Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials.
During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other.
To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.
26 Related JoVE Articles!
Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities
Institutions: University of Iowa Carver College of Medicine, University of Bath.
Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using styryl FM dyes, which reveal membrane turnover. Conventional protocols for the use of FM dyes were designed for analyzing neurons following stimulated (evoked) synaptic activity. Recently, protocols have become available for analyzing the FM signals that accompany weaker synaptic activities, such as spontaneous or miniature synaptic events. Analysis of these small changes in FM signals requires that the imaging system is sufficiently sensitive to detect small changes in intensity, yet that artifactual changes of large amplitude are suppressed. Here we describe a protocol that can be applied to evoked, spontaneous, and miniature synaptic activities, and use cultured hippocampal neurons as an example. This protocol also incorporates a means of assessing the rate of photobleaching of FM dyes, as this is a significant source of artifacts when imaging small changes in intensity.
Neuroscience, Issue 85, Presynaptic Terminals, Synaptic Vesicles, Microscopy, Biological Assay, Nervous System, Endocytosis, exocytosis, fluorescence imaging, FM dye, neuron, photobleaching
A Guided Materials Screening Approach for Developing Quantitative Sol-gel Derived Protein Microarrays
Institutions: McMaster University .
Microarrays have found use in the development of high-throughput assays for new materials and discovery of small-molecule drug leads. Herein we describe a guided material screening approach to identify sol-gel based materials that are suitable for producing three-dimensional protein microarrays. The approach first identifies materials that can be printed as microarrays, narrows down the number of materials by identifying those that are compatible with a given enzyme assay, and then hones in on optimal materials based on retention of maximum enzyme activity. This approach is applied to develop microarrays suitable for two different enzyme assays, one using acetylcholinesterase and the other using a set of four key kinases involved in cancer. In each case, it was possible to produce microarrays that could be used for quantitative small-molecule screening assays and production of dose-dependent inhibitor response curves. Importantly, the ability to screen many materials produced information on the types of materials that best suited both microarray production and retention of enzyme activity. The materials data provide insight into basic material requirements necessary for tailoring optimal, high-density sol-gel derived microarrays.
Chemistry, Issue 78, Biochemistry, Chemical Engineering, Molecular Biology, Genetics, Bioengineering, Biomedical Engineering, Chemical Biology, Biocompatible Materials, Siloxanes, Enzymes, Immobilized, chemical analysis techniques, chemistry (general), materials (general), spectroscopic analysis (chemistry), polymer matrix composites, testing of materials (composite materials), Sol-gel, microarray, high-throughput screening, acetylcholinesterase, kinase, drug discovery, assay
Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography
Institutions: Baylor College of Medicine, Baylor College of Medicine, University of California at San Diego, Baylor College of Medicine.
Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the structure of neurites is critical to understanding how these processes move materials and signals that support synaptic communication. Electron microscopy (EM) has been traditionally used to assess the ultrastructural features within neurites; however, the exposure to organic solvent during dehydration and resin embedding can distort structures. An important unmet goal is the formulation of procedures that allow for structural evaluations not impacted by such artifacts.
Here, we have established a detailed and reproducible protocol for growing and flash-freezing whole neurites of different primary neurons on electron microscopy grids followed by their examination with cryo-electron tomography (cryo-ET). This technique allows for 3-D visualization of frozen, hydrated neurites at nanometer resolution, facilitating assessment of their morphological differences. Our protocol yields an unprecedented view of dorsal root ganglion (DRG) neurites, and a visualization of hippocampal neurites in their near-native state. As such, these methods create a foundation for future studies on neurites of both normal neurons and those impacted by neurological disorders.
Neuroscience, Issue 84, Neurons, Cryo-electron Microscopy, Electron Microscope Tomography, Brain, rat, primary neuron culture, morphological assay
Methods for the Modulation and Analysis of NF-κB-dependent Adult Neurogenesis
Institutions: University of Bielefeld, University of Bielefeld.
The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-κB plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-κB activity can be used.
In this study, methods are described for the analysis of NF-κB-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.
Neuroscience, Issue 84, NF-κB, hippocampus, Adult neurogenesis, spatial pattern separation-Barnes maze, dentate gyrus, p65 knock-out mice
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates
Institutions: University of California Riverside, University of California Riverside, University of California Riverside.
Close to two decades of research has established that astrocytes in situ
and in vivo
express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+
indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+
events using confocal microscopy. In essence, a “calcium roadmap” is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.
Neuroscience, Issue 85, astrocyte, plasticity, mGluRs, neuronal Firing, electrophysiology, Gq GPCRs, Bolus-loading, calcium, microdomains, acute slices, Hippocampus, mouse
Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons
Institutions: Charité Universitätmedizin.
GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.
