Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

Podsumowanie

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

Streszczenie

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed 'organoids' or 'spheroids', for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures ('asteroids') are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

Wprowadzenie

Astrocytes are a highly abundant glial cell type within the central nervous system (CNS) with a variety of functional responsibilities beyond structural support. Through secretion of soluble synaptogenic factors and extracellular matrix (ECM) components, astrocytes aid in the establishment and clustering of mature synapses during development¹. They also play a critical role in maintaining the health and plasticity of synapses through extracellular signaling^2,3,4,5, and contribute to long-term stability of homeostatic environments by regulating extracellular potassium and glutamate, as well as the secretion of energy substrates and ATP^6,7,8. Finally, they can contribute to neurotransmission by influencing extrasynaptic currents⁹, and can indirectly influence activity through other cell types such as promoting myelination¹⁰. Importantly, because abnormality or dysfunction of astrocytes can lead to many neurodevelopmental syndromes and adult neuropathology, there is an obvious need to include astrocytes alongside neurons within engineered neural networks in order for an improved model of the endogenous brain environment. An integral characteristic of astrocytes is their ability to form dynamic interactions with neuronal synapses^1,11,12. In the absence of glia, neurons form a limited number of synapses, which in general also lack functional maturity¹³.

Human astrocytes display morphological, transcriptional, and functional characteristics — such as increased size and complexity of branching, as well as species-specific genes — that are not recapitulated in rodents^12,14,15. As a result, studies utilizing human pluripotent stem cell (hPSC)-derived neural cells have become widely accepted as a means of examining CNS-related diseases in vitro while developing novel therapies, injury models, and culture paradigms^16,17. Furthermore, hPSCs permit the study of human synapse formation and function without the need for primary tissue^18,19.

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models of the human brain. There is a need for an appropriate platform to recapitulate its synaptic networks with high fidelity and reproducibility. Recently, interest has emerged in the production of 3D culture systems (broadly known as 'organoids,' 'spheroids,' or 'mini brains')²⁰ to model complex three-dimensional (3D) structures at the cellular and macro levels. 3D culture systems retain ECM and cell-cell interactions that are normally absent or limited during typical 2D coculture paradigms^21,22. An abundance of techniques exist for culturing 3D neural spheroids^23,24,25; however, many require lengthy culture periods (months to years) for spontaneous development and layer preservation, with the user exhibiting very little control over the output.

Here, we illustrate a systematic method to rapidly and consistently bioengineer neural interactions among multiple cell types (pre-differentiated neurons and astrocytes) derived from hPSCs by assembling cells into sphere cocultures ('asteroids')²⁶ that recapitulate human-specific morphological complexities in 3D. This high-density neural system generates evenly-dispersed neural subtypes that take on mature properties over time and can be screened or assayed in a high-throughput manner. We demonstrate for the first time that human astrocytes induce synaptic network burst activity in these 3D cocultures. In addition, this protocol is easily adaptable to generate spheres of different sizes, to utilize cells specified to different regional identities of the CNS, and to study interactions of multiple other cell types as desired.

Protokół

1. Cell Culture and Reagent Preparation

NOTE: The protocols in this section are written in the order in which they appear in the differentiation protocol (section 2). See the Table of Materials for materials and catalog numbers.

