We acknowledge funding from the European Research Council (FP7/260916), the Alexander von Humboldt-Stiftung (Sofja Kovalevskaja), and the Deutsche Forschungsgemeinschaft (DE1951 and SFB889).

All animal experiments described here were performed in accordance with the guidelines for German animal welfare.
NOTE: The timeline and main steps of the protocol are illustrated in Figure 1.
1. Preparations
<ol>
	<li>Prepare the following solutions.
	<ol>
		<li>Prepare DMEM+ for culturing polygonal MD astrocytes: DMEM with 10% FCS, 100 U penicillin and 100 &#181;g/mL streptomycin.</li>
		<li>Prepare NB+H for culturing stellate astrocytes: neurobasal medium with&#160;5 ng/mL HBEGF, 1x B-27 supplement, 1x glutamine supplement, 100 U penicillin and 100 &#181;g/mL streptomycin. 
		NOTE: Aliquots of higher concentrations of HBEGF can be prepared and stored at -20 &#176;C to be added to the medium whenever needed.</li>
		<li>Prepare the dissection medium (10 mM HEPES in HBSS).</li>
		<li>Prepare aliquots of 6-7 mL of 0.05% trypsin-EDTA and 6-7 mL 0.25% trypsin-EDTA in 15 mL tubes. These can be stored at -20 &#176;C and thawed in a 37 &#176;C water bath.</li>
	</ol>
	</li>
	<li>The day before starting the protocol, coat 10 cm tissue culture-treated dishes with 5 mL of 0.04% polyethylenimine (PEI) each.</li>
	<li>The next day, remove the PEI and wash the dishes with deionized, distilled water twice. Add 12 mL of DMEM+ to each plate and pre-equilibrate in the incubator at 37 &#176;C and 5% CO2 for at least 30 min before plating cells.</li>
	<li>Prepare the dissection area: In a laminar flow dissection hood, place two 10 cm Petri dishes filled with dissection medium on ice. For each brain area to be isolated (e.g., cortex, hippocampus), prepare a 15-&#160; tube filled with 14 mL dissection medium and keep on ice. Spray dissection tools (fine scissors, Dumont #3 and #5 forceps) with 70% ethanol and place next to dissection microscope in the hood.</li>
</ol>
2. Rat or Mouse Brain Dissection
NOTE: Both mice and rats can be used. Animals older than P8 may also be used (we tested up to P12), but removing the meninges will become increasingly difficult. Further, the cell yield and health will diminish, since brain cells from older animals will have formed intricate processes and connections that may break during tissue dissociation, leading to increased cell death. Cortical preparations are described here, but other brain regions can also be used.
<ol>
	<li>Place 0.05% trypsin-EDTA in a 37 &#176;C water bath.</li>
	<li>Euthanize animals (pregnant rats for E19 pups, or P0-P8 animals) by slowly raising CO2 concentration, and isolate the brains. The following steps require sterile technique.</li>
	<li>If working with embryonic tissue, first remove the embryos from the uterus and open the embryonic sacs, then immediately cut off the heads with scissors. When all heads are cut off, transfer them into pre-cooled dissection medium.
	<ol>
		<li>Once the heads are submerged in the medium, open the skin and skull (both are very soft in young animals) with forceps and pinch off the cerebellum. Carefully lever the rest of the brain up and out of the skull. 
		NOTE: Two to four E19 embryos yield enough tissue for at least eight 10 cm dishes of cortical astrocytes plated at 500,000 cells per dish on the day of dissection.</li>
	</ol>
	</li>
	<li>For newborn or older pups, cut off the heads with scissors, and isolate the brains by medially cutting the skin in a straight line from the back of the head to the tip of the nose. Pull the skin aside and carefully cut the skull left and right, then lift the skull bone up. Cut off the cerebellum, and lever the rest of the brain up and out of the skull. 
	NOTE: Two to three P0-P8 pups yield enough tissue for at least&#160;eight 10 cm dishes of cortical astrocytes plated at&#160;500,000 cells per dish on the day of dissection.</li>
	<li>Separate the cortex from the underlying midbrain structure. Place the forceps within the longitudinal fissure between the hemispheres, then move the forceps in between the cortex and midbrain structures of one hemisphere (perpendicular to the longitudinal fissure) to pinch each hemisphere free.</li>
	<li>Using the forceps, remove all meninges from each hemisphere (and its hippocampus), then separate the hippocampi. If hippocampal astrocytes are required, move each hippocampus into a 15 mL tube filled with 14 mL dissection medium on ice.</li>
	<li>Remove further areas from each hemisphere&#39;s cortex if specific regions are required (e.g., frontal cortex). Cut any cortical tissue to be used for generating astrocytes into pieces no larger than 1 mm3. Collect all pieces of cortical tissue in a separate 15-mL tube filled with dissection medium on ice.</li>
</ol>
3. Tissue Digestion and Dissociation
<ol>
	<li>Once all tissue pieces are collected, wait for them to settle to the bottom of the tube, then aspirate as much medium as possible.</li>
	<li>Add 2-3 mL of 0.05% trypsin-EDTA using a serological pipette, and incubate the tubes in a 37 &#176;C water bath for 20 min. 
	NOTE: Release all trypsin-EDTA from the pipette swiftly to mix the tissue pieces and allow to settle again.</li>
	<li>Carefully aspirate the trypsin-EDTA, leaving tissue pieces in as little liquid as possible, at the bottom of the tube.</li>
