The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with specific functions (e.g. carbon sequestration, soil quality improvements, or contaminant sorption). In 2013, the International Biochar Initiative (IBI) made publically available their Standardized Product Definition and Product Testing Guidelines (Version 1.1) which set standards for physical and chemical characteristics for biochar. Six biochars made from three different feedstocks and at two temperatures were analyzed for characteristics related to their use as a soil amendment. The protocol describes analyses of the feedstocks and biochars and includes: cation exchange capacity (CEC), specific surface area (SSA), organic carbon (OC) and moisture percentage, pH, particle size distribution, and proximate and ultimate analysis. Also described in the protocol are the analyses of the feedstocks and biochars for contaminants including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and mercury as well as nutrients (phosphorous, nitrite and nitrate and ammonium as nitrogen). The protocol also includes the biological testing procedures, earthworm avoidance and germination assays. Based on the quality assurance / quality control (QA/QC) results of blanks, duplicates, standards and reference materials, all methods were determined adequate for use with biochar and feedstock materials. All biochars and feedstocks were well within the criterion set by the IBI and there were little differences among biochars, except in the case of the biochar produced from construction waste materials. This biochar (referred to as Old biochar) was determined to have elevated levels of arsenic, chromium, copper, and lead, and failed the earthworm avoidance and germination assays. Based on these results, Old biochar would not be appropriate for use as a soil amendment for carbon sequestration, substrate quality improvements or remediation.
26 Related JoVE Articles!
Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue
Institutions: College of William and Mary.
The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16
. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and Müller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23
. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33
. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39
. Xenopus laevis,
a classic model system for the study of early neural development 19,27,29,31-32,40-42
, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43
. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis
that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1
Developmental Biology, Issue 70, Neuroscience, Cellular Biology, Surgery, Anatomy, Physiology, Ophthalmology, retina, primary cell culture, dissection, confocal microscopy, calcium imaging, fluorescent in situ hybridization, FISH, Xenopus laevis, animal model
Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica
Institutions: Case Western Reserve University , Case Western Reserve University , Case Western Reserve University .
In animals with large identified neurons (e.g.
mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4
. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5
. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5
, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia
) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7
. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g.
in the suspended buccal mass preparation8
or in vivo9
. This process can also be applied in other motor pools10,11,12
or in other animal systems2,3,13,14
Neuroscience, Issue 73, Physiology, Biomedical Engineering, Anatomy, Behavior, Neurobiology, Animal, Neurosciences, Neurophysiology, Electrophysiology, Aplysia, Aplysia californica, California sea slug, invertebrate, feeding, buccal mass, ganglia, motor neurons, neurons, extracellular stimulation and recordings, extracellular electrodes, animal model
Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells
Institutions: University of Sydney , University of Western Sydney, University of Sydney .
Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3
and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.
Neuroscience, Issue 75, Neurobiology, Biomedical Engineering, Anatomy, Physiology, Molecular Biology, Cellular Biology, Neurons, Retinal Neurons, Retinal Ganglion Cells, Eye, Retina, Neurosciences, retina, ganglion cells, synaptic conductance, artificial conductance, tetrodotoxin (TTX), patch clamp, dynamic clamp, conductance clamp, electrophysiology, mouse, animal model
Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents
Institutions: University of Miami.
The marine gastropod mollusk Aplysia californica
has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.
Neuroscience, Issue 77, Neurobiology, Anatomy, Physiology, Cellular Biology, Molecular Biology, Environmental Sciences, Marine Biology, Receptors, Neurophysiology, Neurotransmitter, Neurotransmitter Agents, Patch Clamp Recordings, Primary Cell Culture, Electrophysiology, L-Glutamate, NMDA, D-Aspartate, dissection, ganglia, buccal ganglion, neurons, invertebrate, Aplysia californica, california sea slug, mollusk, animal model
Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes
Institutions: University of Massachusetts, Amherst, University of Massachusetts, Amherst, University of Massachusetts, Amherst.
RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans
. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans
genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans
Developmental Biology, Issue 79, Caenorhabditis elegans (C. elegans), Gene Knockdown Techniques, C. elegans, dsRNA interference, gene knockdown, large scale feeding screen
Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons
Institutions: The Scripps Research Institute, Florida.
A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica
is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia
have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia
nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia
Neurobiology, Issue 83, intracellular recording, identified neuron, neural circuitry, gene expression, action potential, CREB, Aplysia californica, genomics
Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons
Institutions: VU University Amsterdam.
