Name: isolation and direct neuronal reprogramming of mouse astrocytes
Uploaded: 2022-07-07T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes at JoVE.com

We would like to thank Ines M&#252;hlhahn for cloning the constructs for reprogramming, Paulina Chlebik for viral production, and Magdalena G&#246;tz and Judith Fischer-Sternjak for comments on the manuscript.

The following procedure follows the animal care guidelines of the Helmholtz Zentrum Munich in accordance with the directive 2010/63/EU on the protection of animals used for scientific purposes. Please make sure to comply with the animal care guidelines of the institution where the dissection is performed.
1. Preparation of dissection, dissociation, and culture materials
NOTE: Prepare all culture reagents within a biological safety cabinet and work usi...

<ol>
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Primary cultures of murine astrocytes are a remarkable in vitro model system to study direct neuronal reprogramming. In fact, despite being isolated at a postnatal stage, cells express typical astrocyte markers29, retain the expression of patterning genes28,29, and maintain the capacity to proliferate, similar to in vivo astrocytes at a comparable age38. After MACS-mediated isolation, cells first a...

The mammalian central nervous system (CNS) is highly complex, consisting of hundreds of different cell types, including a vast number of different neuronal subtypes1,2,3,4,5,6. Unlike other organs or tissues7,8,9, the mammalian CNS has a very limited regenerative capacity; neuronal loss following traumatic brain injury or neurodegeneration is irreversible and often results in motor and cognitive deficits10. Aiming to rescue brain functions, different strategies to replace lost neurons are under intense investigation11. Among them, direct reprogramming of somatic cells into functional neurons is emerging as a promising therapeutic approach12. Direct reprogramming, or transdifferentiation, is the process of converting one differentiated cell type into a new identity without passing through an intermediate proliferative or pluripotent state13,14,15,16. Pioneered by the identification of MyoD1 as a factor sufficient to convert fibroblasts into muscle cells17,18, this method has been successfully applied to reprogram several cell types into functional neurons19,20,21.
Astrocytes, the most abundant macroglia in the CNS22,23, are a particularly promising cell type for direct neuronal reprogramming for several reasons. First, they are widely and evenly distributed across the CNS, providing an abundant in loco source for new neurons. Second, astrocytes and neurons are developmentally closely related, as they share a common ancestor during embryonic development, the radial glial cells24. The common embryonic origin of the two cell types seems to facilitate neuronal conversion as compared to reprogramming of cells from different germ layers19,21. Furthermore, patterning information inherited by astrocytes through their radial glia origin is also maintained in adult astrocytes25,26,27, and seems to contribute to the generation of regionally appropriate neuronal subtypes28,29,30. Hence, investigating and understanding the conversion of astrocytes into neurons is an important part of achieving the full potential of this technique for cell-based replacement strategies.
The conversion of in vitro cultured astrocytes into neurons has led to several breakthroughs in the field of direct neuronal reprogramming, including: i) the identification of transcription factors sufficient to generate neurons from astrocytes15,19,31, ii) the unravelling of molecular mechanisms triggered by different reprogramming factors in the same cellular context32, and iii) highlighting the impact of developmental origin of the astrocytes on inducing different neuronal subtypes28,29,33. Furthermore, in vitro direct conversion of astrocytes unravelled several major hurdles that limit direct neuronal reprogramming34,35, such as increased reactive oxygen species (ROS) production34 and differences between the mitochondrial proteome of astrocytes and neurons35. Hence, these observations strongly support the use of primary cultures of astrocytes as a model for direct neuronal reprogramming to investigate several fundamental questions in biology12, related to cell identity maintenance, roadblocks preventing cell fate changes, as well as the role of metabolism in reprogramming.
Here we present a detailed protocol to isolate astrocytes from mice at postnatal (P) age with very high purity, as demonstrated by isolating astrocytes from the murine spinal cord29. We also provide protocols to reprogram astrocytes into neurons via viral transduction or DNA plasmid transfection. Reprogrammed cells can be analyzed at 7 days post-transduction (7 DPT) to assess various aspects, such as reprogramming efficiency and neuronal morphology, or can be maintained in culture for several weeks, to assess their maturation over time. Importantly, this protocol is not specific to spinal cord astrocytes and can be readily applied to isolate astrocytes from various other brain regions, including the cortical gray matter, midbrain, and cerebellum.

