We thank Jenny Gringer Richards for her thorough work in validating our control subjects, from which we generated the control iPSCs. This work was supported by NIH T32 MH019113 (to D.A.M. and K.A.K.), the Nellie Ball Trust (to T.H.W. and A.J.W.), NIH R01 MH111578 (to V.A.M. and J.A.W.), NIH KL2 TR002536 (to A.J.W.), and the Roy J. Carver Charitable Trust (to V.A.M., J.A.W., and A.J.W.).&#160;The figures were created with BioRender.com.

The human subjects research was approved under the University of Iowa Institutional Review Board approval number 201805995 and the University of Iowa Human Pluripotent Stem Cell Committee approval number 2017-02. Skin biopsies were obtained from the subjects after obtaining written informed consent. The fibroblasts were cultured in DMEM with 15% fetal bovine serum (FBS) and 1% MEM-non-essential amino acids solution at 37 &#176;C and 5% CO2. Fibroblasts were reprogrammed using an episomal reprogramming kit following the manufacturer&#39;s protocol (see Table of Materials) using a nucleofector for electroporation. All procedures were performed in a Class II Type A2 biological safety cabinet (&#34;hood&#34; for short). All cell culture media were antibiotic-free; therefore, every component that entered the hood was cleaned with 70% ethanol. All cell culture media and components were sterile filtered or opened in the hood to maintain their sterility.
1. Experimental preparation
<ol>
	<li>Prepare basement membrane matrix (BMM)-coated plates. 
	NOTE: BMM solidifies more quickly at warmer temperatures. Plates must be prepared rapidly and immediately placed at 4 &#176;C for storage.
	<ol>
		<li>Thaw the BMM (see Table of Materials) on ice, at 4 &#176;C for at least 2 h, or overnight.</li>
		<li>Mix DMEM/F12 and BMM to a final concentration of 80 &#181;g/mL. Distribute the BMM solution in tissue culture dishes (1 mL for 35 mm and 2 mL for 60 mm dishes) and incubate at 37 &#176;C for at least 1 h or overnight before plating the cells. 
		NOTE: The unused dishes can be stored at 4 &#176;C for 2 weeks.</li>
	</ol>
	</li>
	<li>Prepare poly-L-ornithine/laminin (PLO/laminin)-coated plates.
	<ol>
		<li>Prepare 20 &#181;g/mL PLO (see Table of Materials) in sterile DPBS+/+ and add 1 mL to each well of a 6-well plate. Incubate overnight at 37 &#176;C in the incubator.</li>
		<li>The following day, aspirate the PLO with a vacuum aspirator and wash two times with DPBS+/+. Air dry in the hood. 
		NOTE: Air-dried plates can be stored at 4 &#176;C for up to 2 weeks, wrapped in aluminum foil, for future use.</li>
		<li>Prepare 10 &#181;g/mL laminin (see Table of Materials) in DPBS+/+ and add 1 mL to each well of a 6-well plate. Incubate at least for 3 h or overnight at 37 &#176;C in the incubator.</li>
		<li>Aspirate the laminin with a vacuum aspirator and add either 1 mL of medium or sterile DPBS+/+. 
		NOTE: The laminin coating must not dry out. PBS or an appropriate culture medium should be added immediately to prevent this. Coated plates can be stored at 4 &#176;C for up to 2 weeks.</li>
	</ol>
	</li>
	<li>Prepare PSC passaging solution. 
	NOTE: An osmometer (see Table of Materials) is required to make PSC passaging solution10.
	<ol>
		<li>Dissolve 11.49 g of potassium chloride (KCl) and 0.147 g of sodium citrate dihydrate (HOC(COONa)(CH2COONa)2*2H2O) in 400 mL of sterile cell culture grade water (see Table of Materials).</li>
		<li>Measure the volume of the solution and record it as the initial volume (Vi).</li>
		<li>Measure the osmolarity and adjust it to 570 mOsm by adding sterile cell culture grade water using the formula Vi &#215; Oi = Vf &#215; 570 mOsm.</li>
		<li>Filter-sterilize the solution with a 0.20 &#181;m filter and make 10 mL aliquots. Store the aliquots at room temperature (RT) for up to 6 months.</li>
	</ol>
	</li>
	<li>Prepare pluripotent stem cell (PSC) medium. 
	NOTE: iPSCs are maintained in a medium containing heat-stable FGF2 (see Table of Materials), providing a weekend-free feeding schedule. Other commercially available PSC media have not been tried for this protocol.
	<ol>
		<li>Thaw the PSC medium supplement overnight at 4 &#176;C.</li>
		<li>Add 10 mL of PSC medium supplement to 500 mL of basal PSC medium. Make 25 mL aliquots and store at &#8722;20 &#176;C. 
		NOTE: Frozen aliquots can be stored at &#8722;20 &#176;C for 6 months. Thawed aliquots must be stored at 4 &#176;C and used within 2 weeks. Cells can be frozen for long-term storage in a PSC medium with 10% (v/v) dimethyl sulfoxide (DMSO).</li>
	</ol>
	</li>
	<li>Prepare PSC thawing medium.
	<ol>
		<li>Supplement PSC medium with 50 nM chroman, 1.5 &#181;M emricasan, 1x polyamine supplement, and 0.7 &#181;M trans-ISRIB (CEPT cocktail11) (see Table of Materials).</li>
		<li>Filter-sterilize with a 0.20 &#181;m filter and store at 4 &#176;C for up to 4 weeks.</li>
	</ol>
	</li>
	<li>Prepare pulled glass pipettes.
	<ol>
		<li>Pull 22.9 cm (9 in) glass Pasteur pipettes into two pieces above a Bunsen burner, ~2 cm below the neck, creating two pipettes, with the thinner side being ~4 cm shorter than the other.</li>
		<li>With the help of the flame, bend the tips of the pulled side to create a smooth &#34;r&#34;.</li>
