as a technique to modulate brain activity with cell-type specificity and with high temporal resolution. Among the challenges associated with this technique is the difficulty to target a spatially restricted neuron population. Indeed, light absorption and scattering in biological tissues make it difficult to illuminate a minute volume, especially in the deep brain, without the use of optical fibers to guide light. This work describes the design and the in vivo application of a side-firing optical fiber adequate for delivering light to specific regions within a brain subcortical st...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Remote Patterning of Transgene Expression Using Near Infrared-Responsive Plasmonic Hydrogels
The development of noninvasive technologies for remote control of gene expression has received increased attention for their therapeutic potential in clinical scenarios, including cancer, neurological disorders, immunology, tissue engineering, as well as developmental biology research. Near-infrared (NIR) light is a suitable source of energy that can be employed to pattern transgene expression in plasmonic cell constructs. Gold nanoparticles tailored to exhibit a plasmon surface band absorption peaking at NIR wavelengths within the so called tissue optical window (TOW) can be used as fillers in fibrin-based hydrogels. Thes...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Inscribing Optical Excitability to Non-Excitable Cardiac Cells: Viral Delivery of Optogenetic Tools in Primary Cardiac Fibroblasts
We describe in detail a method to introduce optogenetic actuation tools, a mutant version of channelrhodopsin-2, ChR2(H134R), and archaerhodopsin (ArchT), into primary cardiac fibroblasts (cFB) in vitro by adenoviral infection to yield quick, robust, and consistent expression. Instructions on adjusting infection parameters such as the multiplicity of infection and virus incubation duration are provided to generalize the method for different lab settings or cell types. Specific conditions are discussed to create hybrid co-cultures of the optogenetically modified cFB and non-transformed cardiomyocytes to obtain light-sensiti...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Optogenetic Light Crafting Tools for the Control of Cardiac Arrhythmias
The control of spatiotemporal dynamics in biological systems is a fundamental problem in nonlinear sciences and has important applications in engineering and medicine. Optogenetic tools combined with advanced optical technologies provide unique opportunities to develop and validate novel approaches to control spatiotemporal complexity in neuronal and cardiac systems. Understanding of the mechanisms and instabilities underlying the onset, perpetuation, and control of cardiac arrhythmias will enable the development and translation of novel therapeutic approaches. Here we describe in detail the preparation and optical mapping...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Optogenetics: Basic Concepts and Their Development
The discovery of light-gated ion channels and their application to controlling neural activities have had a transformative impact on the field of neuroscience. In recent years, the concept of using light-activated proteins to control biological processes has greatly diversified into other fields, driven by the natural diversity of photoreceptors and decades of knowledge obtained from their biophysical characterization. In this chapter, we will briefly discuss the origin and development of optogenetics and highlight the basic concepts that make it such a powerful technology. We will review how these enabling concepts have d...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Optogenetic Engineering of Atrial Cardiomyocytes
Optogenetics is emerging in the cardiology field as a new strategy to explore biological functions through the use of light-sensitive proteins and dedicated light sources. For example, this technology allows modification of the electrophysiological properties of cardiac muscle cells with superb spatiotemporal resolution and quantitative control. In this chapter, the optogenetic modification of atrial cardiomyocytes (aCMCs) from 2-day-old Wistar rats using lentiviral vector (LV) technology and the subsequent activation of the light-sensitive proteins (i.e., ion channels) through light-emitting diodes (LEDs) are described. (...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