Neuroscience, Issue 91, electrophysiology, acute slice, whole-cell patch-clamp recording, neuronal morphology, immunocytochemistry, parvalbumin, hippocampus, inhibition, GABAergic interneurons, synaptic transmission, IPSC, GABA-B receptor
Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue
Institutions: CNRS UMR 7241, INSERM U1050, Collège de France, Paris Descartes University, Sorbonne Paris Cité, CEA, Paris Descartes University, Paris Descartes University, La Pitié-Salpêtrière Hospital, AP-HP, Sorbonne and Pierre and Marie Curie University.
Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo
Medicine, Issue 92, electrophysiology, multi-electrode array, human tissue, slice, epilepsy, neocortex
Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons
Institutions: University of California, San Diego, Shanghai Jiao Tong University, University of California, San Diego, VA San Diego Healthcare System.
BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are increasingly viewed as an early feature of neurodegenerative diseases, including Alzheimer’s disease (AD) and Huntington’s disease (HD). As yet unclear is the mechanism(s) by which axonal injury is induced. We reported the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. Quantum dot-labeled BDNF (QD-BDNF) was produced by conjugating quantum dot 655 to mBtBDNF. A microfluidic device was used to isolate axons from neuron cell bodies. Addition of QD-BDNF to the axonal compartment allowed live imaging of BDNF transport in axons. We demonstrated that QD-BDNF moved essentially exclusively retrogradely, with very few pauses, at a moving velocity of around 1.06 μm/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders.
Neuroscience, Issue 91, live imaging, brain-derived neurotrophic factor (BDNF), quantum dot, trafficking, axonal retrograde transport, microfluidic chamber
Paired Whole Cell Recordings in Organotypic Hippocampal Slices
Institutions: University of Auckland, Stanford University.
Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.
Neuroscience, Issue 91, hippocampus, paired recording, whole cell recording, organotypic slice, synapse, synaptic transmission, synaptic plasticity
Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis
Institutions: The Scripps Research Institute, University of California San Diego, University of California San Diego, University of California San Diego School of Medicine.
Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative analysis of motor/cargo associations at the single vesicle level. The goal of this protocol is to use quantitative fluorescence microscopy to correlate (“map”) the position and directionality of movement of live cargo to the composition and relative amounts of motors associated with the same cargo. “Cargo mapping” consists of live imaging of fluorescently labeled cargoes moving in axons cultured on microfluidic devices, followed by chemical fixation during recording of live movement, and subsequent immunofluorescence (IF) staining of the exact same axonal regions with antibodies against motors. Colocalization between cargoes and their associated motors is assessed by assigning sub-pixel position coordinates to motor and cargo channels, by fitting Gaussian functions to the diffraction-limited point spread functions representing individual fluorescent point sources. Fixed cargo and motor images are subsequently superimposed to plots of cargo movement, to “map” them to their tracked trajectories. The strength of this protocol is the combination of live and IF data to record both the transport of vesicular cargoes in live cells and to determine the motors associated to these exact same vesicles. This technique overcomes previous challenges that use biochemical methods to determine the average motor composition of purified heterogeneous bulk vesicle populations, as these methods do not reveal compositions on single moving cargoes. Furthermore, this protocol can be adapted for the analysis of other transport and/or trafficking pathways in other cell types to correlate the movement of individual intracellular structures with their protein composition. Limitations of this protocol are the relatively low throughput due to low transfection efficiencies of cultured primary neurons and a limited field of view available for high-resolution imaging. Future applications could include methods to increase the number of neurons expressing fluorescently labeled cargoes.
Neuroscience, Issue 92, kinesin, dynein, single vesicle, axonal transport, microfluidic devices, primary hippocampal neurons, quantitative fluorescence microscopy
Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)
Institutions: University of Wuerzburg, Max Planck Institute of Neurobiology, Martinsried, Ludwig-Maximilians University of Munich.
Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+
indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+
indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro
. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+
indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+
indicator and a hydrophilic fluorescent dye/Ca2+
complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0
Cellular Biology, Issue 75, Neurobiology, Neuroscience, Molecular Biology, Biochemistry, Biomedical Engineering, Bioengineering, Virology, Medicine, Anatomy, Physiology, Surgery, Endoplasmic Reticulum, ER, Calcium Signaling, calcium store, calcium imaging, calcium indicator, metabotropic signaling, Ca2+, neurons, cells, mouse, animal model, cell culture, targeted esterase induced dye loading, imaging
Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices
Institutions: Collège de France, Paris Diderot University.
Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum
astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.