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			<td>Life Technologies</td>
			<td>25300054</td>
			<td></td>
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			<td>4&#39;, 6-Diamidino-2-phenyindole, dilactate (DAPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D9564</td>
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			<td>anti-mouse IgG1 Alexa 647</td>
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			<td>anti-rabbit Alexa 546</td>
			<td>Thermo Fisher</td>
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			<td>Cat# 18606-20</td>
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			<td>B27 Supplement</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td></td>
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			<td>BDNF</td>
			<td>Peprotech</td>
			<td>450-02</td>
			<td></td>
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			<td>bFGF</td>
			<td>Life Technologies</td>
			<td>13256029</td>
			<td></td>
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			<td>Bovine Serum Albumine (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# A9418</td>
			<td></td>
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			<td>cAMP</td>
			<td>Sigma Aldrich</td>
			<td>D0260</td>
			<td></td>
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			<td>C-Tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-237</td>
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			<td>DMEM/F12</td>
			<td>Life Technologies</td>
			<td>21331020</td>
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			<td>Dorsomorphin</td>
			<td>Sigma Aldrich</td>
			<td>P5499</td>
			<td></td>
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			<td>EGF</td>
			<td>Life Technologies</td>
			<td>PHG0311</td>
			<td></td>
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			<td>Fetal Bovine Serum</td>
			<td>PAN Biotech</td>
			<td>P30-3302</td>
			<td></td>
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			<td>Forskolin</td>
			<td>Sigma Aldrich</td>
			<td>F6886</td>
			<td></td>
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			<td>GDNF</td>
			<td>Peprotech</td>
			<td>450-10</td>
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			<td>gentleMACS Octo Dissociator</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-427</td>
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			<td>GFAP</td>
			<td>Dako</td>
			<td>Cat# Z0334; RRID: AB_100013482</td>
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			<td>Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G8769</td>
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			<td>GlutaMax</td>
			<td>Life Technologies</td>
			<td>35050038</td>
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			<td>HBSS</td>
			<td>Life Technologies</td>
			<td>14025050</td>
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			<td>Hepes</td>
			<td>Life Technologies</td>
			<td>15630056</td>
			<td></td>
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			<td>Lipofectamine 2000 (Transfection reagent)</td>
			<td>Thermo Fisher</td>
			<td>Cat# 11668019</td>
			<td></td>
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			<td>MACS SmartStrainer 70&#181;m</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-462</td>
			<td></td>
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			<td>MiniMACS Seperator</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
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			<td>Mouse anti-ACSA-2 MicroBeat Kit&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-678</td>
			<td></td>
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			<td>Mouse IgG1 anti-Synaptophysin 1</td>
			<td>Synaptic Systems</td>
			<td>Cat# 101 011 RRID:AB_887824)</td>
			<td></td>
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			<td>Mouse IgG2b anti-Tuj-1 (&#946;III-tub)</td>
			<td>Sigma Aldrich</td>
			<td>T8660</td>
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			<td>MS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
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			<td>N2 Supplement</td>
			<td>Life Technologies</td>
			<td>17502048</td>
			<td></td>
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			<td>Neural Tissue Dissociation Kit&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-628</td>
			<td></td>
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			<td>NT3</td>
			<td>Peprotech</td>
			<td>450-03</td>
			<td></td>
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			<td>octoMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-109</td>
			<td></td>
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			<td>OptiMEM &#8211; GlutaMAX (serum-reduced medium)</td>
			<td>Thermo Fisher</td>
			<td>Cat# 51985-026</td>
			<td></td>
		</tr>
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			<td>Penicillin/Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
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			<td>Poly-D-Lysine</td>
			<td>Sigma Aldrich</td>
			<td>P1149</td>
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		<tr>
			<td>Rabbit anti-RFP</td>
			<td>Rockland</td>
			<td>Cat# 600-401-379; RRID:AB_2209751</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-Sox9</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# AB5535; RRID:AB_2239761</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin Alexa 405</td>
			<td>Thermo Fisher</td>
			<td>Cat# S32351</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# T9284</td>
			<td></td>
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isolation and direct neuronal reprogramming of mouse astrocytes

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through multipotent intermediates. This technique not only holds great promises in the field of disease modeling, as it allows to convert, for example, fibroblasts for patients suffering neurodegenerative diseases into neurons, but also represents a promising alternative for cell-based replacement therapies. In this context, a major scientific breakthrough was the demonstration that differentiated non-neural cells within the central nervous system, such as astrocytes, could be converted into functional neurons in vitro. Since then, in vitro direct reprogramming of astrocytes into neurons has provided substantial insights into the molecular mechanisms underlying forced identity conversion and the hurdles that prevent efficient reprogramming. However, results from in vitro&#160;experiments performed in different labs are difficult to compare due to differences in the methods used to isolate, culture, and reprogram astrocytes. Here, we describe a detailed protocol to reliably isolate and culture astrocytes with high purity from different regions of the central nervous system of mice at postnatal ages via magnetic cell sorting. Furthermore, we provide protocols to reprogram cultured astrocytes into neurons via viral transduction or DNA transfection. This streamlined and standardized protocol can be used to investigate the molecular mechanisms underlying cell identity maintenance, the establishment of a new neuronal identity, as well as the generation of specific neuronal subtypes and their functional properties.