		<li>Place the pulled pipettes in autoclave sleeves and autoclave for sterilization.</li>
	</ol>
	</li>
	<li>Prepare cerebellar differentiation medium (CDM).
	<ol>
		<li>Mix IMDM and Ham&#39;s F12 nutrient mix in a 1:1 ratio. Supplement the mix with 1x L-alanine-L-glutamine supplement, 1% (v/v) chemically defined lipid concentrate, 0.45 mM 1-thio-glycerol, 15 &#181;L/mL apo-transferrin, 5 mg/mL bovine serum albumin (BSA), and 7 &#181;g/mL insulin (see Table of Materials).</li>
		<li>Filter-sterilize with a 0.20 &#181;m filter, store at 4 &#176;C, and use within 1 month.</li>
	</ol>
	</li>
	<li>Prepare cerebellar maturation medium (CMM).
	<ol>
		<li>Supplement neurobasal medium with 1x L-alanine-L-glutamine supplement and 1x N-2 supplement (see Table of Materials).</li>
		<li>Filter-sterilize with a 0.20 &#181;m filter, store at 4 &#176;C, and use within 2 weeks.</li>
	</ol>
	</li>
</ol>
2. Feeder-free iPSC culture
<ol>
	<li>Thaw the cells following the steps below.
	<ol>
		<li>Place a BMM plate (step 1.1) into the 37 &#176;C incubator to gel for at least 1 h or overnight before thawing the cells.</li>
		<li>Pre-warm 10 mL of PSC thawing medium (step 1.5) at 37 &#176;C.</li>
		<li>Transfer the cryovial with cells from liquid nitrogen to a water bath at 37 &#176;C. 
		NOTE: To avoid generating a source of contamination in the cell culture space, using a standard water bath is not recommended. Instead, fresh water can be heated in a small beaker to 37 &#176;C to generate a one-time-use water bath.</li>
		<li>When there is a small piece of ice left in the tube, remove it from the water bath, dry the tube, and spray with 70% ethanol. Transfer the tube to the hood and use a 2 mL or 5 mL serological pipette (see Table of Materials) to gently transfer the cells to a 15 mL conical tube.</li>
		<li>Add 8 mL of PSC thawing medium to the cells dropwise while swirling the conical tube. Centrifuge at 200 x g for 5 min at RT.</li>
		<li>Carefully aspirate the supernatant with a vacuum aspirator without disturbing the cell pellet. Resuspend the cells in 2 mL of PSC thawing medium.</li>
		<li>Aspirate the BMM from the plate and add the resuspended cells dropwise all over the plate for even distribution.</li>
		<li>Place the plate in the incubator at 37 &#176;C, 5% CO2. Refresh the medium the next day with PSC medium. After that, change the medium daily.</li>
	</ol>
	</li>
	<li>Maintain the cells following the steps below.
	<ol>
		<li>Pre-warm the necessary volume of PSC medium at 37 &#176;C (e.g., 1 mL of medium per 35 mm plate).</li>
		<li>Investigate the plates for differentiated cells or colonies and remove the differentiated cells with a pulled glass pipette (step 1.6). 
		NOTE: iPSC colonies have smooth edges with morphologically identical cells.</li>
		<li>Change the spent medium daily and passage every 3-4 days or when 70% confluency is reached.</li>
	</ol>
	</li>
	<li>Passage the cells.
	<ol>
		<li>Place the necessary amount of BMM plates into the incubator for at least 1 h or overnight before passaging. Pre-warm the necessary amount of PSC medium (step 1.3) at 37 &#176;C.</li>
		<li>Using a pulled glass pipette, clean off any differentiated cells or colonies.</li>
		<li>Aspirate the spent medium with a vacuum aspirator and add PSC dissociation medium, 1 mL per 35 mm dish. Incubate at 37 &#176;C for 1-3 min. 
		NOTE: If colonies are lifting off in the PSC passaging solution before adding PSC medium, the incubation time can be reduced.</li>
		<li>Aspirate the PSC passaging solution and add pre-warmed PSC medium.</li>
		<li>Using a sterile 200 &#181;L pipette tip, scratch lines parallel to each other in one direction, turn the plate 90&#176;, and make a second set of scratch lines perpendicular to the previous ones (making a cross-hatched pattern across the plate).</li>
		<li>Aspirate the BMM solution from the set plates. Collect the colonies using a serological pipette and distribute them to new BMM plates.</li>
		<li>Incubate at 37 &#176;C, 5% CO2. Change the medium daily.</li>
	</ol>
	</li>
	<li>Freeze the cells.
	<ol>
		<li>Prepare cryovials by labeling the content and sterilize under ultraviolet (UV) light in the hood (see Table of Materials) for 30 min.</li>
		<li>Prepare PSC freezing medium by supplementing PSC medium with 10% (v/v) DMSO. Follow steps 2.3.2-2.3.5.</li>
		<li>Transfer the cells to a 15 mL conical tube with a 5 mL serological pipette and centrifuge at 200 x g for 5 min at RT.</li>
		<li>Carefully aspirate the medium and resuspend the cell pellet in PSC freezing medium.</li>
		<li>Distribute into cryovials. Place the vials into a freezing container and place the container in a &#8722;80 &#176;C freezer.</li>
		<li>The following day, transfer the cryovials to liquid nitrogen for long-term storage.</li>
	</ol>
	</li>
</ol>
3. Cerebellar differentiation
NOTE: Before starting the differentiation, iPSCs are passaged to six 35 mm dishes and are ready for the differentiation when they are at 70% confluency. Each 35 mm plate will be transferred to one well of the 6-well plate.
<ol>
	<li>On day 0, lift the healthy iPSC colonies for EB formation.
