This work was supported by the Heart and Stroke Foundation, Ontario Institute of Regenerative Medicine, Canada First Research Excellence Fund (Medicine by Design, MbD), and Connaught Innovation Award (University of Toronto).

This protocol was approved by the Animal Care Committee at the University of Toronto and adheres to the Guide to the Care and Use of Experimental Animals (2nd Edition, Canadian Council on Animal Care, 2017). For this study, wild-type (C57BL/6J) and transgenic tdTom-Cre reporter (B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J)22 mouse strains were used. Mice were 7-9 weeks old and included both males and females. Details of the reagents and equipment used are listed in the Table of Materials.
1. Endothelin-1 stroke surgery
NOTE: All surgical instruments and materials are sterilized by autoclave prior to surgery. The stereotaxic frame is sterilized with PREempt. Syringes are sterilized with PREempt, 70% alcohol, and primed with sterile PBS prior to surgery. Endothelin-1 (ET-1) is stored on ice throughout the surgery.
<ol>
	<li>Before starting the surgery, set up the recovery area by placing a clean cage on a heating pad set to 37 &#176;C. Alternatively, if a heating lamp is being used, place only half of the cage under the heat.
	<ol>
		<li>To anesthetize mice using isoflurane, follow the protocol approved by the Animal Care Committee. Place the mouse in the anesthetic induction chamber and turn on the oxygen to 1-1.5 L/min, then adjust the isoflurane vaporizer to 3%-5% to initiate anesthesia.</li>
	</ol>
	</li>
	<li>Remove the animal from the chamber once it lies on its side and can no longer stand, and place it onto a clean surface. Ensure the animal is adequately anesthetized by confirming the absence of a toe pinch reflex before and throughout the surgery.
	<ol>
		<li>Secure the nose cone extended from the anesthetic machine on the animal and adjust isoflurane to 1%-2% for maintenance. Minimize the exposure to isoflurane (5 min at 3%-5% for induction and the remaining time at 1%-2%), as the procedure can take up to 40 min to complete.</li>
	</ol>
	</li>
	<li>Use animal hair trimmers to remove fur from the neck/behind ears to the nose of the animal to expose the skin on the skull for surgery.</li>
	<li>Weigh the animal and administer the appropriate dose of pre-operative analgesic subcutaneously (Meloxicam (2.0 mg/kg)) based on the weight.</li>
	<li>Apply eye lubricant ointment on each eye of the mouse to prevent corneal desiccation during anesthesia.</li>
	<li>Sterilize the fur-clipped skin area (step 1.3) with 70% ethanol, followed by Chlorhexidine alcohol twice to form a surgical field.</li>
	<li>Transfer the mouse onto a mouse stereotaxic frame by first placing its nose in the stereotaxic nose cone, then stabilize the skull with ear bars. Keep the mouse on a heating pad set to 37 &#176;C for thermal support throughout the surgery procedure. Maintain isoflurane at 1%-2% so that the mouse displays a respiratory rate of 1 breath/s.</li>
	<li>Repeat application of eye lubrication to prevent corneal desiccation during the procedure if needed. Apply a sterile surgical drape on the body of the mouse, below the ears, to extend the size of the sterile field.</li>
	<li>Make a vertical incision parallel to the sagittal plane of the animal from behind the mid-point of the eyes to the back of the head (parietal bone) with a #10 scalpel blade. Expose the skull by gently retracting the skin towards the sides.</li>
	<li>Use a Q-tip to disrupt the fascia on the skull and allow the skull to dry to expose bregma.</li>
	<li>Set up manipulator arms with drill holder carrying high-speed stereotaxic drill with a .018&#34; drill bit.</li>
	<li>Drill 3 burr holes (.018&#34; diameter) with a high-speed stereotaxic drill at the coordinates in the right sensorimotor cortex at 2.25 mm lateral to bregma and +0.0 mm rostral, +1.0 mm rostral and -1.0 mm caudal to bregma.</li>
	<li>Replace the drill with a 26 G syringe with a 0.375&#8243; long needle.</li>
	<li>Load 1.5 &#181;L of 400 &#181;M ET-1 dissolved in sterile PBS into the syringe. To ensure good flow, release a small volume of ET-1 until a drop can be observed at the tip of the needle. Use a sterile Q-tip to wipe off the excess before performing an injection.</li>