	<li>Add 5 mL of pre-cooled dissection medium to wash off the remaining trypsin-EDTA. Pipette to the side of the upper part of the tube to achieve mixing of the tissue pieces.</li>
	<li>Aspirate the dissection medium, and leave the tissue pieces in as little liquid as possible. Repeat this washing step with pre-cooled dissection medium twice more.</li>
	<li>After the final washing step, add 1 mL of DMEM+ (warmed to room temperature) using a 1,000 &#181;L pipette tip, and triturate the tissue by pipetting up and down without introducing bubbles. 
	NOTE: It is important to avoid producing bubbles, as this may change the pH of the solution, to which astrocytes are quite sensitive.</li>
	<li>Place a 100 &#181;m cell strainer in the opening of a 50-mL tube and pre-wet the filter with 4.5 mL of pre-cooled DMEM+.</li>
	<li>Pipette the 1 mL of dissociated tissue suspension onto the cell strainer and add another 4.5 mL pre-cooled DMEM+ to wash the cells through the cell strainer.</li>
</ol>
4. Cell Plating for Astrocyte Enrichment by Passaging
<ol>
	<li>Count the cells, e.g., by diluting 10 &#181;L of the cell suspension with 10 &#181;L trypan blue and pipetting the mix onto a hemocytometer.</li>
	<li>Plate 500,000 cells in 12 mL DMEM+ per 10-cm dish. 
	NOTE: DMEM+ is helpful for astrocytic growth, more so than for neurons or microglia. During shaking, the physical forces acting on glass coverslips within wells are much stronger than those acting on the 10-cm dishes (without coverslips). If cells are shaken on 24-well plates, they become unhealthy or die. Only plate astrocytes on 24-well plates after shaking.</li>
	<li>Keep cell cultures in the incubator at 37 &#176;C and 5% CO2 for seven days.</li>
	<li>On day 6, the day before shaking and passaging cells, prepare the PEI-coated culture plates as described in step 5.</li>
</ol>
5. Passaging Cells
<ol>
	<li>The day before passaging, coat cell culture plates with 0.04% PEI.
	<ol>
		<li>For immunocytochemistry, viral transduction, or plasmid transfection, use 24-well or 6-well plates (each containing a sterilized 12 mm or 25 mm glass coverslip, respectively).</li>
		<li>Sterilize the glass coverslips by incubation in 70% ethanol for at least 1 h, then wash three times in deionized, distilled water before placing the coverslips in the wells.</li>
		<li>Add at least 70 &#181;L 0.04% PEI per 12-mm coverslip in 24-well plates, and 600 &#181;L 0.04% PEI per 25 mm coverslip in 6-well plates.</li>
		<li>For Western blot or RNA-Seq analysis, the 10 cm dishes already containing the cells after the dissection can continue to be used. Dishes only need to be shaken, then the cells are washed with 1x PBS and treated with NB+H (do not transfer to new dishes).</li>
	</ol>
	</li>
	<li>On day in vitro (DIV) 7 after the dissection, place the 10-cm dishes with cells in DMEM+ on a laboratory shaker in the incubator and shake the dishes at 110 rpm for 6 h.</li>
	<li>Wash off the PEI from coated coverslips with deionized, distilled water twice. Add NB+H medium to the plates (500 &#181;L per well for 24-well plates and 2 mL per well for 6-well plates). Pre-equilibrate the plates in the incubator at 37 &#176;C and 5% CO2 while the 10 cm dishes are shaking, or for at least 30 min.</li>
	<li>20-30 min before the end of the 6 h shaking, pre-warm 1x PBS, DMEM+, NB+H, and 0.25% trypsin-EDTA to 37 &#176;C in the water bath.</li>
	<li>After 6 h of shaking, take the culture dishes off the shaker and immediately replace the medium (that contains shaken-off neurons, oligodendrocytes, and microglia) with 10 mL pre-warmed 1x PBS per dish. 
	NOTE: It is important to immediately aspirate the old medium containing detached cells to avoid the non-astrocytic cells re-attaching.</li>
	<li>Remove the PBS and add 3 mL pre-warmed trypsin-EDTA per dish. Incubate each dish for 4 min in the incubator at 37 &#176;C and 5% CO2. 
	NOTE: If preparing samples for Western blot or RNA-Seq, do not add trypsin-EDTA. Instead, replace the PBS with 12 mL pre-warmed NB+H, after which cultures can be placed back in the incubator.</li>
	<li>Add 5 mL pre-warmed DMEM+ to the 3 mL trypsin-EDTA in each dish, pipette the cells off the dish using a 5 mL serological pipette, and collect the cell suspension in a 50 mL tube. 
	NOTE: Pipette directly from the bottom of the dish - the floor of the dish should become clearer as the cells detach. Only use serum-containing medium (in place of DMEM+), as serum inactivates trypsin.</li>
	<li>Centrifuge the cell suspension at 3,220 x g at 20 &#176;C for 4 min.</li>
	<li>Remove the supernatant and resuspend the cell pellet in 1 mL pre-warmed NB+H.</li>
</ol>
6. Cell Plating for Final Astrocyte Cultures
<ol>
	<li>Count the cells.</li>
	<li>Plate 20,000 cells per well in 6-well plates (in 2 mL NB+H medium per well) or 5,000 cells per well in 24-well plates (in 500 &#181;L NB+H medium per well).</li>
	<li>Keep the cell cultures in the incubator at 37 &#176;C and 5% CO2 until further treatment or analysis.</li>
	<li>Exchange half the medium once a week. 
	NOTE: In low-nutrient environments, astrocytes stop proliferating but any remaining microglia will proliferate (although in our experience, no microglia appeared in AWESAM cultures). Exchanging half the medium once a week prevents enrichment of any potentially remaining microglia8, and the monocultures remain healthy for at least three weeks.</li>
</ol>