	<ol>
		<li>Add 1 mL of CDM (step 1.7) supplemented with 10 &#181;M Y-27632 and 10 &#181;M SB431542 (see Table of Materials) to each well of a 6-well ultra-low attachment (ULA) plate and place in the incubator until the lifted colonies are ready to be added to the wells.</li>
		<li>Clean the differentiated cells using a pulled glass pipette. Aspirate the medium and add 1 mL of PSC passaging solution for each 35 mm dish. Incubate for 3 min at 37 &#176;C, aspirate, and add 2 mL of CDM supplemented with 10 &#181;M Y-27632 and 10 &#181;M SB431542. 
		NOTE: If colonies are lifting off in the PSC passaging solution before adding CDM, the incubation time can be reduced.</li>
		<li>Under a transmitted-light inverted microscope, gently lift the colonies using the bent edge of the pulled glass pipette using 4x magnification. Once every colony is lifted, gently transfer all to one well of the 6-well ULA plate using a 10 mL serological pipette. Repeat this process for each iPSC plate. Incubate the cells at 37 &#176;C with 5% CO2. 
		NOTE: If the colonies are too large or are merged, they can be sliced with the tip of the pulled glass pipette to create smaller EBs.</li>
	</ol>
	</li>
	<li>On day 2, add FGF2 to each well to a final concentration of 50 ng/mL. 
	NOTE: The FGF2 used for cerebellar differentiation is not heat-stable. This protocol has not been tested with heat-stable FGF2.</li>
	<li>On day 7, do a 1/3 medium change. For 3 mL of total medium in a well, with a 1,000 &#181;L pipettor, gently aspirate 1 mL of spent medium and replace it with 1 mL of fresh CDM. Incubate the EBs for 7 days.</li>
	<li>On day 14, gently aspirate nearly all the spent medium using a 1,000 &#181;L pipettor. To ensure minimal damage to the EBs, swirl the plate to gather all the EBs in the center of the plate, and then tilt the plate and aspirate slowly from the edge.
	<ol>
		<li>As the medium amount decreases, slowly lay the plate flat and continue to aspirate. Leave enough medium to avoid drying the EBs out. Afterward, add 3 mL of fresh CDM supplemented with 10 &#181;M Y-27632. Transfer the EBs using a 10 mL serological pipette to a PLO/laminin-coated dish (step 1.2). 
		NOTE: Depending on the downstream application, the EBs can be transferred to a 6-well PLO/laminin plate or single EBs can be transferred to a single well of a PLO/laminin-coated 24-well plate or coverslip.</li>
	</ol>
	</li>
	<li>On day 15, aspirate the medium and replace it with fresh CDM. 
	NOTE: It is important to add enough medium to the wells to ensure enough medium between feedings as, over time, there will be evaporation (e.g., for a well in a 6-well plate, add 3 mL of medium). If the medium has started to acidify (turn clear and yellow), refresh the medium even if it is not on the feeding schedule until the end of the protocol.</li>
	<li>On day 21, aspirate the spent medium and replace it with CMM. On day 28, change the medium with fresh CMM. On day 35, harvest the cells for further applications.</li>
</ol>
4. Sample preparation for RNA isolation
<ol>
	<li>Aspirate the medium and add 500 &#181;L of cell dissociation reagent (see Table of Materials) to each well of the 6-well plate. Leave it on for a couple of seconds and aspirate.</li>
	<li>Incubate the plate, with the lid on, at room temperature for 2 min. Tap the sides of the plate to detach the cells.</li>
	<li>Add 1 mL of CMM (step 1.8) and collect the cells into a 1.5 mL tube.</li>
	<li>Pellet the cells by centrifugation using a benchtop mini centrifuge (see Table of Materials) for 30 s at RT (max speed 2000 x g), discard the medium, and add 1 mL of 1x PBS pH 7.4 to wash the cell pellet.</li>
	<li>Pellet the cells again using a benchtop mini centrifuge for 30 s at RT and wash with PBS once more.</li>
	<li>Pellet the cells and discard the PBS. Lyse the cells in phenol and guanidine isothiocyanate containing reagent (see Table of Materials). 
	&#8203;NOTE: Cells lysed in phenol and guanidine isothiocyanate&#160;containing&#160;reagent can be stored at &#8722;80 &#176;C for up to 1 year. Cells can be lysed directly in the dish depending on the RNA isolation method and reagents being used. Due to the low number of cells in a dish, isolating RNA using silica spin columns (see Table of Materials) will result in higher yields of RNA.</li>
</ol>
5. Preparing cells for immunofluorescent staining
NOTE: For a 24-well plate, one EB per well is sufficient.
<ol>
	<li>On day 35, aspirate the spent medium and wash the cells by adding 1 mL of DPBS+/+ per well (for one well of a 24-well plate).</li>
	<li>Aspirate the DPBS+/+ and add 500 &#181;L of cold 4% paraformaldehyde (PFA, see Table of Materials) prepared in DPBS+/+ per well. Incubate for 20 min at RT.</li>
	<li>Remove 4% PFA and wash the cells twice with DPBS+/+, as in step 5.1. Add 1 mL of DPBS+/+ and store the cells at 4 &#176;C until staining is done. 
	NOTE: It is advised to do the immunofluorescent staining within 1 week after fixation to avoid cell detachment and the loss of antigens. However, depending on the antibody and the cellular location of the target, staining can be done at later times up to 4 weeks after fixation.</li>
</ol>