	<li>Lower the tip of the needle past the skull in the middle burr hole (in the right sensorimotor cortex at + 0.0 anterior and 2.25 mm lateral to bregma) without puncturing the dura mater. Once aligned, lower the needle 1 mm into the brain.</li>
	<li>Inject 1 &#181;L of ET-1 by dispensing 0.1 &#181;L at a time. Push the plunger of the Hamilton syringe forward to dispense 0.1 &#181;L and wait for a minute before injecting the next 0.1 &#181;L, inject until 1 &#181;L has been dispensed.
	<ol>
		<li>After the final 0.1 &#181;L has been dispensed (0.1 &#181;L x 10 injections = 1 &#181;L total), keep the needle in place for 10 min to prevent backflow and then slowly withdraw upward to remove the syringe.</li>
	</ol>
	</li>
	<li>To ensure the needle does not become blocked during injection, release a small volume of the ET-1 until a drop can be observed at the tip of the needle. Use a sterile Q-tip to wipe off the excess before performing an injection. Sterilize syringes before injecting the next animal with PREempt, followed by 70% alcohol, and then sterile PBS.</li>
	<li>To close the wound, suture the overlying skin on the skull together using a 4-0 polysorb polyester sterile suture.</li>
	<li>Administer antibiotic ointment using a cotton swab on the sutured area to prevent post-operative infection.</li>
	<li>Remove the mouse from the stereotaxic frame and transfer it to a clean, pre-heated animal housing cage in the recovery area. Turn off the isoflurane vaporizer and oxygen.</li>
	<li>Keep the cage on top of a heating pad for the duration of recovery from anesthesia. Alternatively, if using a heating lamp place the mouse on the side of the cage under the lamp so that the mouse can move to the cool side of the cage as it wakes up from anesthesia to prevent overheating. Monitor the mouse as it comes out of anesthesia in the next 5-10 min.</li>
	<li>Monitor mouse over the next 3 days (weight monitoring), provide post-operative care (moist mashed chow to ensure hydration), and analgesics (2 mg/kg meloxicam, 2x daily subcutaneously, for 2-3 days) in accordance with regulations set out by local animal care committee.</li>
</ol>
2. Adeno-Associated Virus (AAV) delivery (7 days - subacute model or 21 days - chronic model) post-stroke
CAUTION: Refer to institutional biosafety guidelines for working with AAVs. As per the guidelines at the University of Toronto, this surgery is performed in a Containment Level 2 Biosafety Cabinet.
NOTE: All surgical instruments and materials are sterilized by autoclave prior to surgery. The stereotaxic frame is sterilized with PREempt. Syringes are sterilized with PREempt, 70% alcohol, and primed with sterile PBS. AAVs are stored on ice throughout the surgery.
<ol>
	<li>Before starting the surgery, set up the recovery area by placing a clean cage on a heating pad set to 37 &#176;C. Alternatively, if a heating lamp is being used, place only half of the cage under the heat.
	<ol>
		<li>To anesthetize mice using isoflurane, place the mouse in the anesthetic induction chamber and turn on the oxygen to 1-1.5 L/min, then adjust the isoflurane vaporizer to 3%-5% to initiate anesthesia (following the instutionally approved protocols).</li>
	</ol>
	</li>
	<li>Remove the animal from the chamber once it lies on its side and can no longer stand on a clean surface. Ensure the animal is adequately anesthetized by confirming the absence of a toe pinch reflex before and throughout the surgery.
	<ol>
		<li>Secure the nose cone extended from the anesthetic machine on the animal and adjust isoflurane to 1%-2% for maintenance. Minimize the exposure to isoflurane (5 min at 3%-5% for induction and the remaining time at 1%-2%), as the procedure can take up to 40 min to complete.</li>
	</ol>
	</li>
	<li>Weigh the animal and administer the appropriate dose of pre-operative analgesic subcutaneously (Meloxicam (2.0 mg/kg)) based on the weight.</li>
	<li>Apply eye lubricant ointment on each eye of the mouse to prevent corneal desiccation from being under anesthesia.</li>
	<li>Sterilize the fur-clipped skin area with 70% ethanol, followed by Chlorhexidine alcohol twice to form a surgical field.</li>
	<li>Transfer the mouse onto a mouse stereotaxic frame by first placing its nose in the stereotaxic nose cone, then stabilize the skull with ear bars. Keep the mouse on a heating pad set to 37 &#176;C for thermal support throughout the surgery procedure. Maintain isoflurane at 1%-2% so that the mouse displays the respiratory rate of 1 breath/s.</li>