The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive functions including decision making, sensory-guided behavior or memory. To understand how such intricate neuronal networks perform such tasks, a crucial step is to determine the function (or electrical activity) of individual cell types within the network, preferentially when the animal is performing a relevant cognitive task. Additionally, it is equally important to determine the anatomical structure of the network and the morphological architecture of the individual neurons to allow reverse engineering the cortical network. Technical breakthroughs available today allow recording cellular activity in awake, behaving animals with the valuable option of post hoc
identifying the recorded neurons. Here, we demonstrate the juxtasomal biocytin labeling technique, which involves recording action potential spiking in the extracellular (or loose-patch) configuration using conventional patch pipettes. The juxtasomal recording configuration is relatively stable and applicable across behavioral conditions, including anesthetized, sedated, awake head-fixed, and even in the freely moving animal. Thus, this method allows linking cell-type specific action potential spiking during animal behavior to reconstruction of the individual neurons and ultimately, the entire cortical microcircuit. In this video manuscript, we show how individual neurons in the juxtasomal configuration can be labeled with biocytin in the urethane-anaesthetized rat for post hoc
identification and morphological reconstruction.
Bioengineering, Issue 84, biocytin, juxtasomal, morphology, physiology, action potential, structure-function, histology, reconstruction, neurons, electrophysiological recordings
Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees
Institutions: University of Würzburg.
In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1
, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2
connecting the AL with the MB. The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi channel wire electrodes is demonstrated. Pairwise differential amplification of the micro wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long term recordings over many hours up to days, which is a clear advantage compared to conventional extra and intracellular in vivo
Neuroscience, Issue 89, honeybee brain, olfaction, extracellular long term recordings, double recordings, differential wire electrodes, single unit, multi-unit recordings
Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology
Institutions: University of Pittsburgh School of Medicine, University of Pittsburgh School of Medicine.
We describe an in vitro
method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo
experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e.
smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo
. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro
smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.
Medicine, Issue 90, Krebs, species differences, in vitro, smooth muscle contractility, neural stimulation
The Swimmeret System of Crayfish: A Practical Guide for the Dissection of the Nerve Cord and Extracellular Recordings of the Motor Pattern
Institutions: University of Cologne.
Here we demonstrate the dissection of the crayfish abdominal nerve cord. The preparation comprises the last two thoracic ganglia (T4, T5) and the chain of abdominal ganglia (A1 to A6). This chain of ganglia includes the part of the central nervous system (CNS) that drives coordinated locomotion of the pleopods (swimmerets): the swimmeret system. It is known for over five decades that in crayfish each swimmeret is driven by its own independent pattern generating kernel that generates rhythmic alternating activity 1-3
. The motor neurons innervating the musculature of each swimmeret comprise two anatomically and functionally distinct populations 4
. One is responsible for the retraction (power stroke, PS) of the swimmeret. The other drives the protraction (return stroke, RS) of the swimmeret. Motor neurons of the swimmeret system are able to produce spontaneously a fictive motor pattern, which is identical to the pattern recorded in vivo 1
The aim of this report is to introduce an interesting and convenient model system for studying rhythm generating networks and coordination of independent microcircuits for students’ practical laboratory courses. The protocol provided includes step-by-step instructions for the dissection of the crayfish’s abdominal nerve cord, pinning of the isolated chain of ganglia, desheathing the ganglia and recording the swimmerets fictive motor pattern extracellularly from the isolated nervous system.
Additionally, we can monitor the activity of swimmeret neurons recorded intracellularly from dendrites. Here we also describe briefly these techniques and provide some examples. Furthermore, the morphology of swimmeret neurons can be assessed using various staining techniques. Here we provide examples of intracellular (by iontophoresis) dye filled neurons and backfills of pools of swimmeret motor neurons. In our lab we use this preparation to study basic functions of fictive locomotion, the effect of sensory feedback on the activity of the CNS, and coordination between microcircuits on a cellular level.
Neurobiology, Issue 93, crustacean, dissection, extracellular recording, fictive locomotion, motor neurons, locomotion
Appetitive Associative Olfactory Learning in Drosophila Larvae
Institutions: University of Konstanz, University of Fribourg.
In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila
larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative
learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4
. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6
. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila
larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10
larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14
. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9
. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9
Neuroscience, Issue 72, Developmental Biology, Neurobiology, Biochemistry, Molecular Biology, Physiology, Behavior, Drosophila, fruit fly, larvae, instar, olfaction, olfactory system, odor, 1-octanol, OCT, learning, reward, sugar, feeding, animal model
An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica
Institutions: Case Western Reserve University , Case Western Reserve University , Case Western Reserve University .