Neuroscience, Issue 69, Physiology, Anatomy, Medicine, hippocampus preparation, acute brain slice, electrophysiology, patch-clamp, neurons, astrocytes, astroglial, neuroglial interactions, glutamate transporter current, potassium current, paired recordings, synaptic activity, synaptically-evoked responses
Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol
Institutions: LSU Health Sciences Center - New Orleans, Medical School and Stanley S. Scott Cancer Center.
We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.
Neuroscience, Issue 63, Cell culture, neuron, cortex, rat, brain, isolation
Sexual Development and Ascospore Discharge in Fusarium graminearum
Institutions: Michigan State University, Michigan State University, Michigan State University, Michigan State University.
has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum
most easily produces fruiting bodies, called perithecia, on carrot agar. Perithecia contain numerous tissue types, produced at specific stages of perithecium development. These include (in order of appearance) formation of the perithecium initials (which give rise to the ascogenous hyphae), the outer wall, paraphyses (sterile mycelia which occupy the center of the perithecium until the asci develop), the asci, and the ascospores within the asci14
. The development of each of these tissues is separated by approximately 24 hours and has been the basis of transcriptomic studies during sexual development12,8
. Refer to Hallen et al.
(2007) for a more thorough description of development, including photographs of each stage. Here, we present the methods for generating and harvesting synchronously developing lawns of perithecia for temporal studies of gene regulation, development, and physiological processes. Although these methods are written specifically to be used with F. graminearum
, the techniques can be used for a variety of other fungi, provided that fruiting can be induced in culture and there is some synchrony to development. We have recently adapted this protocol to study the sexual development of F. verticillioides
. Although individual perithecia must be hand picked in this species, because a lawn of developing perithecia could not be induced, the process worked well for studying development (Sikhakolli and Trail, unpublished).
The most important function of fungal fruiting bodies is the dispersal of spores. In many of the species of Ascomycota (ascus producing fungi), spores are shot from the ascus, due to the generation of turgor pressure within the ascus, driving ejection of spores (and epiplasmic fluid) through the pore in the ascus tip2,7
. Our studies of forcible ascospore discharge have resulted in development of a "spore discharge assay", which we use to screen for mutations in the process. Here we present the details of this assay.
is homothallic, and thus can form fruiting bodies in the absence of a compatible partner. The advantage of homothallism is that crossing is not necessary to generate offspring homozygous for a particular trait, a facet that has facilitated the study of sexual development in this species14,7
. However, heterothallic strains have been generated that can be used for crossing5,9
. It is also possible to cross homothallic strains to obtain mutants for several genes in one strain1
. This is done by coinoculating one Petri dish with 2 strains. Along the meeting point, the majority of perithecia will be recombinant (provided a mutation in one of the parent strains does not inhibit outcrossing). As perithecia age, they exude ascospores en masse instead of forcibly discharging them. The resulting spore exudate (called a cirrhus) sits at the tip of the perithecium and can easily be removed for recovery of individual spores. Here we present a protocol to facilitate the identification of recombinant perithecia and the recovery of recombinant progeny.
Plant Biology, Issue 61, Ascospores, perithecia, forcible discharge, mycotoxin, conidia, development
Isolation and Culture of Hippocampal Neurons from Prenatal Mice
Institutions: Auburn University .
Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.
Neuroscience, Issue 65, Physiology, Medicine, Brain, Cell Culture, Hippocampal Neurons
Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia
Institutions: Drexel University College of Medicine.
This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox
), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo
. Furthermore, the co-culture can be used to study neuronal-glial interactions1
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3
. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4
, cellular and molecular biology5-8
, imaging and microscopy4,6,7,9,10
. The primary neurons extend axons and dendrites to form functional synapses11
, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13
. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106
cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.
Neuroscience, Issue 57, neuron, rat, brain, co-culture, cortex, glia, glial cells
Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro
Institutions: The University of Chicago Medical Center, The University of Chicago Medical Center.
Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro
using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro
preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers.
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo
counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure
neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic
SD immune signaling.
Neuroscience, Issue 52, innate immunity, hormesis, microglia, T-cells, hippocampus, slice culture, gene expression, laser dissection microscopy, real-time qPCR, interferon-gamma
Strategies for Study of Neuroprotection from Cold-preconditioning
Institutions: The University of Chicago Medical Center.
Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by development of effective, easy to administer and safe preconditioning treatments. We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. Low-level pro-inflammatory mediator signaling changes over time are essential for cold-preconditioning neuroprotection. This signaling is consistent with the basic tenets of physiological conditioning hormesis, which require that irritative stimuli reach a threshold magnitude with sufficient time for adaptation to the stimuli for protection to become evident.