	<li>Repeat application of eye lubrication to prevent corneal desiccation during the procedure if needed. Apply a sterile surgical drape on the body of the mouse, below the ears, to extend the size of sterile field.</li>
	<li>If still present, cut sutures with a pair of surgical scissors and remove the scab with a pair of forceps. Retract the skin to expose the skull. 
	NOTE: For the chronic model it may be necessary to excise the skin with #10 scalpel blade.</li>
	<li>Use a sterile Q-tip to dry the skull and locate burr holes.</li>
	<li>Set up manipulator arms of the stereotaxic apparatus with a needle holder carrying a 26 G Syringe with a 0.375&#8243; long needle.</li>
	<li>Load 3.4 &#181;L of AAV (1 x 109 Genome Copies(GC)/&#181;L for acute study; 1 x 1010 GC/&#181;L for chronic study) into the syringe. To ensure that the flow is good, release a small volume of AAV until a drop can be observed at the tip of the needle. Use a sterile Q-tip to wipe off the excess before performing an injection.</li>
	<li>Lower the tip of the needle past the skull in the first burr hole used for ET-1 (in the right sensorimotor cortex at + 0.0 anterior and 2.25 mm lateral to bregma) without puncturing the dura mater. Once aligned, lower the needle 1 mm into the brain. (0 AP, +2.25 ML, &#8722;1.0 DV from bregma).</li>
	<li>Inject 1 &#181;L of AAV (1 x 109 GC/&#181;L for acute study; 1 x 1010 GC/&#181;L for chronic study) by dispensing 0.1 &#181;L at a time. Push the plunger of the Hamilton syringe forward to dispense 0.1 &#181;L and wait for a minute before injecting the next 0.1 &#181;L; inject until 1 &#181;L has been dispensed. After the final 0.1 &#181;L has been dispensed (0.1 &#181;L x 10 injections = 1 &#181;L total), keep the needle in place for 10 min to prevent backflow and then slowly withdraw upward to remove the syringe.</li>
	<li>To ensure the needle does not become blocked during injection, release a small volume of the AAV until a drop can be observed at the tip of the needle. Use a sterile Q-tip to wipe off the excess before performing an injection.</li>
	<li>Repeat steps 2.11-2.14 for the AAV injections into the two remaining burr holes (in the right sensorimotor cortex at +1 AP, +2.25 ML, &#8722;1.0 DV; and &#8722;1 AP, +2.25 ML, &#8722;1.0 DV mm from bregma. Sterilize syringes before injecting the next animal with PREempt, followed by 70% alcohol, and finally sterile PBS.</li>
	<li>To close the wound, suture the overlying skin on the skull together using a 4-0 polysorb polyester sterile suture.</li>
	<li>Administer antibiotic ointment by using a cotton swab on the sutured area to prevent post-operative infection.</li>
	<li>Remove the mouse from the stereotaxic frame and transfer it to a clean, pre-heated animal housing cage in the recovery area. Turn off the isoflurane vaporizer and oxygen.</li>
	<li>Keep the mouse inside the cage and sterilize the outside of the cage with PREempt and 70% alcohol before placing it on top of a heating pad for recovery from anesthesia.
	<ol>
		<li>Alternatively, if using a heating lamp, place the mouse on the side of the cage under the lamp so that the mouse can move to the cool side of the cage as it wakes up from anesthesia to prevent overheating. Monitor the mouse as it comes out of anesthesia in the next 5-10 min.</li>
	</ol>
	</li>
	<li>Monitor the mouse over the next 3 days (weight monitoring), provide post-operative care (moist mashed chow to ensure hydration), and analgesics (2 mg/kg meloxicam, 2x daily subcutaneously, for 2-3 days) in accordance with regulations set out by local animal care committee.</li>
</ol>
3. Tissue preparation and dissection
<ol>
	<li>To euthanize mouse, intraperitoneally inject the mouse with Tribromoethanol (Avertin) (conc. 12.5 mg/mL, 250 mg/BW(kg)) and allow 2-3 min for complete induction. Ensure the animal is adequately anesthetized by removing the mouse from the cage and confirming the absence of a toe pinch reflex.</li>
	<li>In a fume hood, perform transcardial perfusion26 with saline followed by 4% paraformaldehyde. Spray the head of the euthanized animal with 70% ethanol until soaked.</li>