Primary cultures of astrocytes typically reach 80%-90% confluency between 7 to 10 days after MAC-sorting and plating (Figure 1B). Generally, a single T25 culture flask yields around 1-1.5 x 106 cells, which is sufficient for 20-30 coverslips when seeding cells at a density of 5-5.5 x 104 cells per well. The day after plating, cells typically cover 50%-60% of the coverslip surface (Figure 1C). At this stage, cultures consist almost exclusive...

Watch this Scientific Journal Video about Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes at JoVE.com

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

Here we describe a detailed protocol to generate highly enriched cultures of astrocytes derived from different regions of the central nervous system of postnatal mice and their direct conversion into functional neurons by the forced expression of transcription factors.

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through ...

Astrocytes are an interesting autogenous population to target for direct neuronal programming. This protocol provides a reliable technique to isolating culture astrocytes with high purity from different region or the central nervous system. This protocol is designed and optimized to investigate the ability of astrocytes to be reprogrammed into functional neurons without confounding factors such as differences in astrocytes purity of different startup population.Demonstrating the procedure will be done by Bob Hersbach, Postdoc in the lab, and Tatiana Simon a technical assistance in our laboratory. To begin, place the torso of a euthanized mouse in a 35 millimeter Petri dish and keep it on ice. Open the skin with scissors and remove the vertebra with small scissors.Then extract the spinal cord and place it in a dissection buffer on ice. Under a stereotactic dissection microscope with two times magnification, remove the meninges from the isolated spinal cords using forceps and transferred dissected and cleaned spinal cord tissue to a C tube. Add 50 microliters of enzyme P from the neural tissue dissociation kit and 30 microliters of previously prepared enzyme mix to each C tube.Invert the C tubes and place them on a heated dissociated ensuring that all tissue is collected in the tube's lid. Elude cells by removing the column from the separator adding 800 microliters of astrocyte culture medium and pushing cells out of the column with the included plunger. Plate cells in the previously prepared culture flasks by adding 4.2 milliliters of astrocyte culture medium supplemented with 10 nanograms per milliliters of EGF and bFGF.It is not necessary to coat the culture flasks for astrocytes isolated from other brain regions but it provides a better substrate for astrocytes isolated from the spinal cord. Culture the cells at 37 degrees Celsius and 5%carbon dioxide. Perform astrocyte seating under a biological safety cabinet with a safety level one and prepare 24 well plates with Poly-D-Lysine coated glass cover slips as described in the text manuscript.For plating, isolate six spinal cords from P2 mice yields around 1 million cells. Aspirate media from the T-12.5 culture flasks containing the cultured astrocytes and wash once with 1x PBS. Detach the astrocytes from the culture flask by adding 0.5 milliliters of 0.05%trypsin EDTA and incubate at 37 degrees Celsius for five minutes.Gently tap the side of the flask to release cells from the culture flask surface and check detachment under a brightfield microscope using a 10 times magnification. Stop trypsinazation with 2.5 milliliters of astrocyte culture medium and collect cell suspension in 15 milliliter tubes. Centrifuge at 300 RCF for five minutes at four degrees Celsius.Aspirate supernatant and resend cells in one milliliter of astrocyte culture medium. Calculate cell concentration using a hemocytometer or an automated cell counting system. Based on the number of cells, dilute the cell suspension with fresh astrocyte medium supplemented with EGF and bFGF at 10 nanograms per milliliter per factor.Add 500 microliters of the cell suspension to each well of the previously prepared 24 well plates and culture cells at 37 degrees Celsius and 5%carbon dioxide. Prepare neuronal differentiation medium by adding one milliliter B27 supplement to 49 milliliters of basic culture medium. After 24 to 48 hours, depending on whether cells have been transduced or transfected, replace astrocyte medium with one milliliter neuronal differentiation medium per well.Culture the cells at 37 degrees Celsius with 9%carbon dioxide. To increase the reprogramming efficiency, supplement the neuronal differentiation medium with forskolin and dorsomorphin to a final concentration of 30 micromolar and one micromolar respectively when replacing the astrocyte medium with the differentiation medium. When opting to treat cells with forskolin and dorsomorphin provide a second dose of dorsomorphin two days after the initial treatment.Add this second treatment directly to the culture medium. Primary cultures of astrocytes reached 80 to 90%confluency between seven to 10 days after magnetic assisted cell sorting and plating. The day after plating, cells typically covered 50 to 60%of the cover slip surface.Cultures majorly consisted of astrocytes at this stage, while other cell types such as neuro blasts were virtually absent. At seven days post transduction or transfection, induced neuronal cells were clearly distinguishable from astrocytes. The neuronal cell soma was smaller than unreprogrammed Astrocytes had long processes and was positive for neuronal marker, beta three tubulin, and negative for astrocyte marker GFAP.At 21 days post transduction or transfection, many induced neurons were capable of firing action potentials and were positive for the mature neuronal marker NeuN and the pan synaptic protein Synaptophysin. It is essential to properly dissect the region of interest. Avoiding contamination from surrounding tissue.Care must be taken to remove the meninges and the dorso root ganglia from the isolated spinal cord. This method can be used to isolate astrocytes from various regions of the central nervous system including the cerebral cortex, dorsal midbrain, and cerebellum. In the future, this technique will allow us to expand our knowledge on regional astrocytes and their potential to be reprogrammed into functional neurons.