1. Prepare coated plates for cell culture.
   1. Dilute extracellular matrix (ECM) coating solution with DMEM/F12 media to prepare a 1 mg/mL stock solution. Aliquot the diluted ECM stock solution into 30 conical tubes of 3 mL each and immediately store in -20 °C. For a working solution, resuspend 3 mL of ECM stock into 33 mL of pre-chilled DMEM/F12 media, bringing the total volume to 36 mL for a concentration of 80 µg/mL.
   2. Coat 1 mL per well of a 6-well cell culture plate with ECM working solution. Add an additional 1 mL DMEM/F12 per well (if necessary) to ensure that the surface of the well is fully covered. Allow the coated 6-well cell culture plates to sit at room temperature for at least 1 h before use.
      NOTE: Coated plates may be stored in the incubator or at 4 °C for up to 2 weeks.
2. Prepare media formulations, growth factors, and small molecules.
   1. To prepare 500 mL of human pluripotent stem cell (hPSC) medium²⁷, add 20x and 500x supplements according to manufacturers’ instructions.
   2. To prepare 500 mL of neural medium (NM), add heparin to a final concentration of 2 mg/mL, 1x of antibiotic/antimycotic solution, 1x B27 supplement or 1x N2 supplement to a 500 mL bottle of DMEM/F12 with L-glutamine supplement as previously detailed²⁸.
   3. To prepare a 10 mM (1,000x) stock solution of Rho-Kinase Inhibitor Y27632 (Y), add 10 mg of powder into 3 mL of phosphate buffered saline (PBS). Filter sterilize, aliquot, and store at -20 °C. Use at a 10 µM working concentration in media.
   4. To prepare a 20 mM (10,000x) stock solution of SB431542, add 10 mg of powder into 1.3 mL of dimethyl sulfoxide (DMSO). To prepare a 2 mM (1,000x) stock solution of DMH1, add 10 mg of powder into 13.1 mL of DMSO. Filter sterilize, aliquot, and store each solution at -20 °C. Use each at a 2 µM working concentration in media (NM + SB431542 + DMH1).
      NOTE: This protocol recommends 2 µM working concentrations of both SB431542 and DMH1 based upon our observations and reports from others²⁹ stating that this concentration effectively promotes neural induction from hPSCs. Higher concentrations have also been reported³⁰. Additionally, with alternative media, it has also been shown that small molecules are not necessary for high neural conversion³¹. Thus, working concentrations will vary depending on alternative cell culture environments, and optimal concentrations should be tested by individual researchers.
   5. To prepare individual 100 µg/mL (10,000x) stock solutions of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF2), add 1 mg of each into 10 mL of PBS + 0.1% BSA. Aliquot EGF and FGF2 stock solutions into 50 µL aliquots and store at -80 °C. Use each at a 10 ng/mL working concentration in media; e.g., add 5 µL EGF and 5 µL FGF2 to 50 mL of NM (NM + EGF + FGF2).
   6. To prepare a stock solution of doxycycline hydrochloride (Dox), first dissolve powder in DMSO to 100 mg/mL and store at -20 °C. Further dilute it to make a 2 mg/mL (1,000x) stock solution in PBS and store at -20 °C. Use at 2 µg/mL working concentration in media; e.g., add 50 µL Dox to 50 mL of NM (NM + Dox).
      Note: Do not re-freeze solutions after thawing.

2. Generation of Neural Subtypes from Human Induced Pluripotent Cells (hPSCs)

NOTE: All cell cultures should be maintained in an incubator with 5% CO₂ at 37 °C. These cultures are maintained at room oxygen levels, though lower levels may be utilized.