	<li>Cut off the head at the base of the skull by using a pair of large scissors. Cut open the skin along the midline of the entire skull using straight 4-1/2&#34; iris scissors. Retract the skin to expose the skull.</li>
	<li>From the opening of the spinal cavity at the neck, insert the iris scissors and cut through the skull along the sagittal and inter-frontal suture.</li>
	<li>Then insert scissors into the two eye sockets and cut through the frontal-nasal suture.</li>
	<li>Take particular care not to damage the brain. Pry open the skull plates to the left and right until the brain is exposed.</li>
	<li>Transfer the brain into a scintillation vial with 4% paraformaldehyde. Keep the brain in 4% paraformaldehyde at 4 &#176;C for 4 h.</li>
	<li>The brain is then cryoprotected by replacing 4% paraformaldehyde with 30% sucrose. Keep the brain in 30% sucrose at 4 &#176;C until the brain sinks to the bottom of the vial (~12 h).</li>
</ol>
4. Sectioning frozen brains
<ol>
	<li>Set the temperature of the cryostat chamber between -20 &#176;C to -26 &#176;C.</li>
	<li>Make a coronal cut across the cerebellum and attach the flat, caudal part of the brain onto a cryostat chuck with optimal cutting temperature compound (OCT).</li>
	<li>Place the chuck with the brain in the disk holder and align the olfactory bulb with the knife blade.</li>
	<li>Section the brain into coronal slices with a thickness of 20 &#181;m. To avoid streaks or scratch lines on sectioned brain slices, place a piece of tape on the sectioning platform to create a small gap between the anti-rolling plate and the platform. A paintbrush can be used to adjust the orientation of the section before capturing it on a positively charged glass slide.</li>
	<li>Store slides containing brain sections at -20 &#176;C.</li>
</ol>
5. Immunohistochemistry and quantification
<ol>
	<li>Process the sections for immunohistochemistry using a primary antibody against NeuN, a pan-neuronal marker, according to standard protocols22,26.</li>
	<li>Thaw cryopreserved brain sections for 5 min at room temperature. Wash the sections three times with 1x phosphate-buffered saline (PBS) for 5 min each.</li>
	<li>Incubate the sections in 0.3% Triton X-100 in PBS for 20 min at room temperature to permeabilize.</li>
	<li>Prepare the blocking solution (5% normal goat serum and 0.3% Triton X-100 in PBS) and incubate the sections in the blocking solution for 1 h at room temperature.</li>
	<li>Dilute the primary antibody, rabbit anti-NeuN, at a 1:500 ratio in the blocking solution. Incubate the sections with the primary antibody solution overnight at 4 &#176;C.</li>
	<li>Wash the sections three times with 0.3% Triton X-100 in PBS, for 5 min each wash.</li>
	<li>Dilute the secondary antibody, goat anti-rabbit 568 or 488, at a 1:1000 ratio in PBS. Incubate the sections with the secondary antibody solution for 1 h at room temperature, protected from light.</li>
	<li>Perform three washes with 0.3% Triton X-100 in PBS, for 5 min each. Incubate the sections with DAPI (1:10,000 in PBS) for 5 min at room temperature.</li>
	<li>Wash the sections three more times with PBS, for 5 min each. Mount the stained sections on a glass slide with a fluorescence mounting medium and a glass coverslip. Allow mounted slides to dry overnight.</li>
	<li>Image stained slides using a 20x objective with a confocal microscope to obtain views of Reporter+ cells around the stroke lesion in 3 different coronal sections (between the anatomical regions of the crossing of the corpus callosum anteriorly and the crossing of the anterior commissure posteriorly) for each animal. 
	NOTE: The distance of the AAV spread will vary within the hemisphere. However, based on our experience and post-tissue processing analysis of Reporter+ cells, the AAV spread generally spans approximately +3 mm to -3 mm from bregma. Reporter+ cells around the stroke lesion (as identified by the absence/sparse staining of NeuN) in the area between where the corpus callosum crosses the midline and the anterior commissure connects should be quantified.</li>
	<li>For quantification of transduced neurons, perform colocalization analysis. Manually count the number of double-positive cells (Reporter+NeuN+) and the total number of transduced (Reporter+) cells in brain slices. For results herein, &#62;3 fields of view were counted surrounding the lesion (medial, lateral, and inferior) from 3 coronal sections per mouse.</li>
</ol>