A Multichannel Recording System with Optical Stimulation for Closed-Loop Optogenetic Experiments
Selective perturbation of the activity of specific cell types in the brain tissue is essential in understanding the function of neuronal circuits involved in cognition and behavior and might also provide therapeutic neuromodulation strategies. Such selective neuronal addressing can be achieved through the optical activation of light-sensitive proteins called opsins that are expressed in specific cell populations through genetic methods—hence the name“optogenetics.” In optogenetic experiments, the electrical activity of the targeted cell populations is optically triggered and monitored using arrays of micr...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Optogenetic Control of Fibroblast Growth Factor Receptor Signaling
FGFR1 is a member of the fibroblast growth factor family, which controls diverse cellular functions such as cell proliferation, migration, and differentiation. OptoFGFR1, an optogenetic method to modulate the FGFR signaling pathway with light by utilizing PHR domain of cryptochrome2 and cytoplasmic region of FGFR1, enabled light-guided activation of FGFR to study its effects on downstream signaling pathway and during diverse biological processes such as cell migration. Here, we describe about optogenetic and microscopic methods to spatiotemporally manipulate FGFR signaling in a single cell or group of cells using confocal ...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Origin and Detection of Neonatal Seizures: Animal and Clinical Studies
Neonatal seizures remain a major clinical problem worldwide and are harmful to the developing brain. Seizures are associated with poor neurodevelopmental outcomes and significant risk of death requiring urgent diagnosis and intervention. Current antiepileptic drugs however have limited efficacy and are potentially harmful to the developing newborn brain. Despite this, standard clinical practice for the treatment of neonatal seizures remains unchanged. This chapter describes a clinically relevant neonatal animal model of HI-induced seizures. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - November 9, 2015 Category: Neuroscience Source Type: news

Using Pregnant Sheep to Model Developmental Brain Damage
In order to develop more effective ways of identifying, managing, and treating preterm asphyxial brain injury, stable experimental models are essential. The present review describes the key experimental factors that determine the pattern and severity of brain injury in chronically instrumented fetal sheep, including the depth (“severity”) and duration of asphyxia, and the maturity, and condition of the fetus. These models are valuable to dissect the pathogenesis of key clinical patterns of brain injury in a stable thermal and biochemical environment, and to test therapeutic interventions. (Source: Springer prot...
Source: Springer protocols feed by Neuroscience - November 9, 2015 Category: Neuroscience Source Type: news

Prenatal Determinants of Brain Development: Recent Studies and Methodological Advances
Despite advances in neonatal intensive care, survivors of premature birth remain highly susceptible to unique patterns of developmental brain injury that manifest as cerebral palsy and cognitive-learning disabilities. Whereas preterm infants were previously at high risk for destructive brain lesions that resulted in cystic white matter injury and secondary cortical and subcortical gray matter degeneration, contemporary cohorts of preterm survivors commonly display less severe injury that does not appear to involve pronounced glial or neuronal loss. Cerebral development in fetal sheep shares many anatomical and physiologica...
Source: Springer protocols feed by Neuroscience - November 9, 2015 Category: Neuroscience Source Type: news

Oligodendrocytes: Cells of Origin for White Matter Injury in the Developing Brain
A prominent pattern of brain injury in preterm born infants involves damage to white matter with impaired oligodendrocyte maturation. This results in diffuse deficits in myelination that are associated with later development of cerebral palsy. While numerous experimental animal models of perinatal white matter injury have been developed, they show a spectrum of effects. This review proposes that adopting a more standard approach to defining white matter injury is important for validating experimental findings against the bona fide human condition. This chapter will describe the pathology of perinatal white matter injury an...
Source: Springer protocols feed by Neuroscience - November 9, 2015 Category: Neuroscience Source Type: news

Perinatal and Postnatal Determinants of Brain Development: Recent Studies and Methodological Advances
Perinatal diet is an important factor in programming brain development and susceptibility to obesity. There are currently several elegant and simple prenatal and postnatal animal models in use to mimic the effects of early life overfeeding and to study its impact on brain and metabolic development. In this chapter we will discuss the background to some of these models, with a specific focus on manipulating rodent litter sizes to alter the early life nutritional environment. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - November 9, 2015 Category: Neuroscience Source Type: news

Prenatal Programming of the Mesolimbic Reward Pathway and Food Preferences
The drive to consume palatable foods, high in fat and sugar, goes beyond the need to satisfy hunger and has a strong hedonic component. Studies in rodent models have demonstrated that the preference for these foods can be programmed before birth, and that feeding dams on cafeteria diets during pregnancy and lactation is associated with an increased preference for palatable foods in the offspring after weaning. More recently, attention has turned towards elucidating the biological mechanisms which drive these effects, with studies to date focussing on the impact of maternal cafeteria diets on the development of the mesolimb...
Source: Springer protocols feed by Neuroscience - November 9, 2015 Category: Neuroscience Source Type: news