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+method+for+the+purification+and+culture+of+rodent+astrocytes.">Development of a method for the purification and culture of rodent astrocytes.</a> Neuron. 71 (5), 799-811 (2011).</li><li>Wolfes, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+culturing+stellate+astrocytes+reveals+spatially+distinct+Ca2++signaling+and+vesicle+recycling+in+astrocytic+processes.">A novel method for culturing stellate astrocytes reveals spatially distinct Ca2+ signaling and vesicle recycling in astrocytic processes.</a> J Gen Physiol. 149 (4), (2016).</li><li>McCarthy, K. 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The authors have nothing to disclose.

Four steps within the protocol are critical: 1) preparing the HBEGF concentration to precisely 5 ng/mL, since small increases in the concentration can lead to immature cultures, e.g., 10 ng/mL HBEGF will de-differentiate astrocytes4; 2) avoiding bubbles in the solutions that contain tissue or cells, which can change the pH and impede astrocyte health; 3) pre-equilibrating media to achieve the optimal pH and temperature for growing healthy astrocytes; 4) exchanging only half the medium onc...

Astrocytes influence brain function through trophic support, blood flow, synaptic signaling and plasticity, and intercellular communication - all of which are mechanisms that center around the thin astrocytic processes. The AWESAM protocol described here allows the study of these processes without interference from neurons and other glia, which is useful e.g., because the expression of many proteins and even Ca2+ signaling overlap across different brain cell types. Further, this method overcomes limitations of previously available techniques. In particular, our protocol provides in vivo-like morphology (stellate astrocytes with thin processes) and gene expression in a quick, easy, and cheap manner.
Cell morphology, gene expression, and many other regulatory processes are directly influenced by the environment. In a culture dish, this refers to factors released by surrounding cells, but also the medium in which the cells are grown. For astrocytes, HBEGF was previously reported to induce a stellate morphology1 but was also found to de-differentiate astrocytes2. However, lower concentrations of HBEGF were later used for generating more in vivo-like astrocytes as identified via RNA sequencing in two different protocols3,4. Moreover, astrocyte morphology changes with the medium composition, regardless of the protocol4: Neurobasal medium with a low concentration of HBEGF is optimal for growing stellate astrocytes, while other medium compositions (e.g., serum-containing DMEM) produce polygonal cells (even in astrocytes freshly isolated by immunopanning).
The AWESAM protocol overcomes disadvantages of previous techniques for growing astrocyte monocultures4. Previous techniques for growing astrocyte monocultures have the following disadvantages: polygonal morphology, which is uncharacteristic of stellate astrocytes in vivo (MD method)5; the length and cost of protocol (preparing astrocytes from induced pluripotent stem cells takes three months and requires many reagents, including expensive growth factors)6; the low amounts of material (immunopanning method)3; and the de-differentiation of astrocytes and requirement of a 3D matrix (Puschmann et al.)1,2. In contrast, the AWESAM protocol provides: in vivo-like morphology (stellate astrocytes with thin processes); a quick, easy, and cheap method; large quantities of material; and the most in vivo-like gene expression compared with immunopanned and MD astrocytes. Additionally, 2D culture allows for the study of Ca2+ signaling and vesicle recycling in thin processes, and in events close to the membrane (e.g., TIRF microscopy is not possible in 3D cultures).