<ol>
	<li>Erceg, S., Lukovic, D., Moreno-Manzano, V., Stojkovic, M., Bhattacharya, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+cerebellar+neurons+from+human+pluripotent+stem+cells.">Derivation of cerebellar neurons from human pluripotent stem cells.</a> Current Protocols in Stem Cell Biology. , (2012).</li><li>Erceg, S., 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=Efficient+differentiation+of+human+embryonic+stem+cells+into+functional+cerebellar-like+cells.">Efficient differentiation of human embryonic stem cells into functional cerebellar-like cells.</a> Stem Cells and Development. 19 (11), 1745-1756 (2010).</li><li>Muguruma, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+culture+for+self-formation+of+the+cerebellum+from+human+pluripotent+stem+cells+through+induction+of+the+isthmic+organizer.">3D culture for self-formation of the cerebellum from human pluripotent stem cells through induction of the isthmic organizer.</a> Methods in Molecular Biology. 1597, 31-41 (2017).</li><li>Muguruma, K., Nishiyama, A., Kawakami, H., Hashimoto, K., Sasai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organization+of+polarized+cerebellar+tissue+in+3D+culture+of+human+pluripotent+stem+cells.">Self-organization of polarized cerebellar tissue in 3D culture of human pluripotent stem cells.</a> Cell Reports. 10 (4), 537-550 (2015).</li><li>Holmes, D. B., Heine, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streamlined+3D+cerebellar+differentiation+protocol+with+optional+2D+modification.">Streamlined 3D cerebellar differentiation protocol with optional 2D modification.</a> Journal of Visualized Experiments: JoVE. (130), e56888 (2017).</li><li>Holmes, D. B., Heine, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+3D+protocol+capable+of+generating+early+cortical+neuroepithelium.">Simplified 3D protocol capable of generating early cortical neuroepithelium.</a> Biology Open. 6 (3), 402-406 (2017).</li><li>Silva, T. P., 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=Maturation+of+human+pluripotent+stem+cell-derived+cerebellar+neurons+in+the+absence+of+co-culture.">Maturation of human pluripotent stem cell-derived cerebellar neurons in the absence of co-culture.</a> Frontiers in Bioengineering and Biotechnology. 8, 70 (2020).</li><li>Nayler, S., Agarwal, D., Curion, F., Bowden, R., Becker, E. B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+transcriptional+landscape+of+xeno-free+human+induced+pluripotent+stem+cell-derived+cerebellar+organoids.">High-resolution transcriptional landscape of xeno-free human induced pluripotent stem cell-derived cerebellar organoids.</a> Scientific Reports. 11, 12959 (2021).</li><li>Salero, E., Hatten, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+ES+cells+into+cerebellar+neurons.">Differentiation of ES cells into cerebellar neurons.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (8), 2997-3002 (2007).</li><li>Nie, Y., Walsh, P., Clarke, D. L., Rowley, J. A., Fellner, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scalable+passaging+of+adherent+human+pluripotent+stem+cells.">Scalable passaging of adherent human pluripotent stem cells.</a> PLoS One. 9 (1), 88012 (2014).</li><li>Chen, Y., 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+versatile+polypharmacology+platform+promotes+cytoprotection+and+viability+of+human+pluripotent+and+differentiated+cells.">A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells.</a> Nature Methods. 18 (5), 528-541 (2021).</li><li>Machold, R., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Math1+is+expressed+in+temporally+discrete+pools+of+cerebellar+rhombic-lip+neural+progenitors.">Math1 is expressed in temporally discrete pools of cerebellar rhombic-lip neural progenitors.</a> Neuron. 48 (1), 17-24 (2005).</li><li>Wang, V. Y., Rose, M. F., Zoghbi, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Math1+expression+redefines+the+rhombic+lip+derivatives+and+reveals+novel+lineages+within+the+brainstem+and+cerebellum.">Math1 expression redefines the rhombic lip derivatives and reveals novel lineages within the brainstem and cerebellum.</a> Neuron. 48 (1), 31-43 (2005).</li><li>Hoshino, M., 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=Ptf1a,+a+bHLH+transcriptional+gene,+defines+GABAergic+neuronal+fates+in+cerebellum.">Ptf1a, a bHLH transcriptional gene, defines GABAergic neuronal fates in cerebellum.</a> Neuron. 47 (2), 201-213 (2005).</li><li>Mizuhara, E., 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=Purkinje+cells+originate+from+cerebellar+ventricular+zone+progenitors+positive+for+Neph3+and+E-cadherin.">Purkinje cells originate from cerebellar ventricular zone progenitors positive for Neph3 and E-cadherin.</a> Developmental Biology. 338 (2), 202-214 (2010).</li><li>Ferreira, A., Caceres, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+Class+III+&#946;-tubulin+isotype+in+developing+neurons+in+culture.">Expression of the Class III &#946;-tubulin isotype in developing neurons in culture.</a> Journal of Neuroscience Research. 32 (4), 516-529 (1992).</li><li>Sullivan, K. F., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+conserved+isotype-defining+variable+region+sequences+for+four+vertebrate+beta+tubulin+polypeptide+classes.">Identification of conserved isotype-defining variable region sequences for four vertebrate beta tubulin polypeptide classes.