1. Generate and maintain astrocyte progenitors (hAstros) from hPSCs.
   NOTE: This section provides a brief and simplified differentiation protocol. For a detailed description (with embryoid body formation, rosette selection and regional patterning steps) refer to the previously described protocol²⁸ (Figure 1A, dotted green box).
   1. Seed clusters of hPSCs (Figure 1B) onto ECM-coated 6-well plates (see step 1.1) with 2 mL of hPSC medium + Y per well. Feed the stem cells every day until they are about 50% confluent and split as needed.
      1. To split hPSCs, add 1 mL of passaging reagent to wells and aspirate after 1 min. Incubate cells at room temperature for 5 min, add 1 mL of hPSC medium, and split aggregates 1:10 onto new ECM-coated wells in 2 mL of hPSC medium + Y per well.
   2. Maintain the stem cells until they are about 50% confluent, then change media to NM + SB431542 + DMH1 to induce neural differentiation (day 0). When cells are about 95% confluent, split 1:6 into new ECM-coated wells in the same medium.
   3. On day 14, dissociate the cells with detachment solution and transfer to a non-coated flask with Y to promote formation of aggregates.
      NOTE: The addition of Y is utilized to promote cell survival and sphere formation but is not included during every feed.
   4. For the generation of neuronal progenitors and neurons (hNeurons), use these day-14 cells as described below. Alternatively, utilize these cells at later time points from monolayer or sphere cultures before neuronal maturation occurs or gliogenesis commences.
      NOTE: By default, this protocol produces dorsal-cortical astrocytes. However, astrocyte subtypes can be regionally specified by the addition of patterning morphogens if desired^28,32,33. Retinoic acid (RA) may be added to caudalize cells into spinal cord phenotypes, while sonic hedgehog (SHH), smoothened agonist (SAG), or purmorphamine will produce ventral phenotypes.
   5. For the generation of astrocyte progenitors and astrocytes in spontaneously-formed 3D aggregates (hAstrospheres), switch to NM + EGF + FGF2 and feed weekly (or as needed to ensure stable pH) as previously detailed³⁴.
      1. Gently dissociate hAstro aggregates with detachment solution when dark centers appear and remove spheres that spontaneously attach. Break spheres gently once a week to maintain sphere health and to avoid necrotic cores.
         NOTE: Do not exceed 5 min of treatment with detachment solution.
      2. After 4-6 months of expansion, confirm cell identity and either freeze in cryopreservation medium (according to manufacturer’s instructions) or alternative storage conditions for long-term preservation in liquid nitrogen, or use immediately for experimentation.
   6. To thaw a frozen stock of hAstrospheres, quickly thaw a vial at room temperature, transfer the contents to an empty 15 mL conical tube, and centrifuge at 300 x g for 1 min. Carefully aspirate supernatant, wash with DMEM/F12, and repeat for a total of two wash steps. Add to a T25 flask with a total volume of 6 mL of NM + EGF + FGF2 + Y (or scale up as needed).
2. Generate and maintain inducible neurons (iNeurons) from hPSCs.
   1. Seed transgenic hPSCs (with a stable doxycycline-inducible neurogenin 2 transgene³⁵) on ECM-coated 6-well plates with 2 mL of hPSC medium + Y per well (as described above for non-transgenic lines). Feed everyday with 2 mL of media per well until they are ready to lift at 70% confluency. Split cells as described in step 2.1.2.
   2. When cells are about 35% confluent, add NM + Dox to induce differentiation into iNeurons. Maintain as a monolayer culture with NM + Dox for 2 days before proceeding to step 3.2.
      NOTE: Cells will transition into neuronal progenitors but will not yet extend neurites (Figure 1C).

3. Preparation and Maintenance of 3D Sphere Cocultures

1. Dissociate hAstros from 3D aggregates in suspension (as described in step 2.1.5.1).
2. Dissociate hNeurons or iNeurons from a 2D monolayer.
   1. Remove media from 6-well plate and add 500 µL of detachment solution to each well containing monolayer cultures. Incubate at 37 °C for 5 min. Gently add 2-3 mL DMEM/F12 to remove attached cells.
   2. Collect cells and media in a 15 mL conical tube and centrifuge at 300 x g for 1 min. Aspirate the supernatant, add 1 mL of fresh media, and pipette up and down gently with a 1,000 µL micropipette to achieve a single cell suspension.
3. Form 3D spheres using microwell culture plates.
   1. Prepare plate by adding 0.5 mL of anti-adherence rinsing solution to each well of the microwell plate. Centrifuge plate at 2,000 x g for 5 min. Aspirate rinsing solution, add 1 mL of DMEM/F12 to each well to wash, and aspirate again.
      NOTE: Always ensure that the centrifuge is balanced during use.
   2. Count each cell type using a hemocytometer or automated cell counter. Add desired ratio of dissociated hAstros and hNeurons or iNeurons to each well in a total volume of 2 mL of NM + Y (include Dox if iNeurons are utilized). Centrifuge plate at 100 x g for 3 min and return to the incubator.
      NOTE: 24-well microwell plates (see the Table of Materials) contain 300 microwells per well. Add a minimum of 6 x 10⁵ cells (for spheres of 2 x 10³ cells each) and a maximum of 6 x 10⁶ cells (for spheres of 2 x 10⁴ cells each) in 2 mL of media per well. For viable spheres of a specific density within this range, use a defined quantity of cells and divide by 300 to calculate the number of cells per sphere. Alternative sphere forming methods and tools may also be utilized if desired. Follow manufacturer’s instructions for acceptable ranges of cell densities.
   3. Allow cells to self-assemble into densely packed spheres within microwell plates over the next 2 days (Figure 2A). Replace 50% media if the culture is yellowing.
      NOTE: Exchange only half media and pipette gently when removing and adding media in order not to disturb spheres. Spheres may lift up from microwells and fuse together with higher force.
   4. After 2 days, gently remove spheres from microwells with a 1,000 µL micropipette (Figure 1D). Lightly apply force to the bottom of microwells with media to remove any additional adhered spheres. Let spheres settle in a 15 mL conical tube, aspirate the old media, and add fresh media.
4. Set up the spinner flask bioreactor system for forming 3D spheres.
   1. Clean the surface of a magnetic stir plate with 70% ethanol, place it into a cell culture incubator. Autoclave individual spinner flasks to sterilize.
   2. Add spheres with a minimum of 50–60 mL media to each spinner flask (Figure 1D, inset). Place the flask on the magnetic stir plate set at 60 rpm. Culture spheres in spinner flasks until they are ready for data collection.
      NOTE: A minimum of 3 weeks in culture is needed for synapse formation. If the spinner flask bioreactor system is not desired, spheres may be cultured in stationary conditions in cell culture flasks or dishes. Spheres may also be embedded in ECM or hydrogel.