The MD method has been extensively used since its publication in 19805, offering a simple and fast technique for polygonal astrocyte monocultures. In brief, the MD method entails growing mixed brain cells in fetal calf serum (FCS)-containing DMEM, followed by shaking steps that enrich for astrocytes (as all other brain cell types detach from the dish) and further culture in the same medium. DMEM supplemented with FCS is used for many other cell types, ranging from fibroblasts and adipocytes to different cancer cell lines, all of which share the polygonal morphology exhibited by MD astrocytes in culture. Until the early 2000s7, little thought had been given to media tailored to astrocytes, specifically to favor their typical stellate morphology found in vivo. One protocol published in 2011 does achieve such stellate morphology: Called the immunopanning method, it employs serum-free medium optimized for culturing astrocytes3. Using this method, freshly isolated brain cells are exposed to a series of dishes coated with antibodies that target cell type-specific cell surface proteins, to enrich for astrocytes. Despite the more in vivo-like morphology and expression profile of immunopanned astrocytes, the majority of in vitro studies still rely on the MD astrocyte method. The MD method is simple and fast, while the immunopanning method comprises more complex and time-consuming steps on the first day of culture (such as longer enzyme digestion periods, careful layering of solutions of different density, immunopanning itself, and several centrifugation steps - all before plating). However, by using more specialized medium, the AWESAM method offers both the speed of the MD method and the in vivo-like morphology of the immunopanning protocol.
Overall, the AWESAM protocol is useful for studying more in vivo-like astrocytes in 2D monocultures isolated from neurons and other glia (as previously characterized4 by immunostainings, immunoblots, and RNA sequencing). It allows for the study of thin astrocytic processes, and provides great accessibility for visualizing spontaneous Ca2+ signaling and events close to the membrane (e.g., imaged by TIRF microscopy).

<table border="0" cellpadding="0" cellspacing="0" width="898">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">0.05% trypsin-EDTA</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:344px;">25300054</td>
			<td style="width:167px;">warm in 37 &#186;C waterbath before use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.25% trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>warm in 37 &#186;C waterbath before use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">B-27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>50X stock</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>100X stock</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Penicillin / streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>50 stock; penicillin: 5000 U/ml; streptomycin: 5000 &#181;g/ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>without L-glutamine</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DMEM</td>
			<td>Gibco</td>
			<td>41966029</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HBEGF</td>
			<td>Sigma</td>
			<td>4643</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEPES</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HBSS</td>
			<td>Gibco</td>
			<td>14170112</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">FCS</td>
			<td>Gibco</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PBS</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td>1X</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">100 &#956;m nylon cell strainer</td>
			<td>BD</td>
			<td style="width:344px;">352360</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trypan Blue</td>
			<td>Sigma</td>
			<td>T8154&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell culture incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>Hera Cell 240i cell culture incubator</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Laboratory shaker</td>
			<td>Heidolph</td>
			<td>Rotamax 120&#160;</td>
			<td>use inside cell culture incubator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810 R&#160;</td>
			<td></td>
		</tr>
	</tbody>
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culturing in vivo-like murine astrocytes using the fast, simple, and inexpensive awesam protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Astrocytes that are co-cultured with neurons and grown in NB+ medium appear stellate after 2 weeks of culture (Figure 2). In addition to NB+ medium, the co-cultured astrocytes are also exposed to unknown neuron-derived factors that likely contribute to their survival and morphology. In contrast, MD astrocytes grown in DMEM+ as monocultures (after shaking off other cell types before passage) appear polygonal after 2 weeks. AWESAM astrocytes grown in NB+H after...