isolation-and-direct-neuronal-reprogramming-of-mouse-astrocytes

Research

JoVE Journal

Neuroscience

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies indicate that the severity of brain injury appear to be sex dependent, and that the male neonates are more susceptible to the effects of HI-related brain injury, resulting in more severe neurological outcomes as compared to females with comparable brain injuries. The development of reliable methods to isolate and maintain highly enriched populations of sexed hippocampal astrocytes is essential to understand the cellular basis of sex differences in the pathological consequences of neonatal HI. In this study, we describe a method for creating sex specific hippocampal astrocyte cultures that are subjected to a model of in-vitro ischemia, oxygen-glucose deprivation, followed by reoxygenation. Subsequent reactive astrogliosis was examined by immunostaining for the Glial Fibrillary Acidic Protein (GFAP) and S100B. This method provides a useful tool to study the role of male and female hippocampal astrocytes following neonatal HI, separately.

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrocytes are one of the most important key players in the central nervous system (CNS). Here, we are reporting a practical method of sexed hippocampal astrocyte culture protocol in order to study the mechanisms underlying the astrocyte function in male and female neonate pups after in-vitro ischemia.

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative cell replacement therapies while also creating tools to study cell fate in situ. To date, it has been possible to obtain neurons via in vivo reprogramming, but the precise phenotype of these neurons or how they mature has not been analyzed in detail. In this protocol, we describe a more efficient conversion and cell-specific identification of the in vivo reprogrammed neurons, using an AAV-based viral vector system. We also provide a protocol for functional assessment of the reprogrammed cells&#8217; neuronal maturation. By injecting flip-excision (FLEX) vectors, containing the reprogramming and synapsin-driven reporter genes&#160;to specific cell types in the brain that serve as the target for cell reprogramming. This technique allows for the easy identification of newly reprogrammed neurons. Results show that the obtained reprogrammed neurons functionally mature over time, receive synaptic contacts and show electrophysiological properties of different types of interneurons. Using the transcription factors Ascl1, Lmx1a and Nurr1, the majority of the reprogrammed cells have properties of fast-spiking, parvalbumin-containing interneurons.

This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo.

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.

In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes

Astrocytes tile the cerebral cortex uniformly, making the analysis of their complex morphology challenging at the cellular level. The protocol provided here uses multicolor labeling based on in utero electroporation to single out cortical astrocytes and analyze their volume and morphology with a user-friendly image analysis pipeline.

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are optimized to minimize their impact on behavior. This is first achieved by using a holder that minimizes movement hindrances. The back of the fly&#8217;s head is glued to this holder at an angle that allows optical access to the whole brain while retaining the fly&#8217;s ability to walk, groom, smell, taste and see. The back of the head is dissected to remove tissues in the optical path and muscles responsible for head movement artefacts. The fly brain can subsequently be imaged to record brain activity, for instance using calcium or voltage indicators, during specific behaviors such as walking or grooming, and in response to different stimuli. Once the challenging dissection, which requires considerable practice, has been mastered, this technique allows to record rich data sets relating whole brain activity to behavior and stimulus responses.

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method specifically tailored to image the whole brain of adult Drosophila during behavior and in response to stimuli. The head is positioned to allow optical access to the whole brain, while the fly can move its legs and the antennae, the tip of the proboscis, and the eyes can receive sensory stimuli.

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are ...

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 ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.