Multifunctionality, the ability of one peripheral structure to generate multiple, distinct behaviors1
, allows animals to rapidly adapt their behaviors to changing environments. The marine mollusk Aplysia californica
provides a tractable system for the study of multifunctionality. During feeding, Aplysia
generates several distinct types of behaviors using the same feeding apparatus, the buccal mass. The ganglia that control these behaviors contain a number of large, identified neurons that are accessible to electrophysiological study. The activity of these neurons has been described in motor programs that can be divided into two types, ingestive and egestive programs, based on the timing of neural activity that closes the food grasper relative to the neural activity that protracts or retracts the grasper2
. However, in isolated ganglia, the muscle movements that would produce these behaviors are absent, making it harder to be certain whether the motor programs observed are correlates of real behaviors. In vivo
, nerve and muscle recordings have been obtained corresponding to feeding programs2,3,4
, but it is very difficult to directly record from individual neurons5
. Additionally, in vivo
, ingestive programs can be further divided into bites and swallows1,2
, a distinction that is difficult to make in most previously described in vitro
The suspended buccal mass preparation (Figure 1
) bridges the gap between isolated ganglia and intact animals. In this preparation, ingestive behaviors - including both biting and swallowing - and egestive behaviors (rejection) can be elicited, at the same time as individual neurons can be recorded from and stimulated using extracellular electrodes6
. The feeding movements associated with these different behaviors can be recorded, quantified, and related directly to the motor programs. The motor programs in the suspended buccal mass preparation appear to be more similar to those observed in vivo
than are motor programs elicited in isolated ganglia. Thus, the motor programs in this preparation can be more directly related to in vivo
behavior; at the same time, individual neurons are more accessible to recording and stimulation than in intact animals. Additionally, as an intermediate step between isolated ganglia and intact animals, findings from the suspended buccal mass can aid in interpretation of data obtained in both more reduced and more intact settings. The suspended buccal mass preparation is a useful tool for characterizing the neural control of multifunctionality in Aplysia
Neuroscience, Issue 70, Physiology, Biomedical Engineering, Anatomy, Marine Biology, Aplysia, Aplysia californica, California sea slug, invertebrate, feeding, neurobiology, buccal mass, semi-intact preparation, extracellular electrodes, extracellular recording, neurons, animal model
Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution
Institutions: The University of Texas at Austin.
Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1
. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2
. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (>97% spike detection efficiency), has a low rate of false positive spike detection (< 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3
. This optical method of spike detection can be used to record neural activity in vitro
and in anesthetized animals in vivo3,4
Neuroscience, Issue 67, functional calcium imaging, spatiotemporal patterns of activity, dithered random-access scanning
Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil
Institutions: Oxitec Ltd, Universidade de São Paulo, Universidade de São Paulo, Moscamed Brasil, University of Oxford, Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM).
New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the development of new and innovative methods of mosquito control based around the Sterile Insect Technique (SIT)1-3
. A control method known as RIDL (Release of Insects carrying a Dominant Lethal)4
, is based around SIT, but uses genetic methods to remove the need for radiation-sterilization5-8
. A RIDL strain of Ae. aegypti
was successfully tested in the field in Grand Cayman9,10
; further field use is planned or in progress in other countries around the world.
Mass rearing of insects has been established in several insect species and to levels of billions a week. However, in mosquitoes, rearing has generally been performed on a much smaller scale, with most large scale rearing being performed in the 1970s and 80s. For a RIDL program it is desirable to release as few females as possible as they bite and transmit disease. In a mass rearing program there are several stages to produce the males to be released: egg production, rearing eggs until pupation, and then sorting males from females before release. These males are then used for a RIDL control program, released as either pupae or adults11,12
To suppress a mosquito population using RIDL a large number of high quality male adults need to be reared13,14
. The following describes the methods for the mass rearing of OX513A, a RIDL strain of Ae. aegypti 8,
for release and covers the techniques required for the production of eggs and mass rearing RIDL males for a control program.
Basic Protocol, Issue 83, Aedes aegypti, mass rearing, population suppression, transgenic, insect, mosquito, dengue
Propagation of Human Embryonic Stem (ES) Cells
Institutions: MGH - Massachusetts General Hospital.
Cellular Biology, Issue 1, ES, embryonic stem cells, tissue culture
Ole Isacson: Development of New Therapies for Parkinson's Disease
Institutions: Harvard Medical School.