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Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

culturing-vivo-like-murine-astrocytes-using-fast-simple-inexpensive

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Neuroscience

8.3K Views.  University of Oxford. The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

Video: Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Analyzing the Size, Shape, and Directionality of Networks of Coupled Astrocytes

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network level. Astrocytes are strongly connected to each other through gap junctions and coupling through these junctions is dynamic and highly regulated. An emerging concept is that astrocytic functions are specialized and adapted to the functions of the neuronal circuit with which they are associated. Therefore, methods to measure various parameters of astrocytic networks are needed to better describe the rules governing their communication and coupling and to further understand their functions.
Here, using the image analysis software (e.g., ImageJFIJI), we describe a method to analyze confocal images of astrocytic networks revealed by dye-coupling. These methods allow for 1) an automated and unbiased detection of labeled cells, 2) calculation of the size of the network, 3) computation of the preferential orientation of dye spread within the network, and 4) repositioning of the network within the area of interest.
This analysis can be used to characterize astrocytic networks of a particular area, compare networks of different areas associated to different functions, or compare networks obtained under different conditions that have different effects on coupling. These observations may lead to important functional considerations. For instance, we analyze the astrocytic networks of a trigeminal nucleus, where we have previously shown that astrocytic coupling is essential for the ability of neurons to switch their firing patterns from tonic to rhythmic bursting1. By measuring the size, confinement, and preferential orientation of astrocytic networks in this nucleus, we can build hypotheses about functional domains that they circumscribe. Several studies suggest that several other brain areas, including the barrel cortex, lateral superior olive, olfactory glomeruli, and sensory nuclei in the thalamus and visual cortex, to name a few, may benefit from a similar analysis.

Here we present a protocol to assess the organization of astrocytic networks. The described method minimizes bias to provide descriptive measures of these networks such as cell count, size, area, and position within a nucleus. Anisotropy is assessed with a vectorial analysis.

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network ...

An Emerging Target Paradigm to Evoke Fast Visuomotor Responses on Human Upper Limb Muscles

To reach towards a seen object, visual information has to be transformed into motor commands. Visual information such as the object&#8217;s color, shape, and size are processed and integrated within numerous brain areas, then ultimately relayed to the motor periphery. In some instances, a reaction is needed as fast as possible. These fast visuomotor transformations, and their underlying neurological substrates, are poorly understood in humans as they have lacked a reliable biomarker. Stimulus-locked responses (SLRs) are short latency (&#60;100 ms) bursts of electromyographic (EMG) activity representing the first wave of muscle recruitment influenced by visual stimulus presentation. SLRs provide a quantifiable output of rapid visuomotor transformations, but SLRs have not been consistently observed in all subjects in past studies. Here we describe a new, behavioral paradigm featuring the sudden emergence of a moving target below an obstacle that consistently evokes robust SLRs. Human participants generated visually guided reaches toward or away from the emerging target using a robotic manipulandum while surface electrodes recorded EMG activity from the pectoralis major muscle. In comparison to previous studies that investigated SLRs using static stimuli, the SLRs evoked with this emerging target paradigm were larger, evolved earlier, and were present in all participants. Reach reaction times (RTs) were also expedited in the emerging target paradigm. This paradigm affords numerous opportunities for modification that could permit systematic study of the impact of various sensory, cognitive, and motor manipulations on fast visuomotor responses. Overall, our results demonstrate that an emerging target paradigm is capable of consistently and robustly evoking activity within a fast visuomotor system.

Presented here is a behavioral paradigm that elicits robust fast visuomotor responses on human upper limb muscles during visually guided reaches.

To reach towards a seen object, visual information has to be transformed into motor commands. Visual information such as the object&#8217;s color, shape, ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of mitochondrial Ca2+ contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Here we present a cell-type specific and mitochondria targeting molecular approach for mitochondrial Ca2+ imaging in astrocytes and neurons in vitro and in vivo. We constructed DNA plasmids encoding mitochondria-targeting genetically encoded Ca2+ indicators (GECIs) GCaMP5G or GCaMP6s (GCaMP5G/6s) with astrocyte- and neuron-specific promoters gfaABC1D and CaMKII and mitochondria-targeting sequence (mito-). For in vitro mitochondrial Ca2+ imaging, the plasmids were transfected in cultured astrocytes and neurons to express GCaMP5G/6s. For in vivo mitochondrial Ca2+ imaging, adeno-associated viral vectors (AAVs) were prepared and injected into the mouse brains to express GCaMP5G/6s in mitochondria in astrocytes and neurons. Our approach provides a useful means to image mitochondrial Ca2+ dynamics in astrocytes and neurons to study the relationship between cytosolic and mitochondrial Ca2+ signaling, as well as astrocyte-neuron interactions.

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators GECIs

This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of ...