4. Measurement of Live Synaptic Physiology with Multielectrode Arrays (MEAs)

1. Prepare MEAs for cell culture.
   1. Clean surface of each MEA with 1 mL of detergent for 1 h. Rinse with sterile deionized (DI) water, rinse with 70% ethanol to sterilize, and then air dry in biosafety cabinet under UV light.
      NOTE: MEAs can be stored in DI water at 4 °C under sterile conditions until use.
   2. Prepare a 0.5 mg/mL solution of poly-ornithine (PLO) by dissolving 50 mg of PLO into 100 mL of boric acid buffer. Coat the surface of each MEA with 1 mL of PLO to render the surface hydrophilic and incubate at 37 °C for a minimum of 4 h. Remove the PLO, wash the surface with DI water, add 1 mL of ECM (see step 1.1.1), and incubate at 37 °C for a minimum of 4 h.
   3. Remove ECM and place spheres in 1.5 mL of media on a MEA surface, ensuring that the spheres are positioned on top of the electrode array (Figures 3A, 3A’). Allow to adhere for 2 days. Change half of the media every 2–3 days (or more often if needed), ensuring that the spheres are not disturbed.
      NOTE: Addition of growth factors may improve culture survival and maturation.
2. Measure electrical activity of neural spheres.
   1. Set up a multielectrode array (MEA) system with a temperature-controlled headstage. Create a software program with a 5 Hz high-pass filter, and a 200 Hz low-pass filter, and a spike threshold of 5x standard deviation (SD).
   2. Place the MEA on headstage and record spontaneous electrical activity (Figure 3B, B’). Save raw data including spike frequency and amplitude for statistical analysis.
      NOTE: A perfusion system may be utilized with the MEA to add pharmacological agents or to increase flow of fresh media for long-term recording. Additional post-processing of spikes may be performed as desired^36,37.