Medicine, Issue 3, Parkinson' disease, Neuroscience, dopamine, neuron, L-DOPA, stem cell, transplantation
Preparation of Aplysia Sensory-motor Neuronal Cell Cultures
Institutions: University of California, Los Angeles, University of California, Los Angeles, University of California, Los Angeles.
The nervous system of the marine mollusk Aplysia californica
is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1
. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3
. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia
culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5
. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia
, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.
Neuroscience, Issue 28, Aplysia Californica, Synaptic Plasticity, Sensory Motor Neuronal Cultures, Invertebrates, Short-Term Facilitation, Monosynaptic, Intermediate-Term Facilitation, Ganglia, Long-Term Depression, Autapses, Sirnas, Glutamatergic Synapses, Somata
Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals
Institutions: Case Western Reserve University, Case Western Reserve University, Case Western Reserve University.
Recording from key nerves and muscles of Aplysia
during feeding behavior allows us to study the patterns of neural control in an intact animal. Simultaneously recording from multiple nerves and muscles gives us precise information about the timing of neural activity. Previous recording methods have worked for two electrodes, but the study of additional nerves or muscles required combining and averaging the recordings of multiple animals, which made it difficult to determine fine details of timing and phasing, because of variability from response to response, and from animal to animal. Implanting four individual electrodes has a very low success rate due to the formation of adhesions that prevent animals from performing normal feeding movements. We developed a new method of electrode fabrication that reduces the bulk of the electrodes inside the animal allowing for normal feeding movements. Using a combination of glues to attach the electrodes results in a more reliable insulation of the electrode which lasts longer, making it possible to record for periods as long as a week. The fabrication technique that we describe could be extended to incorporate several additional electrodes, and would be applicable to vertebrate animals.
Neuroscience, Issue 40, in vivo electrodes, Aplysia, neurobiology, chronic recording, extracellular recording
Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors
Institutions: School of Medicine, University of California, Davis.
Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (>80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV
1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.
Neurobiology, Issue 39, pluripotent stem cell, oligodendrocyte precursor cells, differentiation, myelin, neuroscience, brain
Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat
Institutions: University of Lethbridge.
Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of behaviors from the spiking activity of its neurons. One of the most effective methods to monitor spiking activity from a large number of neurons in multiple local neuronal circuits simultaneously is by using silicon electrode arrays1-3
Action potentials produce large transmembrane voltage changes in the vicinity of cell somata. These output signals can be measured by placing a conductor in close proximity of a neuron. If there are many active (spiking) neurons in the vicinity of the tip, the electrode records combined signal from all of them, where contribution of a single neuron is weighted by its 'electrical distance'. Silicon probes are ideal recording electrodes to monitor multiple neurons because of a large number of recording sites (+64) and a small volume. Furthermore, multiple sites can be arranged over a distance of millimeters, thus allowing for the simultaneous recordings of neuronal activity in the various cortical layers or in multiple cortical columns (Fig. 1). Importantly, the geometrically precise distribution of the recording sites also allows for the determination of the spatial relationship of the isolated single neurons4
. Here, we describe an acute, large-scale neuronal recording from the left and right forelimb somatosensory cortex simultaneously in an anesthetized rat with silicon probes (Fig. 2).
Neuroscience, Issue 56, neuronal ensembles, silicon probes, spiking, local field potentials, tetrode, acute recordings, rat
Using Affordable LED Arrays for Photo-Stimulation of Neurons
Institutions: Institut Pasteur and Centre National de la Recherche Scientifique (CNRS).
Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single cells in response to electrical or pharmacological manipulations1,2
. With the invention of methods to optically control genetically targeted neurons (optogenetics), researchers now have an unprecedented level of control over specific groups of neurons in the standard slice preparation. In particular, photosensitive channelrhodopsin-2 (ChR2) allows researchers to activate neurons with light3,4
. By combining careful calibration of LED-based photostimulation of ChR2 with standard slice electrophysiology, we are able to probe with greater detail the role of adult-born interneurons in the olfactory bulb, the first central relay of the olfactory system. Using viral expression of ChR2-YFP specifically in adult-born neurons, we can selectively control young adult-born neurons in a milieu of older and mature neurons. Our optical control uses a simple and inexpensive LED system, and we show how this system can be calibrated to understand how much light is needed to evoke spiking activity in single neurons. Hence, brief flashes of blue light can remotely control the firing pattern of ChR2-transduced newborn cells.
Neuroscience, Issue 57, Adult neurogenesis, Channelrhodopsin, Neural stem cells, Plasticity, Synapses, Electrophysiology
Designing and Implementing Nervous System Simulations on LEGO Robots
Institutions: Northeastern University, Bremen University of Applied Sciences.