Studies on the Effects Prenatal Immune Activation on Postnatal Behavior: Models of Developmental Origins of Schizophrenia
Human epidemiological studies have indicated an association between infection during pregnancy and an increased risk of neurodevelopmental disorders such as schizophrenia in offspring. As infections arising from various causes have a similar debilitating effect in later life, it is thought that the maternal response, common to most infections, may be the critical factor altering fetal brain development. In this chapter, we discuss various animal models of prenatal exposure to an infection, that have aimed to cause neurobiological, pharmacological and behavioral abnormalities in offspring comparable to those seen in schizop...
Source: Springer protocols feed by Neuroscience - November 9, 2015 Category: Neuroscience Source Type: news

Placental Transport and Metabolism: Implications for the Developmental Effects of Selective Serotonin Reuptake Inhibitors (SSRI) Antidepressants
A host of neurodevelopmental processes are modulated by serotonin (5-HT), a molecule also implicated in the etiology of diverse psychiatric disorders. Prenatal exposures that affect serotonergic signaling and the developing 5-HT system are increasingly associated with multiple long-term repercussions for the offspring. Both maternal depression and antidepressant treatments have been shown to affect fetal neurodevelopment during pregnancy, possibly through alterations of 5-HT levels that are otherwise precisely set by placental and endogenous sources. The result of such dysregulation impacts a variety of critical signaling ...
Source: Springer protocols feed by Neuroscience - November 9, 2015 Category: Neuroscience Source Type: news

Models of Perinatal Compromises in the Guinea Pig: Their Use in Showing the Role of Neurosteroids in Pregnancy and the Newborn
Placental progesterone production during late gestation has a major role in maintaining elevated neurosteroid levels during pregnancy. These levels of key neurosteroids, including allopregnanolone, are critical for optimal brain development during late gestation and the early neonatal period. The long gestation period (~70), in utero brain development and placental progesterone synthesis of the guineas pig makes this species very suitable for studying the mechanisms by which pregnancy compromises impact neurosteroid pathways. We have used models of intrauterine growth restriction and preterm birth to show that these challe...
Source: Springer protocols feed by Neuroscience - November 9, 2015 Category: Neuroscience Source Type: news

Maternal Obesity in Pregnancy: Consequences for Brain Function in the Offspring
It is perhaps not surprising that an inhospitable intrauterine environment can result in neurodevelopmental disorders, given the enormous changes in brain development that occur during gestation. Here we discuss: (1) Obesity is a state of low-grade inflammation and is thus a candidate for having an unfavorable impact on brain function in the offspring. (2) Maternal obesity has recently been associated with offspring attention deficit hyperactivity disorder and autism spectrum disorder. A recent study found differences in amniotic fluid mRNA for 20 genes in fetuses of obese versus lean women, and several of these genes impa...
Source: Springer protocols feed by Neuroscience - November 9, 2015 Category: Neuroscience Source Type: news

Circadian Rhythms in the Fetus and Newborn: Significance of Interactions with Maternal Physiology and the Environment
Timing of balanced and precise daily delivery of oxygen, nutrients, hormones, and biophysical cues from mother to fetus is essential for fetal growth and successful transition to extrauterine life. Such timing is provided by an arrangement of biological clocks operating in the mother and fetus. However, adverse intrauterine conditions including effects of altering the photoperiod (chronodisruption) during gestation on fetal growth/development and postnatal physiology may translate into adult disease, in which the role played by fetal circadian system remains unclear. Here we review the development of the circadian system, ...
Source: Springer protocols feed by Neuroscience - November 9, 2015 Category: Neuroscience Source Type: news

Fetal Sleep and Spontaneous Behavior In Utero: Animal and Clinical Studies
The term fetal behavior includes fetal sleep states, which in precocial mammalian species (i.e., those born relatively mature at birth) develop in late gestation, along with the fetal physiological processes, such as fetal body movements and breathing activity, cardiovascular function, blood gas, and acid-base status, which are linked to fetal sleep states. Since ~1970, as a result of technological and technical advances, there has been significant advances in our understanding of fetal behavior in man and animals, and also the development of fetal monitoring techniques in human pregnancy that are based on the changes in f...
Source: Springer protocols feed by Neuroscience - November 9, 2015 Category: Neuroscience Source Type: news