5. Measurement of Synaptic Density with Immunocytochemistry

1. Prepare the reagents.
   1. To make a 500 mL stock solution of 4% paraformaldehyde (PFA), add 400 mL of 1x PBS to a glass beaker or a stir plate in a ventilated hood. Add stir bar and heat to 60 °C while stirring; do not boil. Add 20 g of PFA powder to the heated PBS solution and raise the pH to 6.9 by slowly adding 1 N NaOH dropwise.
      NOTE: 4% PFA solution can be aliquoted and stored at 4 °C for up to one month.
   2. Add 20 g or 30 g of sucrose to 100 mL of PBS to make 20% and 30% solutions of sucrose, respectively.
   3. Add 1 mL of detergent to 10 mL of PBS to prepare a 10% stock solution. Invert several times to homogenize.
   4. Add 250 µL of 10% detergent stock solution (final concentration of 0.25%) and 500 µL each of goat and donkey serums (final concentration of 5% each) to 10 mL PBS to prepare the primary blocking buffer.
   5. Add 100 µL each of goat and donkey serums (final concentration of 1% each) to 10 mL PBS to prepare the secondary blocking buffer.
2. Prepare the samples.
   1. Transfer the spheres into a 15 mL conical tube, allow them to settle at the bottom of the tube, and aspirate the old media. Rinse spheres with 500 µL of PBS, let settle, and aspirate PBS. Add 500 µL of 4% PFA (or enough to cover spheres) and incubate at 4 °C for 30 min.
   2. Aspirate the PFA and gently wash twice with PBS. Add 20% sucrose solution and incubate at 4 °C for several hours or overnight. Carefully aspirate, add 30% sucrose solution, and incubate at 4 °C for several hours or overnight.
      NOTE: Samples may be stored at 4 °C in either PBS or sucrose for up to 1 week before proceeding to the next step. If not slicing, maintain as 3D spheres (Figure 4A-C) in a 1.5 mL tube and proceed to step 5.3.
   3. If slicing the spheres, carefully transfer spheres to an embedding cryogenic mold on top of dry ice. Aspirate the 30% sucrose solution and slowly pour tissue embedding solution into the mold until it fully covers the sample, avoiding air bubbles. Wait until solution freezes and store at -80 °C until cryostat slicing.
   4. Using a cryostat at -20 °C, slice the embedded blocks into 30 µm sections (or as desired) and transfer to glass slides. Store at -80 °C until ready to stain.
3. Immunostain the spheres.
   1. Outline the edges of the slides with a hydrophobic PAP pen to prevent liquid spillage. Prepare primary and secondary blocking solutions with antibodies (see the Table of Materials). Add 500 µL of primary blocking buffer to each slide or tube and incubate at room temperature for 30 min.
   2. Remove the liquid, add 500 µL of fresh primary blocking buffer with diluted primary antibodies to each slide or tube, and incubate overnight at 4 °C. Remove primary antibody solution and wash 3x with PBS for 10 min each.
   3. Add 500 µL of secondary blocking buffer with diluted secondary antibodies to each slide or tube and incubate at 4 °C for 1 h. Remove secondary antibody solution and wash 3x with PBS for 10 min each.
      NOTE: A list of recommended antibodies is provided in the Table of Materials. Other antibodies or dyes may be used as desired. Do not expose fluorescent samples to light (step 5.3.3 onward).
   4. Add 50 µL of mounting solution dropwise to cover the surface and carefully place a coverslip over the slide, avoiding bubbles. Allow the slides to dry at room temperature for 24 h and store at 4 °C.
4. Image the slides.
   1. Use a confocal or two-photon fluorescent microscope with a 63X oil immersion objective to image pre- and post-synaptic protein abundance and colocalization within sphere slices (Figure 4D). Use a 20X or 40X objective to visualize morphological features of the hAstros.
   2. Open fluorescence image files with ImageJ software. Count pre- and post- synaptic puncta manually using the Cell Counter plug-in, using an unbiased counting method as previously detailed³⁸, or with alternative methods.

Wyniki

When performed properly, this protocol will produce defined populations of functional cocultures of astrocytes^28,33,34 and neurons³⁵ generated from hPSCs (Figure 1A-1C), as detailed previously²⁶ and described here in steps 2.1–2.2. This stepwise procedure, with the use of microwell plates, is expe...

Dyskusje

In this protocol, we describe a systematic method for the production of 3D spheres of neural cocultures. The spheres are composed of astrocytes and neurons, which are derived independently from hPSCs. Though not the focus of this protocol, the generation of pure populations of astrocytes from hPSCs²⁸ is a critical step and can be technically challenging if performed without prior experience. This first step in the generation of these synaptic microcircuits should be performed with meticulous timin...