AAV Systems and Mouse Models to Study Ectopic Neurod1 Expression Post-Stroke

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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=Ectopic+expression+of+neurod1+is+sufficient+for+functional+recovery+following+a+sensory-motor+cortical+stroke.">Ectopic expression of neurod1 is sufficient for functional recovery following a sensory-motor cortical stroke.</a> Biomedicines. 12 (3), 663 (2024).</li><li>Tai, W., Xu, X. M., Zhang, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+through+in+vivo+cell+fate+reprogramming+for+neural+repair.">Regeneration through in vivo cell fate reprogramming for neural repair.</a> Front Cell Neurosci. 14, 107 (2020).</li><li>Wu, Z., 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=Gene+therapy+conversion+of+striatal+astrocytes+into+gabaergic+neurons+in+mouse+models+of+Huntington's+disease.">Gene therapy conversion of striatal astrocytes into gabaergic neurons in mouse models of Huntington's disease.</a> Nat Commun. 11 (1), 1105 (2020).</li><li>Roome, R. B., 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+reproducible+endothelin-1+model+of+forelimb+motor+cortex+stroke+in+the+mouse.">A reproducible endothelin-1 model of forelimb motor cortex stroke in the mouse.</a> J Neurosci Methods. 233, 34-44 (2014).</li><li>Islam, R., 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=Inhibition+of+apoptosis+in+a+model+of+ischemic+stroke+leads+to+enhanced+cell+survival,+endogenous+neural+precursor+cell+activation+and+improved+functional+outcomes.">Inhibition of apoptosis in a model of ischemic stroke leads to enhanced cell survival, endogenous neural precursor cell activation and improved functional outcomes.</a> Int J Mol Sci. 25 (3), 1786 (2024).</li><li>Balkaya, M., Krober, J. M., Rex, A., Endres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+post-stroke+behavior+in+mouse+models+of+focal+ischemia.">Assessing post-stroke behavior in mouse models of focal ischemia.</a> J Cereb Blood Flow Metab. 33 (3), 330-338 (2013).</li><li>Guo, 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=High-titer+AAV+disrupts+cerebrovascular+integrity+and+induces+lymphocyte+infiltration+in+adult+mouse+brain.">High-titer AAV disrupts cerebrovascular integrity and induces lymphocyte infiltration in adult mouse brain.</a> Mol Ther Methods Clin Dev. 31, 101102 (2023).</li><li>Ortinski, P. I., 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=Selective+induction+of+astrocytic+gliosis+generates+deficits+in+neuronal+inhibition.">Selective induction of astrocytic gliosis generates deficits in neuronal inhibition.</a> Nat Neurosci. 13 (5), 584-591 (2010).</li><li>Jiang, M. Q., 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=Conversion+of+reactive+astrocytes+to+induced+neurons+enhances+neuronal+repair+and+functional+recovery+after+ischemic+stroke.">Conversion of reactive astrocytes to induced neurons enhances neuronal repair and functional recovery after ischemic stroke.</a> Front Aging Neurosci. 13, 612856 (2021).</li><li>Aschauer, D. F., Kreuz, S., Rumpel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+transduction+efficiency,+tropism+and+axonal+transport+of+AAV+serotypes+1,+2,+5,+6,+8+and+9+in+the+mouse+brain.">Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain.</a> PLoS One. 8 (9), e76310 (2013).</li><li>Su, 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=Expression+specificity+of+GFAP+transgenes.">Expression specificity of GFAP transgenes.</a> Neurochem Res. 29 (11), 2075-2093 (2004).</li><li>Kofoed, R. H., 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=Efficacy+of+gene+delivery+to+the+brain+using+AAV+and+ultrasound+depends+on+serotypes+and+brain+areas.">Efficacy of gene delivery to the brain using AAV and ultrasound depends on serotypes and brain areas.</a> J Control Release. 351, 667-680 (2022).</li><li>He, T., 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+influence+of+murine+genetic+background+in+adeno-associated+virus+transduction+of+the+mouse+brain.">The influence of murine genetic background in adeno-associated virus transduction of the mouse brain.</a> Hum Gene Ther Clin Dev. 30 (4), 169-181 (2019).</li><li>Hu, N. 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=Expression+patterns+of+inducible+cre+recombinase+driven+by+differential+astrocyte-specific+promoters+in+transgenic+mouse+lines.">Expression patterns of inducible cre recombinase driven by differential astrocyte-specific promoters in transgenic mouse lines.</a> Neurosci Bull. 36 (5), 530-544 (2020).</li><li>Botterill, J. J., 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=Off-target+expression+of+cre-dependent+adeno-associated+viruses+in+wild-type+c57bl/6j+mice.">Off-target expression of cre-dependent adeno-associated viruses in wild-type c57bl/6j mice.</a> eNeuro. 8 (6), ENEURO.0363-ENEURO.0421 (2021).</li><li>Hasel, P., Rose, I. V. L., Sadick, J. S., Kim, R. D., Liddelow, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinflammatory+astrocyte+subtypes+in+the+mouse+brain.">Neuroinflammatory astrocyte subtypes in the mouse brain.</a> Nat Neurosci. 24 (10), 1475-1487 (2021).</li><li>Kim, R. D., 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=Temporal+and+spatial+analysis+of+astrocytes+following+stroke+identifies+novel+drivers+of+reactivity.">Temporal and spatial analysis of astrocytes following stroke identifies novel drivers of reactivity.</a> bioRxiv. , (2023).</li><li>Matias, I., Morgado, J., Gomes, F. C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+heterogeneity:+Impact+to+brain+aging+and+disease.">Astrocyte heterogeneity: Impact to brain aging and disease.</a> Front Aging Neurosci. 11, 59 (2019).</li><li>Miller, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+heterogeneity+in+the+adult+central+nervous+system.">Astrocyte heterogeneity in the adult central nervous system.</a> Front Cell Neurosci. 12, 401 (2018).</li><li>Early, A. N., Gorman, A. A., Van Eldik, L. J., Bachstetter, A. D., Morganti, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+advanced+age+upon+astrocyte-specific+responses+to+acute+traumatic+brain+injury+in+mice.">Effects of advanced age upon astrocyte-specific responses to acute traumatic brain injury in mice.</a> J Neuroinflammation. 17 (1), 115 (2020).</li><li>Donocoff, R. S., Teteloshvili, N., Chung, H., Shoulson, R., Creusot, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+tamoxifen-induced+cre+activity+and+its+effect+on+immune+cell+populations.">Optimization of tamoxifen-induced cre activity and its effect on immune cell populations.</a> Sci Rep. 10 (1), 15244 (2020).</li><li>Gresita, 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=Very+low+efficiency+of+direct+reprogramming+of+astrocytes+into+neurons+in+the+brains+of+young+and+aged+mice+after+cerebral+ischemia.">Very low efficiency of direct reprogramming of astrocytes into neurons in the brains of young and aged mice after cerebral ischemia.</a> Front Aging Neurosci. 11, 334 (2019).</li></ol>