Cerebral Blood Flow Measurements in the Neonatal Brain
Cerebrovascular lesions and hypoxic-ischaemic brain injury are important causes of acquired neonatal brain injury in term and preterm newborn infants, which lead to significant morbidity and long-term mortality. Improved understanding of the cerebral hemodynamics and metabolism in the immature brain, and blood flow responses to physiological and external stimuli would aid understanding of the pathogenesis of neonatal brain injury. There has been increasing research interest and clinical demand to study the neonatal brain, with the exploration of the bedside and real-time measurement of cerebral hemodynamics in guiding ther...
Source: Springer protocols feed by Neuroscience - November 9, 2015 Category: Neuroscience Source Type: news

Advanced EEG and MRI Measurements to Study the Functional Development of the Newborn Brain
In this chapter we review the methodological progress that has recently been made for studying brain development in infants using noninvasive techniques. In particular, we focus on methodological platforms based on electroencephalography (EEG) and functional magnetic resonance imaging (fMRI). Key aspects of experimental setup, data acquisition, data preprocessing, and analysis are described and discussed with emphasis on recordings performed on the infant brain. The measurement and estimation of large-scale brain network connectivity using fMRI and EEG has become an important tool to study brain development. To this end, w...
Source: Springer protocols feed by Neuroscience - November 9, 2015 Category: Neuroscience Source Type: news

Isolation of GABAergic Cortical Neurons and Implications for Cell Transplantation Strategies in the Nervous System
The correct layer placement of interneurons and pyramidal neurons during corticogenesis is required for precise neuronal activity and subsequent functions of the neocortex. Interneurons are generated in the medial ganglionic eminence (MGE) and migrate from the ventral to dorsal telencephalon to reside within the developing cortical plate. Transplantation strategies are valuable in understanding the cellular and molecular basis of interneuron development, and more recently in the potential for cell-based therapies of neurological disorders. With the advancement of transgenic technologies, interneurons can be fluorescently l...
Source: Springer protocols feed by Neuroscience - November 9, 2015 Category: Neuroscience Source Type: news

In Utero Electroporation Methods in the Study of Cerebral Cortical Development
Research in the field of cortical development has benefited from technical advances in recent years, and tools are now available to label, monitor, and modulate cohorts of cerebral cortical neurons using in vivo approaches. Substantial populations of cerebral cortical neurons are generated in a specific sequence by the radial glia progenitors that line the ventricular surface during development. These radial progenitors self-renew and generate intermediate progenitors or neurons in a precisely choreographed fashion. Electroporation or electropermeabilization is a method that uses electric pulses to deliver molecules into c...
Source: Springer protocols feed by Neuroscience - November 9, 2015 Category: Neuroscience Source Type: news

Investigating Early Formation of the Cerebral Cortex by In Utero Electroporation: Methods and Protocols
Cortical development requires a strict balance between neuronal proliferation, differentiation, and cellular migration within restricted developmental stages. The precise spatiotemporal gene manipulation used in developmental studies can be achieved by in vitro or ex vivo experiments or by the generation of transgenic animals. However, these approaches have significant limitations when trying to investigate the origin and molecular regulation of early cortical neurons. In utero electroporation (IUE) is an informative cell labeling technique that provides the ability to label neural progenitor cells and their progeny in viv...
Source: Springer protocols feed by Neuroscience - November 9, 2015 Category: Neuroscience Source Type: news

The Rabbit as a Model of Cerebral Palsy
Rabbits, like humans, are perinatal brain developers. We have developed a model of fetal hypoxia-ischemia that results in postnatal rabbit kits with a phenotype analogous to that observed in humans with cerebral palsy. This chapter gives a practical approach to performing the model and shows the range methods available for study using the model. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - September 21, 2015 Category: Neuroscience Source Type: news

The Sheep as a Model of Brain Injury in the Premature Infant
We describe a global cerebral ischemia preparation that replicates major features of acute and chronic human cerebral injury and which has provided access to complex clinically relevant studies of cerebral blood flow and neuro-imaging that are not feasible in smaller laboratory animals. Despite the higher costs and technical challenges of instrumented preterm fetal sheep models, they allow an integrated analysis of the spectrum of insults that appear to contribute to cerebral injury in human preterm infants. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - September 21, 2015 Category: Neuroscience Source Type: news

