Name: generation of neurospheres from mixed primary hippocampal and cortical neurons isolated from e14-e16 sprague dawley rat embryo
Uploaded: 2019-08-31T13:00:00
Description: Watch this Scientific Journal Video about Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo at JoVE.com

We thank CSIR-IICB animal facility. G. D. thanks ICMR, J. K. and V. G. thank DST Inspire, and D. M. thanks DBT, India for their fellowships. S. G. kindly acknowledges SERB (EMR/2015/002230) India for providing financial support.

All experimental procedures involving animal were approved by the Institutional Animal Ethics Committee of CSIR-Indian Institute of Chemical Biology (IICB/AEC/Meeting/Apr/2018/1).
1. Reagent and media preparation
<ol>
	<li>Poly-D-lysine (PDL) solution: prepare PDL solutions at concentrations of 0.1 mg/mL in deionized water and store in 4 &#176;C until use.</li>
	<li>Dissociation medium: To 1 L of sterile, filtered deionized water, combine the following components in the respective concentrat...

<ol>
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This protocol describes that how by altering the&#160;cell plating densities of primary neurons, two variable neuronal platforms&#160;are obtained. Though this is a simple method, each step must be meticulously performed to achieve the desired results. Other previous methods have either reported long-term primary neuron cultures or neurosphere cultures. Most primary neuron culture protocols have involved the culturing of hippocampal neurons for 3-5 weeks, but most have failed, as the neurons die and wither away due to lo...

The brain is an intricate circuitry of neuronal and non-neuronal cells. For years, scientists have been trying to gain insight into this complex machinery. To do so, neuroscientists initially resorted to various transformed nerve-based cell lines for investigations. However, the inability of these clonal cell lines to form strong synaptic connections and proper axons or dendrites have shifted scientific interest to primary neuron cultures1,2. The most exciting aspect of primary neuron culture is that it creates an opportunity to observe and manipulate living neurons3. Moreover, it is less complex compared to neural tissue, which makes it an ideal candidate for studying the function and transport of various neuronal proteins. Recently, several developments in the fields of microscopy, genomics, and proteomics have generated new opportunities for neuroscientists to exploit neuron cultures4.
Primary cultures have allowed neuroscientists to explore the molecular mechanisms behind neural development, analyze various neural signaling pathways, and develop a more coherent understanding of synapsis. Though a number of methods have reported cultures from primary neurons (mostly from the hippocampal origin5,6,7), a unified protocol with a chemically defined medium that enables long-term culture of neurons is still needed. However, neurons plated at a low density are most often observed, which do not survive long-term, likely due to the lack of trophic support8 that is provided by the adjacent neurons and glial cells. Some methods have even suggested co-culturing of the primary neurons with glial cells, wherein the glial cells are used as a feeder layer9. However, glial cells pose a lot of problems due to their overgrowth, which sometimes override the neuronal growth10. Hence, considering the problems above, a simpler and more cost-effective primary neural culture protocol is required, which can be used by both neurobiologists and neurochemists for investigations.
A primary neuron culture is essentially a form of 2D culture and does not represent the plasticity, spatial integrity, or heterogeneity of the brain. This has given rise to the need for a more believable 3D model called neurospheres11,12.&#160;Neurospheres present a novel platform to neuroscientists, with a closer resemblance to the real, in vivo brain13. Neurospheres are non-adherent 3D clusters of cells that are rich in neural stem cells (NSCs), neural progenitor cells (NPCs), neurons, and astrocytes. They are an excellent source for the isolation of neural stem cells and neural progenitor cells, which can be used to study differentiation into various neuronal and non-neuronal lineages. Again, variability within neurosphere cultures produced using the previously reported protocols presents a barrier to the formulation of a unified neurosphere culture protocol14.
This manuscript presents a protocol in which it is possible to generate both 2D and 3D platforms by alternating cell plating densities from a mixed cortical and hippocampal culture. It is observed that within 7 days free-floating neurospheres are obtained from&#160;high-density plated neurons isolated from E14-E16 Sprague Dawley rat embryo, which upon further culture, form bridges and interconnections through radial glial-like extensions. Similarly, in the low density plated neurons, a primary neuron culture that can be maintained for up to 30 days is obtained by changing the maintenance medium twice per week.