The authors declare no competing interests.

This protocol details two AAV systems and mouse models for investigating the ectopic expression of Neurod1 in the context of a mild-moderate ET-1 cortical stroke model. A number of critical steps relating to the ET-1 stroke are important to consider for the reproducibility and consistency of the injury. Burr holes must be carefully drilled without puncturing the dura mater to prevent unintentional surgical injury. It is important to use consistent landmarks, in this case bregma, to ensure a similar region of int...

Stroke is a leading cause of disability worldwide1. A stroke happens when blood flow to the brain is disrupted. This can occur either through a hemorrhagic stroke (~15% of cases), where a blood vessel in the brain bursts, or through an ischemic stroke, where blood flow to the brain is blocked1. Ischemic strokes are most prevalent and account for ~85% of stroke cases1. A stroke reduces the delivery of glucose and oxygen to the brain, leading to swift neuronal cell death and impaired neural function.
Ischemic stroke leads to the loss of cells within minutes in the core of the lesion through mechanisms of excitotoxicity, oxidative and nitrosative stress, calcium dysregulation, cortical spreading depolarization, edema, disruption of the blood-brain barrier (BBB), and inflammation2,3. Cells in the peri-infarct regions undergo apoptotic cell death within hours, and for days, post-stroke4. The peri-infarct environment is exposed to pro- and anti-inflammatory signals that lead to the activation of microglia and astrocytes (&#34;reactive&#34; astrocytes), that play diverse roles after injury5,6,7,8. Reactive astrocytes undergo a number of phenotypic changes that include the upregulation of glial fibrillary acidic protein (GFAP), and the formation of a border around the lesion within days post-stroke that helps to limit the extent of the lesion as well as impacts neuroplasticity within the injured tissue8,9,10.
Direct cellular reprogramming allows for the conversion of one mature cell type into a different mature cell type without going through a pluripotent state11. Turning resident brain cells into new neurons to substitute those lost to injury presents an appealing avenue for brain restoration. Stroke is an ideal target for cell-based treatments, given the necessity for therapeutic methods to replace lost neurons, broaden the therapeutic timeframe, and ultimately facilitate functional recovery. To this end, there have been many papers demonstrating astrocyte to neuron (AtN) reprogramming in vitro and in vivo, using a variety of basic- helix-loop-helix (bHLH) neurogenic transcription factors, including Ascl1, Neurog2, Neurod1 and more recently, mutated versions of Ascl1 (Ascl1-SA6) which demonstrated improved reprogramming efficacy11,12,13,14. Important to consider are the many experimental design factors that can impact outcomes, interpretation, and comparisons between studies. Ghazale et al. demonstrated that AtN reprogramming efficiency varied between different mouse strains, AAV serotypes, and promoters when using the same bHLH transcription factor14. Other considerations, such as the injury model, brain region, viral dosing, timing of gene delivery, the reprogramming factor, and the mode of delivery, will impact the outcomes. Recent reports have demonstrated that pre-existing transduced neurons have been misinterpreted as reprogrammed neurons15,16. While the reports do not necessarily negate that AtN conversion occurs, these studies have highlighted the importance of well-controlled experiments, and several papers have put forth recommendations for the next steps in the field15,17,18,19. What is apparent from the literature is that the ectopic expression of bHLH transcription factors can improve functional outcomes in a variety of animal models of neurodegenerative disease/injury13,20,21,22,23,24.
Herein, this protocol Neurod1, a transcription factor of interest, is ectopically expressed in transduced cells13,22. The well-established endothelin-1 (ET-1) model of ischemic stroke to the sensorimotor cortex in adult mice is used. Injection of ET-1 leads to local vasoconstriction mimicking the pathophysiology of ischemic stroke, including functional impairment22,23,25. The commonly employed short-GFAP promoter (gfaABC(1)D) is used to drive the ectopic expression of Neurod1 at the injury site and perilesional region using two viral strategies: (1) AAV5-CAG-Flex::GFP+AAV5-GFAP-Neurod1-Cre delivery in wild-type mice and (2) AAV5-GFAP-Neurod1-Cre delivery in tdTom-Cre reporter mice. To further understand the potential for ectopic expression of Neurod1 in the stroke-injured brain, AAVs were delivered in the subacute phase (7 days), or in the chronic phase (21 days), post-stroke. In both paradigms, significantly more labeled neurons were found within weeks following ectopic Neurod1 expression.