Studies of Perinatal Asphyxial Brain Injury in the Fetal Sheep
In order to develop more effective ways of identifying, managing, and treating perinatal asphyxial brain injury, stable experimental models are essential. Although the outcome of clinical asphyxia is highly variable, modern imaging studies have distinguished two major patterns of injury in term infants, involving primary damage in either the parasagittal cortex or in the basal ganglia respectively. The present review describes the experimental preparation in detail, and the key experimental factors that determine the pattern and severity of brain injury in chronically instrumented fetal sheep, including the depth (“s...
Source: Springer protocols feed by Neuroscience - September 21, 2015 Category: Neuroscience Source Type: news

Fetal Brain Activity in the Sheep Model with Intrauterine Hypoxia
The fetal and early neonatal period is a time of rapid brain growth and development, dependent upon adequate oxygenation. Fetal hypoxia, whether chronic due to placental insufficiency or intermittent cord compression, or acute during labor, may give rise to aberrant development and neurologic sequelae. This chapter reviews the metabolic activities and behavioral states of the brain throughout the late fetal period, and the methods for studying them using the chronically instrumented ovine fetus. Also outlined are methods for studying hypoxia in the ovine fetus through embolization of the placenta, and occlusion of the umbi...
Source: Springer protocols feed by Neuroscience - September 21, 2015 Category: Neuroscience Source Type: news

Models of Perinatal Brain Injury in Premature and Term Newborns Resulting from Gestational Inflammation Due to Inactivated Group B Streptococcus (GBS), or Lipopolysaccharide (LPS) from E. coli and/or Immediately Postnatal Hypoxia-Ischemia (HI)
It is known that gestational and/or perinatal inflammation combined or not with hypoxia-ischemia (HI) is a risk factor for brain injuries, but the mechanisms underlying are still unclear. This chapter discusses about animal models mimicking those conditions, allowing scientists to uncover mechanisms involved and to study the adverse effects on the offspring. Here is presented a model of maternal inflammation induced by inactivated Group B Streptococcus (Sect. 2) and two experimental designs using LPS. One explores the effects of prenatal LPS administration and/or immediately postnatal HI (Sect. 3) and the second one, the i...
Source: Springer protocols feed by Neuroscience - September 21, 2015 Category: Neuroscience Source Type: news

Preterm Rabbit Model of Glycerol-Induced Intraventricular Hemorrhage
Intraventricular hemorrhage (IVH) remains a major complication of prematurity. We have developed a model of IVH in preterm rabbit pups. We deliver rabbit pups prematurely by C-section at 29-day gestational age (term = 32 days) and inject intraperitoneal glycerol at 3 h age to induce IVH. About 80 % of glycerol-treated pups develop IVH within 8 h age, which can be detected accurately with head ultrasound. These pups are reared in an infant incubator and gavage fed. At postnatal day 14, they exhibit hypomyelination, gliosis, and impaired neurological function. Hence, this is a novel animal model of IVH that can...
Source: Springer protocols feed by Neuroscience - September 21, 2015 Category: Neuroscience Source Type: news

Perinatal Intracerebral Hemorrhage Model and Developmental Disability
Perinatal intracerebral hemorrhage, also known as germinal matrix hemorrhage (GMH), refers to the bleeding that arises from the sub-ependymal (or periventricular) germinal region of the immature brain. Intraventricular hemorrhage (IVH) refers to the bleeding that extends into the ventricles, usually as an extension of GMH. Clinical studies have shown that infants who experience GMH/IVH may develop hydrocephalus or suffer from long-term neurological dysfunctions, including cerebral palsy, seizures, and learning disabilities. Understanding the pathogenesis of subsequent brain damage is important for the prevention and manage...
Source: Springer protocols feed by Neuroscience - September 21, 2015 Category: Neuroscience Source Type: news

Bilateral Uterine Artery Ligation (BUAL): Placental Insufficiency Causing Fetal Growth Restriction and Cerebral Palsy
Placental insufficiency is the leading cause of intrauterine growth restriction in the western world. The fetus, when exposed to a compromised environment, is vulnerable to a number of disorders later in life, as a consequence of the reduction in oxygen and nutrition during gestation and the resulting fetal growth restriction. These conditions include neurological disabilities such as cerebral palsy (CP), intellectual disability, epilepsy, and mental health issues in childhood (Autism and ADHD) and in later life (schizophrenia). Certainly, fetal growth restriction as a result of placental insufficiency has been strongly as...
Source: Springer protocols feed by Neuroscience - September 21, 2015 Category: Neuroscience Source Type: news