Materiały

Name	Company	Catalog Number	Comments
6 well plate	Fisher Scientific	08-772-1B
15 mL conical tubes	Olympus Plastics	28-101
Accutase	Sigma	A6964-100ML	Detachment solution
AggreWell plate	Stemcell Technologies	34850
Anti-Adherence Rinsing Solution	Stemcell Technologies	7010	Prevent cell adhesion to microwell plates
Anti/anti	Thermofisher	15240062
B27	Thermofisher	17504044	Media Supplement
BrainPhys neuronal medium	Stemcell Technologies	5790	Neurophysiological basal medium alternative
Circular glass coverslips	Neuvitro	GG-12-oz
Cryostor CS10	Stemcell Technologies	7930	Cryopreservation medium with 10% DMSO
DMEM/F12	Thermofisher	10565-042	With GlutaMAX supplement
DMH-1	Stemcell Technologies	73634	HAZARD: Toxic if swallowed. Working concentration: 2 μM
Donkey serum	Lampire Biological Laboratories	7332100	Working concentration: 5% in primary blocking buffer, 1% in secondary blocking buffer
Doxycycline Hydrochloride (Dox)	Sigma	D3072-1ml	HAZARD: Toxic for pregnant women. Working concentration: 2 μg/mL
Epidermal growth factor (EGF)	Peprotech	AF-100-15	Working concentration: 10 ng/mL
Fibroblast growth factor-2 (FGF)	Peprotech	100-18B	Working concentration: 10 ng/mL
Fluoromount-G mounting solution	Southern Biotech	0100-01
Glass slides	Fisherbrand	22-037-246
Goat serum	Lampire Biological Laboratories	7332500	Working concentration: 5% in primary blocking buffer, 1% in secondary blocking buffer
Hemacytometer or automatic cell counter	Life Technologies	AMQAX1000
Heparin	Sigma	H3149-50KU	Working concentration: 2 mg/mL
Magnetic plate	DLAB	8030170200
Matrigel membrane matrix	Corning	354230	ECM coating solution. Working concentration: 80 μg/ml. Prepare on ice and ensure that pipettes, tubes, and media are pre-chilled.
MEA 2100 System	Multichannel Systems	MEA2100
Mounting solution
N2	Thermofisher	17502048	Media Supplement
OCT	Tissue-Tek	4583	Tissue embedding solution for cryosectioning
Pap Pen (Aqua Hold)	Scientific Device Laboratory	9804-02
Paraformaldehyde (PFA)	Acros Organics	169650025	HAZARD: Toxic if inhaled. Working concentration: 4% in PBS
Phosphate buffered saline (PBS)	Stemcell Technologies	CA008-300
Poly-l-ornithine (PLO)	Sigma	P3655-100MG	Working concentration: 0.5 mg/mL
Rectangular glass cover slips	Fisherfinest Premium Superslip	12-545-88
ReLeSR	Stemcell Technologies	5872	Detachment and passaging reagent
Rho-Kinase Inhibitor Y27632- (Y)	Tocris	1254	Working concentration: 10 uM
SB431542	Stemcell Technologies	72234	Working concentration: 2 μM
Spinner flasks	Fisher Scientific	4500-125
Sucrose	Fisher Chemical	S5-3	Working concentration: 20% or 30% in PBS
T25 Culture Flask	Olympus Plastics	25-207	Vented caps
T75 Culture Flask	Olympus Plastics	25-209	Vented caps
Terg-A-zyme	Sigma	Z273287-1EA	Detergent. Working concentration: 1%
TeSR-E8 basal medium	Stemcell Technologies	5940	Human pluripotent stem cell (hPSC) medium
TeSR-E8 supplements	Stemcell Technologies	5940	Supplements for human pluripotent stem cell medium
TritonX-100	Sigma	X100-500ML	Detergent for cell permeabilization. Working concentration: 0.25% in blocking buffer
Trypan blue	Invitrogen	T10282
Antibodies
AlexaFluor 488	Thermofisher	A-11029	Secondary antibody
AlexaFluor 594	Thermofisher	A-11037	Secondary antibody
Ezrin	Thermofisher	MA5-13862	Primary antibody; astrocytes perisynaptic
GFAP	Chemicon	MAB360	Primary antibody; astrocytes
GFP	Aves	GFP-1020	Primary antibody; astrocytes
Glt1	Gift from Dr. Jeffrey Rothstein	n/a	Primary antibody; astrocytes
Homer	Synaptic Systems	160 011	Primary antibody; neurons, post-synaptic
MAP2	Synaptic Systems	188 004	Primary antibody; neurons
PSD95	Abcam	ab2723	Primary antibody; neurons, post-synaptic
S100	Abcam	ab868	Primary antibody; astrocytes
Synapsin 1	Synaptic Systems	106 103	Primary antibody; neurons, pre-synaptic
TuJ1/β3-tubulin (TUBB3)	Covance	MMS-435P	Primary antibody; neurons