<table><tbody><tr><td>#77 Drill Bit (.018&amp;rdquo;)</td><td>David Kopf Instruments</td><td>8177</td><td></td></tr><tr><td>AAV5-GFAP(0.7)-mNeurod1-2A-iCre</td><td>Vector&amp;nbsp;Biolabs</td><td></td><td></td></tr><tr><td>Absorbable Suture with Needle Polysorb&amp;trade; Polyester CV-15 3/8 Circle Taper Point Needle Size 4 - 0 Braided</td><td>Covidien</td><td>GL-881</td><td></td></tr><tr><td>Anti-NeuN Antibody (rabbit)&amp;nbsp;</td><td>Millipore Sigma</td><td>ABN78&amp;nbsp;</td><td></td></tr><tr><td>C57BL/6 Mice</td><td>Charles River</td><td>027</td><td></td></tr><tr><td>Chlorhexidine Solution</td><td>Partnar</td><td>PCH-020</td><td></td></tr><tr><td>Contec&amp;trade;&amp;nbsp;PREempt&amp;trade; RTU Disinfectant Solution</td><td>Fisher Scientific</td><td>29-636-6212</td><td></td></tr><tr><td>Cryostat</td><td>Thermo Scientific&amp;nbsp;</td><td>HM525 NX&amp;nbsp;</td><td></td></tr><tr><td>Endothelin 1</td><td>Millipore Sigma</td><td>05-23-3800-0.5MG</td><td></td></tr><tr><td>Feather Safety Razor Microtome Blades</td><td>Feather</td><td>12-631P</td><td></td></tr><tr><td>Fisherbrand&amp;nbsp;Cover Glasses: Rectangles</td><td>Fisher Scientific</td><td>12-545M&amp;nbsp;</td><td></td></tr><tr><td>Fluorescence Mounting Medium</td><td>Agilent Technologies</td><td>S3023&amp;nbsp;</td><td></td></tr><tr><td>Hamilton Syringe</td><td>Hamilton Company</td><td>7634-01</td><td></td></tr><tr><td>High Speed Stereotaxic Drill</td><td>David Kopf Instruments</td><td>1474</td><td></td></tr><tr><td>Metacam Solution (Meloxicam)</td><td>Boehringer Ingelheim</td><td></td><td></td></tr><tr><td>O.C.T Compound</td><td>Fisher Scientific</td><td>23-730-571</td><td></td></tr><tr><td>pAAV2/5-GFAP-iCre</td><td>Vector Builder</td><td>P190924-1001suq</td><td></td></tr><tr><td>Polyderm Ointment USP</td><td>TARO</td><td>2181908</td><td></td></tr><tr><td>SOMNI Scientific&amp;trade;&amp;nbsp;The Animal Temperature Heating Pad</td><td>Fisher Scientific</td><td>04-777-177</td><td></td></tr><tr><td>Stereotaxic Instruments</td><td>David Kopf Instruments</td><td>Model 902</td><td></td></tr><tr><td>Superfrost Plus Microscope Slides, white</td><td>Fisherbrand</td><td>12-550-15</td><td></td></tr><tr><td>tdTomato Reporter Mice&amp;nbsp;</td><td>The Jackson Laboratory</td><td>007914</td><td></td></tr><tr><td>V-1 Tabletop Laboratory Animal Anesthesia System</td><td>VetEquip</td><td>901806</td><td></td></tr></tbody></table>

aav systems and mouse models for investigating ectopic expression of neurod1 in transduced cells at subacute and chronic times post-ischemic stroke