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generation of neurospheres from mixed primary hippocampal and cortical neurons isolated from e14-e16 sprague dawley rat embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

In this protocol, a simple strategy has been elucidated in which variable cell plating densities from two different neural screening platforms are obtained. Figure 1A,B illustrates the adherence of cells after 4 h of plating the neurons in high and low density plated cells, respectively. On observing the proper adherence of the neurons as shown in Figure 1, the plating medium was replaced by maintenance medium in...

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Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

The overall goal of this procedure is to demonstrate the development of neurospheres from mixed primary hippocampal and cortical neurons isolated from E14-E16 Sprague Dawley rat embryo, in order to offer a robust and low-cost platform for studying the effect of various neurotherapeutic leads. As you know, that brain is a complex network of neurons associated with large number of supporting cells. To understand the brain function, often most of the research is re-initiated from in vitro experiments, which involves commercially available neural lineage derived cell lines.However, this cell lines are transformed cell lines that suffer from genotypic and phenotypic variations. To address this issues, primary neuron culture is the suitable approach. Here, we illustrated a simple and cost-effective protocol to culture primary neurons and built a robust screening platform for various neural therapeutics.The main advantage of this protocol is that just by modulating the cell plating densities, we can showcase two contrasting neuron culture platforms where low-density leads to prolonged primary neuron culture, and high-density generates spontaneous neurospheres. Take two 24-well plates. One for high-density, and another for low-density plating.Transfer 12 mm of sterile glass coverslips in the 24-well plates. Pour 300 microliters of PDL solution in each of the wells in a way that it fully covers the surface of the entire coverslip. Prepare PDL solution at a concentration of 0.1 mg/mL in deionized water.Wrap the plates with aluminum foils to prevent drying, and keep it in the CO2 incubator overnight. Next day, before plating, aspirate the PDL solution. Wash properly with sterile water for two to three times.Then add plating media, and return the plates to the incubator until plating. Sacrifice an E14-E16 timed pregnant Sprague-Dawley rat, Make a V-shaped cut in the abdominal area using sterile forceps and a pair of blunt-end scissors. Remove all the fetuses and carefully place them on the petri plate with cold HBSS solution.Take the embryos out of the embryonic sacs and wash them carefully in a separate petri dish in fresh, cold HBSS. Decapitate the head with the help of sterile scissors. Transfer the heads in the sterile 90 mm petri plates with cold HBSS.Under the stereo microscope, hold the head from the snout region with the sterile serrated forceps, and take out the brain by cutting the skin and the skull open. Remove all the meninges from the hemispheres and the midbrain by holding the brainstem. Carefully remove the intact hemispheres, resembling mushroom caps that contains the hippocampus and the cortex.Collect the hemispheres containing cortex and intact hippocampus in a 15 mL conical tube containing 10 mL of dissociation media. Allow the collected tissues to settle down, and aspirate the dissociation medium, leaving five to ten percent of medium in it. Again, add 10 mL of fresh dissociation medium to it, and repeat this step twice.Add 4.5 mL of dissociation medium and 0.5 mL of trypsin-EDTA solution. Keep it in the incubator at 37 degrees Celsius for 20 minutes for the digestion to proceed. Aspirate the medium and wash with 10 mL of dissociation medium and plating medium, consecutively.Resuspend them in 2.5 mL of plating medium. Keep a 90 mm sterile dish over the inverted cover of the dish and pour it in the base of 90 mm sterile dish. Titrate the digested tissues in the corner base of the dish, using 1000 microliter pipette tip, so as to occupy least volume.Pass the obtained cell suspension through the 70 micrometer cell strainer, excluding any chunks of tissue. Determine the density of viable cells using trypan blue dye exclusion, and count the number of cells in an automated cell counter. Dilute the number of cells obtained in a manner so as to plate 1.5 into 10 to the power of 5 cells per mL for high-density plating, and 20, 000 cells per mL for low-density plating in two separate tubes containing 30 mL of plating medium.Aspirate the previously added plating medium and plate 500 microliters of cells dispersed in plating medium in each well. After that, return the plates to the incubator for four hours. Four hours after plating, examine the cells for adherence under the microscope.After four hours of plating, in both high and low density plates, the neurons show good adherence to the plates. Replace the medium in each well with 500 microliters of fresh