We present a method to use the commercially available LEGO Mindstorms NXT robotics platform to test systems level neuroscience hypotheses. The first step of the method is to develop a nervous system simulation of specific reflexive behaviors of an appropriate model organism; here we use the American Lobster. Exteroceptive reflexes mediated by decussating (crossing) neural connections can explain an animal's taxis towards or away from a stimulus as described by Braitenberg and are particularly well suited for investigation using the NXT platform.1
The nervous system simulation is programmed using LabVIEW software on the LEGO Mindstorms platform. Once the nervous system is tuned properly, behavioral experiments are run on the robot and on the animal under identical environmental conditions. By controlling the sensory milieu experienced by the specimens, differences in behavioral outputs can be observed. These differences may point to specific deficiencies in the nervous system model and serve to inform the iteration of the model for the particular behavior under study. This method allows for the experimental manipulation of electronic nervous systems and serves as a way to explore neuroscience hypotheses specifically regarding the neurophysiological basis of simple innate reflexive behaviors. The LEGO Mindstorms NXT kit provides an affordable and efficient platform on which to test preliminary biomimetic robot control schemes. The approach is also well suited for the high school classroom to serve as the foundation for a hands-on inquiry-based biorobotics curriculum.
Neuroscience, Issue 75, Neurobiology, Bioengineering, Behavior, Mechanical Engineering, Computer Science, Marine Biology, Biomimetics, Marine Science, Neurosciences, Synthetic Biology, Robotics, robots, Modeling, models, Sensory Fusion, nervous system, Educational Tools, programming, software, lobster, Homarus americanus, animal model
Voltage Biasing, Cyclic Voltammetry, & Electrical Impedance Spectroscopy for Neural Interfaces
Institutions: Purdue University, University of Wisconsin-Madison, University of Michigan , Purdue University.
Electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measure properties of the electrode-tissue interface without additional invasive procedures, and can be used to monitor electrode performance over the long term. EIS measures electrical impedance at multiple frequencies, and increases in impedance indicate increased glial scar formation around the device, while cyclic voltammetry measures the charge carrying capacity of the electrode, and indicates how charge is transferred at different voltage levels. As implanted electrodes age, EIS and CV data change, and electrode sites that previously recorded spiking neurons often exhibit significantly lower efficacy for neural recording. The application of a brief voltage pulse to implanted electrode arrays, known as rejuvenation, can bring back spiking activity on otherwise silent electrode sites for a period of time. Rejuvenation alters EIS and CV, and can be monitored by these complementary methods. Typically, EIS is measured daily as an indication of the tissue response at the electrode site. If spikes are absent in a channel that previously had spikes, then CV is used to determine the charge carrying capacity of the electrode site, and rejuvenation can be applied to improve the interface efficacy. CV and EIS are then repeated to check the changes at the electrode-tissue interface, and neural recordings are collected. The overall goal of rejuvenation is to extend the functional lifetime of implanted arrays.
Neuroscience, Issue 60, neuroprosthesis, electrode-tissue interface, rejuvenation, neural engineering, neuroscience, neural implant, electrode, brain-computer interface, electrochemistry
A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo
Institutions: University of Colorado Boulder, University of Colorado Boulder.
Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g.
, channelrhodopsin-2, ChR2) or silence (e.g.
, halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2
transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e.
, pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo
preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.
Neuroscience, Issue 79, Genetic Techniques, Genetics, Behavioral, Biological Science Disciplines, Neurosciences, genetics (animal and plant), Investigative Techniques, Behavior and Behavior Mechanisms, Behavioral Disciplines and Activities, Natural Science Disciplines, Optogenetics, prefrontal cortex, subiculum, virus injection, in vivo recording, Neurophysiology, prelimbic, optrode, molecular neurogenetics, Gene targeting
Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia
Institutions: Mt. Sinai School of Medicine, Phase Five Communications Inc..
It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1
. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica
. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia
and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 °C) that are natural for Aplysia
, preparations are stable for long periods of time.
To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2
which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3
and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4
. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations.
An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia
this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.
Neuroscience, Issue 65, Molecular Biology, Marine Biology, calcium imaging, intracellular recording, invertebrate, mollusc, Aplysia, Calcium Orange, facilitation, synaptic plasticity
ES Cell-derived Neuroepithelial Cell Cultures
Institutions: Harvard Medical School.
ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).
Cellular Biology, issue 1, embryonic stem (ES) cells, rosettes, neuroepithelial precursors, stromal cells, differentiation