Accordingly, delineation of the immune signaling involved in cold-preconditioning neuroprotection requires that biological systems and experimental manipulations plus technical capacities are highly reproducible and sensitive. Our approach is to use hippocampal slice cultures as an in vitro
model that closely reflects their in vivo
counterparts with multi-synaptic neural networks influenced by mature and quiescent macroglia / microglia. This glial state is particularly important for microglia since they are the principal source of cytokines, which are operative in the femtomolar range. Also, slice cultures can be maintained in vitro
for several weeks, which is sufficient time to evoke activating stimuli and assess adaptive responses. Finally, environmental conditions can be accurately controlled using slice cultures so that cytokine signaling of cold-preconditioning can be measured, mimicked, and modulated to dissect the critical node aspects. Cytokine signaling system analyses require the use of sensitive and reproducible multiplexed techniques. We use quantitative PCR for TNF-α to screen for microglial activation followed by quantitative real-time qPCR array screening to assess tissue-wide cytokine changes. The latter is a most sensitive and reproducible means to measure multiple cytokine system signaling changes simultaneously. Significant changes are confirmed with targeted qPCR and then protein detection. We probe for tissue-based cytokine protein changes using multiplexed microsphere flow cytometric assays using Luminex technology. Cell-specific cytokine production is determined with double-label immunohistochemistry. Taken together, this brain tissue preparation and style of use, coupled to the suggested investigative strategies, may be an optimal approach for identifying potential targets for the development of novel therapeutics that could mimic the advantages of cold-preconditioning.
Neuroscience, Issue 43, innate immunity, hormesis, microglia, hippocampus, slice culture, immunohistochemistry, neural-immune, gene expression, real-time PCR
Gene-gun Transfection of Hippocampal Neurons
Institutions: Brown University.
Neuroscience, Issue 1, brain, hippocampus, neuron, transfection, gene-gun
BioMEMS: Forging New Collaborations Between Biologists and Engineers
Institutions: University of California, Irvine (UCI).
This video describes the fabrication and use of a microfluidic device to culture central nervous system (CNS) neurons. This device is compatible with live-cell optical microscopy (DIC and phase contrast), as well as confocal and two photon microscopy approaches. This method uses precision-molded polymer parts to create miniature multi-compartment cell culture with fluidic isolation. The compartments are made of tiny channels with dimensions that are large enough to culture neurons in well-controlled fluidic microenvironments. Neurons can be cultured for 2-3 weeks within the device, after which they can be fixed and stained for immunocytochemistry. Axonal and somal compartments can be maintained fluidically isolated from each other by using a small hydrostatic pressure difference; this feature can be used to localize soluble insults to one compartment for up to 20 h after each medium change. Fluidic isolation enables collection of pure axonal fraction and biochemical analysis by PCR. The microfluidic device provides a highly adaptable platform for neuroscience research and may find applications in modeling CNS injury and neurodegeneration.
Neuroscience, Issue 9, Microfluidics, Bioengineering, Neuron
Primary Culture of Hippocampal Neurons from P0 Newborn Rats
Institutions: Michigan State University (MSU).
The physiological properties of hippocampal neurons are commonly investigated, especially because of the involvement of the hippocampus in learning and memory. Primary hippocampal cell culturing allows neuroscientists to examine the activity and properties of neurons at the individual cell and single synapse level. In this video, we will demonstrate how to isolate and grow primary hippocampal cells from newborn rats. The hippocampus may be isolated from each newborn animal in as short as 2 to 3 minutes, and the cultures can be maintained for up to two weeks. We will also briefly demonstrate how to use these hippocampal neurons for ratiometric calcium imaging. While this protocol describes the process for the hippocampus, with little to no modification, it can be applied to other regions of the brain.
Neuroscience, issue 19, brain, neurons, hippocampus, mouse
The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury
Institutions: Stanford University School of Medicine.
Organotypic hippocampal slice culture is an in vitro
method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1
. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo
preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.
Neuroscience, Issue 44, Organotypic slice culture, excitotoxicity, NMDA
Organotypic Hippocampal Slice Cultures
Institutions: University of Washington School of Medicine.
The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1
. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2
. In addition, the well defined anatomy and connectivity of the hippocampus 3
has made it a classical model system to study synaptic transmission and synaptic plasticity4
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6
or biolistics 7
. In addition, slices can be easily recovered for biochemical assays 8
, or transferred to microscopes for imaging 9
or electrophysiological experiments 10
Neuroscience, Issue 48, Hippocampus, Hippocampal formation, Brain Slices, Organotypic Cultures, Synaptic Transmission, Synaptic Physiology
Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons
Institutions: National Institutes of Health, Bethesda.
FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green
fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.
Neuroscience, Issue 50, Spine, FRAP, hippocampal neurons, live cell imaging, protein mobility
Propagation of Human Embryonic Stem (ES) Cells
Institutions: MGH - Massachusetts General Hospital.
Cellular Biology, Issue 1, ES, embryonic stem cells, tissue culture