</a> Proceedings of the National Academy of Sciences of the United States of America. 83 (12), 4327-4331 (1986).</li><li>Joyner, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engrailed,+Wnt+and+Pax+genes+regulate+midbrain-hindbrain+development.">Engrailed, Wnt and Pax genes regulate midbrain-hindbrain development.</a> Trends in Genetics. 12 (1), 15-20 (1996).</li><li>Joyner, A. L., Liu, A., Millet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Otx2,+Gbx2+and+Fgf8+interact+to+position+and+maintain+a+mid-hindbrain+organizer.">Otx2, Gbx2 and Fgf8 interact to position and maintain a mid-hindbrain organizer.</a> Current Opinion in Cell Biology. 12 (6), 736-741 (2000).</li><li>Minaki, Y., Nakatani, T., Mizuhara, E., Inoue, T., Ono, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+transcriptional+corepressor,+Corl2,+as+a+cerebellar+Purkinje+cell-selective+marker.">Identification of a novel transcriptional corepressor, Corl2, as a cerebellar Purkinje cell-selective marker.</a> Gene Expression Patterns. 8 (6), 418-423 (2008).</li><li>Aldinger, K. A., 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=Spatial+and+cell+type+transcriptional+landscape+of+human+cerebellar+development.">Spatial and cell type transcriptional landscape of human cerebellar development.</a> Nature Neuroscience. 24 (8), 1163-1175 (2021).</li><li>Fossat, N., Courtois, V., Chatelain, G., Brun, G., Lamonerie, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+usage+of+Otx2+promoters+during+mouse+development.">Alternative usage of Otx2 promoters during mouse development.</a> Developmental Dynamics. 233 (1), 154-160 (2005).</li><li>Akbarian, S., 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=The+PsychENCODE+project.">The PsychENCODE project.</a> Nature Neuroscience. 18 (12), 1707-1712 (2015).</li><li>Aijaz, S., 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=Expression+analysis+of+SIX3+and+SIX6+in+human+tissues+reveals+differences+in+expression+and+a+novel+correlation+between+the+expression+of+SIX3+and+the+genes+encoding+isocitrate+dehyhrogenase+and+cadherin+18.">Expression analysis of SIX3 and SIX6 in human tissues reveals differences in expression and a novel correlation between the expression of SIX3 and the genes encoding isocitrate dehyhrogenase and cadherin 18.</a> Genomics. 86 (1), 86-99 (2005).</li><li>Conte, I., Morcillo, J., Bovolenta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+Six3+and+Six6+distribution+in+the+developing+and+adult+mouse+brain.">Comparative analysis of Six3 and Six6 distribution in the developing and adult mouse brain.</a> Developmental Dynamics. 234 (3), 718-725 (2005).</li><li>Andreasen, N. C., Pierson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+cerebellum+in+schizophrenia.">The role of the cerebellum in schizophrenia.</a> Biological Psychiatry. 64 (2), 81-88 (2008).</li><li>Nopoulos, P. C., Ceilley, J. W., Gailis, E. A., Andreasen, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+MRI+study+of+cerebellar+vermis+morphology+in+patients+with+schizophrenia:+Evidence+in+support+of+the+cognitive+dysmetria+concept.">An MRI study of cerebellar vermis morphology in patients with schizophrenia: Evidence in support of the cognitive dysmetria concept.</a> Biological Psychiatry. 46 (5), 703-711 (1999).</li><li>Jacobsen, L. K., 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=Quantitative+morphology+of+the+cerebellum+and+fourth+ventricle+in+childhood-onset+schizophrenia.">Quantitative morphology of the cerebellum and fourth ventricle in childhood-onset schizophrenia.</a> American Journal of Psychiatry. 154 (12), 1663-1669 (1997).</li><li>Saywell, V., Cioni, J. -. M., Ango, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+gene+expression+profile+of+axon+guidance+cues+in+Purkinje+cells+during+cerebellar+circuit+formation.">Developmental gene expression profile of axon guidance cues in Purkinje cells during cerebellar circuit formation.</a> The Cerebellum. 13 (3), 307-317 (2014).</li><li>Kim, D., Ackerman, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+UNC5C+netrin+receptor+regulates+dorsal+guidance+of+mouse+hindbrain+axons.">The UNC5C netrin receptor regulates dorsal guidance of mouse hindbrain axons.</a> Journal of Neuroscience. 31 (6), 2167-2179 (2011).</li><li>Maier, V., 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=Semaphorin+4C+and+4G+are+ligands+of+Plexin-B2+required+in+cerebellar+development.">Semaphorin 4C and 4G are ligands of Plexin-B2 required in cerebellar development.</a> Molecular and Cellular Neuroscience. 46 (2), 419-431 (2011).</li><li>Telley, L., 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=Dual+function+of+NRP1+in+axon+guidance+and+subcellular+target+recognition+in+cerebellum.">Dual function of NRP1 in axon guidance and subcellular target recognition in cerebellum.</a> Neuron. 91 (6), 1276-1291 (2016).</li><li>Wang, S., 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=Differentiation+of+human+induced+pluripotent+stem+cells+to+mature+functional+Purkinje+neurons.">Differentiation of human induced pluripotent stem cells to mature functional Purkinje neurons.</a> Scientific Reports. 5, 9232 (2015).</li><li>Shabanipour, S., 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=Primary+culture+of+neurons+isolated+from+embryonic+mouse+cerebellum.">Primary culture of neurons isolated from embryonic mouse cerebellum.</a> Journal of Visualized Experiments. (152), e60168 (2019).</li></ol>