maintenance medium, and incubate it in an incubator at 37 degrees Celsius. We have to culture these neurons plated for at high-and low-density.While the low-density plated neurons can be used for prolonged cultures, up to 30 days, by changing the maintenance medium twice a week, the high-density plated neurons starts to spontaneously generate neurospheres, which came day after we maintained in ultra-low attachment plate. After 24 hours of plating, both high-and low-density plated neurons are observed to display healthy morphology. A phase contrast image of the low-density plated neurons, cultured for about seven days, is displayed here.The neurons display healthy morphology, and can be maintained up to 30 days with more vigorous routing and interconnections. The bar diagram here shows around 90 percent viability of the low-density plated neurons even after 30 days of culture, as determined by the MTT assay. The primary neurons here have been characterized by immunostaining with Tuj 1, a marker of differentiated neurons, and tau, a marker of neuronal axons.The purity of the neurons is confirmed by the absence of staining in non-neuronal markers GFAP for astrocytes and O4 for oligodendrocytes. In all the characterization studies, nuclei have been stained with Hoechst 33258. Here, the low-density seeded cells have been stained with astrocytic marker GFAP and neuronal marker Tuj 1 to check the purity of the culture up to seven days.It is observed that this protocol supports the preferential growth of neurons over astrocytes as indicated through the quantitative analysis. Nuclei have been stained with Hoechst 33258. Similarly, in the high-density seeded cells, the astrocytes have been stained with GFAP and neurons by Tuj 1.Here, in the quantitative analysis, around 17 percent astrocytes are observed as compared to 83 percent neurons. The nuclei have been stained with Hoechst 33258. After seven days, spontaneously formed multiple neurospheres in the high-density neurons are observed.Around eighth to tenth day of culture, in the high-density plated neurons, the neurospheres starts to form large projections and bridges consisting of radial glial-like extensions. The black arrows indicate the bridge between two newly formed neurospheres. Alive and dead cell assay have been performed on the neurospheres for 15 days.In dense Calcein AM staining, with absolutely no propidium iodide staining, indicates almost 100 percent viability of the neurospheres in culture. The line graph here indicates an expansion in the size of the neurospheres with the passage of number of days. This overly stained image of neurosphere indicates a rich population of neural progenitor cells through increased expression of NPC markers Nestin and Tuj 1.The neurospheres here show high amounts of astrocytes marked by the strong expression of GFAP, accompanied by an even higher expression of neuronal marker Tuj 1. The nuclei have been stained with Hoechst 33258. So I think our protocol is quite exciting and interesting, considering the fact that from a single strategy we are able to get two platforms:one 2-D and one 3-D.And this will be a great platform for screening different neurotherapeutic tracks. So I guess it's really really useful to all neuroscientists. Also another important aspect is that the neurospheres we opt in are extremely rich in neural progenitor cells, the NPCs.And they can be used to differentiate them into neurons and non-neuronal lineages. So, I feel if, really, people can master this technique by taking help from this video, it will be really really useful for everyone. Thank you.

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Research

JoVE Journal

Neuroscience

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

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, biochemistry5, 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.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

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 ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

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.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract of the medulla. The cell bodies of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority are located in the former. The nodose ganglia contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that can modify afferent vagus nerve activity. Modifying vagal afferents through systemic peripheral drug treatments targeted at the receptors on nodose ganglia has the potential of treating diseases such as sleep apnea, gastroesophageal reflux disease, or chronic cough. The protocol here describes a method of injection neurochemicals directly into the nodose ganglion. Injecting neurochemicals directly into the nodose ganglia allows study of effects solely on cell bodies that modulate afferent nerve activity, and prevents the complication of involving the central nervous system as seen in systemic neurochemical treatment. Using readily available and inexpensive equipment, intranodose ganglia injections are easily done in anesthetized Sprague-Dawley rats.

Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...