The authors have no conflicts of interest to declare.

The ability to model human cerebellar development in vitro is important for disease modeling as well as furthering the understanding of normal brain development. Less complicated and cost-effective protocols create more opportunities for replicable data generation and broad implementation across multiple scientific labs. A cerebellar differentiation protocol is described here using a modified method of generating EBs that does not require enzymes or dissociation agents, using growth factors reported by Muguruma ...

Understanding human cerebellar development and the critical time windows of this process is important not only for decoding the possible causes of neurodevelopmental disorders but also for identifying new targets for therapeutic intervention. Modeling human cerebellar development in vitro has been challenging, yet over time, many protocols differentiating human embryonic stem cells (hESCs) or iPSCs with cerebellar lineage fates have emerged1,2,3,4,5,6,7,8. Furthermore, it is important to develop protocols that generate reproducible results, are relatively simple (to reduce error), and are not heavy on monetary costs.
The first protocols for cerebellar differentiation were generated from 2D cultures from plated embryoid bodies (EBs), inducing cerebellar fate with various growth factors similar to in vivo development, including WNT, BMPs, and FGFs1,9. More recent published protocols induced differentiation primarily in 3D organoid culture with FGF2 and insulin, followed by FGF19 and SDF1 for rhombic lip-like structures3,4, or used a combination of FGF2, FGF4, and FGF85. Both cerebellar organoid induction methods resulted in similar 3D cerebellar organoids as both protocols reported similar cerebellar marker expression at identical time points. Holmes and Heine extended their 3D protocol5 to show that 2D cerebellar cells can be generated from hESCs and iPSCs, which start as 3D aggregates. In addition, Silva et al.7 demonstrated that cells representing mature cerebellar neurons in 2D can be generated with a similar approach to Holmes and Heine, using a different time point for switching from 3D to 2D and extending the time of growth and maturation.
The current protocol induces cerebellar fate in feeder-free iPSCs by generating free-floating embryoid bodies (EBs) using insulin and FGF2 and then plating the EBs on PLO/laminin-coated dishes on day 14 for 2D growth and differentiation. By day 35, cells with cerebellar identity are obtained. The ability to recapitulate the early stages of cerebellar development, especially in a 2D environment, allows researchers to answer specific questions requiring experiments with a monolayer structure. This protocol is also amenable to further modifications such as micropatterned surfaces, axonal outgrowth assays, and cell sorting to enrich the desired cell populations.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL Serological pipette</td>
			<td>Fisher Scientific</td>
			<td>13-678-26D</td>
			<td></td>
		</tr>
		<tr>
			<td>1-thio-glycerol</td>
			<td>Sigma</td>
			<td>M6145</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Serological pipette</td>
			<td>Fisher Scientific</td>
			<td>13-678-26B</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL Filter Unit, 0.2 &#181;m aPES, 50 mm Dia</td>
			<td>Fisher Scientific</td>
			<td>FB12566502</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Easy Grip Tissue Cluture Dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>4D Nucleofector core unit</td>
			<td>Lonza</td>
			<td>276885</td>
			<td>Nucleofector</td>
		</tr>
		<tr>
			<td>5 mL Serological pipette</td>
			<td>Fisher Scientific</td>
			<td>13-678-25D</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Easy Grip Tissue Culture Dish</td>
			<td>Falcon</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well ultra-low attachment plates</td>
			<td>Corning</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>9&#34; Disposable Pasteur Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20D</td>
			<td></td>
		</tr>
		<tr>
			<td>Apo-transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture grade water</td>
			<td>Cytiva</td>
			<td>SH30529.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemically defined lipid concentrate</td>
			<td>Gibco</td>
			<td>11905031</td>
			<td></td>
		</tr>
		<tr>
			<td>Chroman 1</td>
			<td>Cayman</td>
			<td>34681</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II, Type A2, Biological safety Cabinet</td>
			<td>NuAire, Inc.</td>
			<td>NU-540-600</td>
			<td>Hood, UV light</td>
		</tr>
		<tr>
			<td>Costar 24-well plate, TC treated</td>
			<td>Corning</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 6-well plate, TC treated</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI solution</td>
			<td>Thermo Scientific</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO (Dimethly sulfoxide)</td>
			<td>Sigma</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS+/+</td>
			<td>Gibco</td>
			<td>14040133</td>
			<td></td>
		</tr>
		<tr>
			<td>Emricasan</td>
			<td>Cayman</td>
			<td>22204</td>
			<td></td>
		</tr>
		<tr>
			<td>Epi5 episomal iPSC reprogramming kit</td>
			<td>Life Technologies</td>
			<td>A15960</td>
			<td></td>
		</tr>
		<tr>
			<td>Essential 8-Flex</td>
			<td>Gibco</td>
			<td>A2858501</td>
			<td>PSC medium with heat-stable FGF2</td>
		</tr>
		<tr>
			<td>EVOS XL Core Imaging system</td>
			<td>Life Technologies</td>
			<td>AMEX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum - Premium Select</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>L-alanine-L-glutamine supplement</td>
		</tr>
		<tr>
			<td>Ham&#39;s F12 Nutrient Mix</td>
			<td>Gibco</td>
			<td>11765054</td>
			<td></td>
		</tr>
		<tr>
			<td>HERAcell VIOS 160i CO2 incubator</td>
			<td>Thermo Scientific</td>
			<td>50144906</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Anti-EN2, mouse</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-293311</td>
			<td></td>
		</tr>
		<tr>
			<td>Human anti-Ki67/MKI67, rabbit</td>
			<td>R&#38;D Systems</td>
			<td>MAB7617</td>
			<td></td>
		</tr>
		<tr>
			<td>Human anti-PTF1a, rabbit</td>
			<td>Novus Biologicals</td>
			<td>NBP2-98726</td>
			<td></td>
		</tr>
		<tr>
			<td>Human anti-TUBB3, mouse</td>
			<td>Biolegend</td>
			<td>801213</td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Gibco</td>
			<td>12440053</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Gibco</td>
			<td>12585</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin Mouse Protein</td>
			<td>Gibco</td>
			<td>23017015</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Matrix</td>
			<td>Corning</td>
			<td>354234</td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>MEM-NEAA</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Centrifuge</td>
			<td>Labnet International</td>
			<td>C1310</td>
			<td>Benchtop mini centrifuge</td>
		</tr>
		<tr>
			<td>Monarch RNA Cleanup Kit (50 &#181;g)</td>
			<td>New England BioLabs</td>
			<td>T2040</td>
			<td>Silica spin columns</td>
		</tr>
		<tr>
			<td>Monarch Total RNA Miniprep Kit</td>
			<td>New England BioLabs</td>
			<td>T2010</td>
			<td>Silica spin columns</td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA 16%</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyamine supplement</td>
			<td>Sigma</td>
			<td>P8483</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Ornithine (PLO)</td>
			<td>Sigma</td>
			<td>3655</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma</td>
			<td>746436</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Sigma</td>
			<td>54317</td>
			<td></td>
		</tr>
		<tr>
			<td>See through self-sealable pouches</td>
			<td>Steriking</td>
			<td>SS-T2 (90x250)</td>
			<td>Autoclave pouches</td>
		</tr>
		<tr>
			<td>Sodium citrate dihydrate&#160;</td>
			<td>Fisher Scientific</td>
			<td>S279-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filters, sterile, PES 0.22 &#181;m, 30 mm Dia</td>
			<td>Research Products International</td>
			<td>256131</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-ISRIB</td>
			<td>Cayman</td>
			<td>16258</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Invitrogen</td>
			<td>15596018</td>
			<td>Phenol and guanidine isothiocyanate</td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme (1x)</td>
			<td>Gibco</td>
			<td>12604039</td>
			<td>Cell dissociation reagent&#160;</td>
		</tr>
		<tr>
			<td>Vapor pressure osmometer</td>
			<td>Wescor, Inc.</td>
			<td>Model 5520</td>
			<td>Osmometer</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Biogems</td>
			<td>1293823</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling human cerebellar development in vitro in 2d structure

The precise and timely development of the cerebellum is crucial not only for accurate motor coordination and balance but also for cognition. In addition, disruption in cerebellar development has been implicated in many neurodevelopmental disorders, including autism, attention deficit-hyperactivity disorder (ADHD), and schizophrenia. Investigations of cerebellar development in humans have previously only been possible through post-mortem studies or neuroimaging, yet these methods are not sufficient for understanding the molecular and cellular changes occurring in vivo during early development, which is when many neurodevelopmental disorders originate. The emergence of techniques to generate human-induced pluripotent stem cells (iPSCs) from somatic cells and the ability to further re-differentiate iPSCs into neurons have paved the way for in vitro modeling of early brain development. The present study provides simplified steps toward generating cerebellar cells for applications that require a 2-dimensional (2D) monolayer structure. Cerebellar cells representing early developmental stages are derived from human iPSCs via the following steps: first, embryoid bodies are made in 3-dimensional (3D) culture, then they are treated with FGF2 and insulin to promote cerebellar fate specification, and finally, they are terminally differentiated as a monolayer on poly-l-ornithine (PLO)/laminin-coated substrates. At 35 days of differentiation, iPSC-derived cerebellar cell cultures express cerebellar markers including ATOH1, PTF1&#945;, PAX6, and KIRREL2, suggesting that this protocol generates glutamatergic and GABAergic cerebellar neuronal precursors, as well as Purkinje cell progenitors. Moreover, the differentiated cells show distinct neuronal morphology and are positive for immunofluorescence markers of neuronal identity such as TUBB3. These cells express axonal guidance molecules, including semaphorin-4C, plexin-B2, and neuropilin-1, and could serve as a model for investigating the molecular mechanisms of neurite outgrowth and synaptic connectivity. This method generates human cerebellar neurons useful for downstream applications, including gene expression, physiological, and morphological studies requiring 2D monolayer formats.

Overview of the 3D to 2D cerebellar differentiation 
Cerebellar cells are generated starting from iPSCs. Figure 1A shows the overall workflow and the addition of major components for differentiation. On day 0, EBs are made by gently lifting the iPSC colonies (Figure 1B) using a pulled glass pipette in CDM containing SB431542 and Y-27632 and placed into ultra-low-attachment plates. FGF2 is added on day 2. On day 7, one-third of the medium is...

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Modeling Human Cerebellar Development In Vitro in 2D Structure

The present protocol explains the generation of a 2D monolayer of cerebellar cells from induced pluripotent stem cells for investigating the early stages of cerebellar development.

Modeling Human Cerebellar Development In Vitro in 2D Structure

The precise and timely development of the cerebellum is crucial not only for accurate motor coordination and balance but also for cognition. In addition, ...

modeling-human-cerebellar-development-in-vitro-in-2d-structure

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1.6K Views.  University of Iowa. The present protocol explains the generation of a 2D monolayer of cerebellar cells from induced pluripotent stem cells for investigating the early stages of cerebellar development. 

Video: Modeling Human Cerebellar Development In Vitro in 2D Structure

Operant Sensation Seeking in the Mouse

Operant methods are powerful behavioral tools for the study of motivated behavior. These 'self-administration' methods have been used extensively in drug addiction research due to their high construct validity. Operant studies provide researchers a tool for preclinical investigation of several aspects of the addiction process. For example, mechanisms of acute reinforcement (both drug and non-drug) can be tested using pharmacological or genetic tools to determine the ability of a molecular target to influence self-administration behavior1-6. Additionally, drug or food seeking behaviors can be studied in the absence of the primary reinforcer, and the ability of pharmacological compounds to disrupt this process is a preclinical model for discovery of molecular targets and compounds that may be useful for the treatment of addiction3,7-9. One problem with performing intravenous drug self-administration studies in the mouse is the technical difficulty of maintaining catheter patency. Attrition rates in these experiments are high and can reach 40% or higher10-15. Another general problem with drug self-administration is discerning which pharmacologically-induced effects of the reinforcer produce specific behaviors. For example, measurement of the reinforcing and neurological effects of psychostimulants can be confounded by their psychomotor effects. Operant methods using food reinforcement can avoid these pitfalls, although their utility in studying drug addiction is limited by the fact that some manipulations that alter drug self-administration have a minimal impact on food self-administration. For example, mesolimbic dopamine lesion or knockout of the D1 dopamine receptor reduce cocaine self-administration without having a significant impact on food self-administration 12,16. 
Sensory stimuli have been described for their ability to support operant responding as primary reinforcers (i.e. not conditioned reinforcers)17-22. Auditory and visual stimuli are self-administered by several species18,21,23, although surprisingly little is known about the neural mechanisms underlying this reinforcement. The operant sensation seeking (OSS) model is a robust model for obtaining sensory self-administration in the mouse, allowing the study of neural mechanisms important in sensory reinforcement24. An additional advantage of OSS is the ability to screen mutant mice for differences in operant behavior that may be relevant to addiction. We have reported that dopamine D1 receptor knockout mice, previously shown to be deficient in psychostimulant self-administration, also fail to acquire OSS24. This is a unique finding in that these mice are capable of learning an operant task when food is used as a reinforcer. While operant studies using food reinforcement can be useful in the study of general motivated behavior and the mechanisms underlying food reinforcement, as mentioned above, these studies are limited in their application to studying molecular mechanisms of drug addiction. Thus, there may be similar neural substrates mediating sensory and psychostimulant reinforcement that are distinct from food reinforcement, which would make OSS a particularly attractive model for the study of drug addiction processes. The degree of overlap between other molecular targets of OSS and drug reinforcers is unclear, but is a topic that we are currently pursuing. While some aspects of addiction such as resistance to extinction may be observed with OSS, we have found that escalation 25 is not observed in this model24. Interestingly, escalation of intake and some other aspects of addiction are observed with self-administration of sucrose26. Thus, when non-drug operant procedures are desired to study addiction-related processes, food or sensory reinforcers can be chosen to best fit the particular question being asked. 
In conclusion, both food self-administration and OSS in the mouse have the advantage of not requiring an intravenous catheter, which allows a higher throughput means to study the effects of pharmacological or genetic manipulation of neural targets involved in motivation. While operant testing using food as a reinforcer is particularly useful in the study of the regulation of food intake, OSS is particularly apt for studying reinforcement mechanisms of sensory stimuli and may have broad applicability to novelty seeking and addiction.

In this protocol we describe a method of operant learning using sensory stimuli as a reinforcer in the mouse. It requires no prior training or food restriction, and it allows the study of motivated behavior without the use of a pharmacological or natural reinforcer such as food.

Operant methods are powerful behavioral tools for the study of motivated behavior.  These 'self-administration' methods have been used extensively in drug ...

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. 
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

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 ...

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, and spinal muscular atrophy, which are all associated with muscular atrophy. Primary cultures of spinal MNs have been used widely to demonstrate the involvement of specific genes in such diseases and characterize the cellular consequences of their mutations. This protocol models a primary MN culture derived from the seminal work of Henderson and colleagues more than twenty years ago. First, we detail a method of dissecting the anterior horns of the spinal cord from a mouse embryo and isolating the MNs from neighboring cells using a density gradient. Then, we present a new way of efficiently transfecting MNs with expression plasmids using magnetofection. Finally, we illustrate how to fix and immunostain primary MNs. Using neurofilament mutations that cause Charcot-Marie-Tooth disease type 2, this protocol demonstrates a qualitative approach to expressing proteins of interest and studying their involvement in MN growth, maintenance, and survival.

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

The goal of this technique is to prepare a highly enriched culture of primary motoneurons (MNs) from murine spinal cord. To evaluate the consequences of mutations causing MN diseases, we describe here the isolation of these isolated MNs and their transfection by magnetofection.

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...