Ectopic expression of neurogenic factors in vivo has emerged as a promising approach for replacing lost neurons in disease models. The use of neural basic helix-loop-helix (bHLH) transcription factors via non-propagating virus-like particle systems, including retrovirus, lentivirus, and adeno-associated virus (AAV), has been extensively reported. For in vivo experiments, AAVs are increasingly used due to their low pathogenicity and potential for translatability. This protocol describes two AAV systems for investigating the ectopic expression of transcription factors in transduced cells post-ischemic stroke. In both systems, Neurod1 expression is controlled by the short GFAP (gfaABC(1)D) promoter, which is upregulated in reactive astrocytes post-stroke as well as in endogenous neurons when combined with neurogenic factor expression. In the ischemic stroke model described, focal ischemia is induced by injecting endothelin-1 (ET-1) into the motor cortex of mice, creating a lesion surrounded by reactive GFAP-expressing astrocytes and surviving neurons. Intracerebral injections of AAV are performed to ectopically induce the expression of Neurod1 in the subacute (7 days) and chronic (21 days) phases post-stroke. Within weeks following AAV injection, a significantly higher number of neurons among transduced cells are identified in mice ectopically expressing Neurod1 compared to mice receiving AAV control viruses. The AAV-based strategies used replicated observed outcomes of increased numbers of neurons expressing the reporter gene in a model of mild-to-moderate cortical stroke. This protocol establishes a standard platform for exploring the effects of ectopic expression of transcription factors delivered with AAV-based systems, contributing to the understanding of neurogenic factor expression in the context of stroke.

To examine the cellular outcomes of short GFAP (gfaABC(1)D) promoter driven, ectopic Neurod1 expression in wild-type (C57BL/6J) and in transgenic tdTom-Cre reporter strains (specifically, B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J)22 two AAV5-based systems were utilized in ET-1 stroke injured mice (Figure 1A-C). In a subacute model of stroke, Neurod1 was&#160;packaged in the Cre-Flex AAV system and deliver...

AAV Systems and Mouse Models for Investigating Ectopic Expression of Neurod1 in Transduced Cells at Subacute and Chronic Times Post-Ischemic Stroke

This protocol describes the viral-mediated ectopic expression of Neurod1 following cortical ischemic stroke. Neurod1 is delivered (1) using the Cre-Flex AAV system in wild-type mice during the subacute phase post-stroke (7 days) and (2) using a single AAV vector in conditional reporter mice during the chronic phase post-stroke (21 days).

Ectopic expression of neurogenic factors in vivo has emerged as a promising approach for replacing lost neurons in disease models. The use of neural basic ...

aav-systems-mouse-models-for-investigating-ectopic-expression-neurod1

Research

JoVE Journal

Neuroscience

396 Views.  University of Toronto. We onderzoeken AAV-gemedieerde ectopische expressie van neurogene transcriptiefactoren als een mogelijke behandeling voor ischemische beroerte. Onze focus is om te zien of neurod1-expressie in cellen die GFAP tot expressie brengen tijdens de subacute en chronische fasen na een beroerte het aantal getransduceerde neuronen verhoogt en correleert met verbeteringen in de motorische functie. Recente bevindingen tonen aan dat genexpressie in de cortex varieert per ...

Video: AAV-systemen en muismodellen voor het onderzoeken van ectopische expressie van neurod1 in getransduceerde cellen op subacute en chronische tijden na ischemische be

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Developmental Biology

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and contributes significantly to the development of vascular dementia. Few models of focal white matter stroke exist and this lack of appropriate models has hampered understanding of the neurobiologic mechanisms involved in injury response and repair after this type of stroke. The main limitation of other subcortical stroke models is that they do not focally restrict the infarct to the white matter or have primarily been validated in non-murine species. This limits the ability to apply the wide variety of murine research tools to study the neurobiology of white matter stroke. Here we present a methodology for the reliable production of a focal stroke in murine white matter using a local injection of an irreversible eNOS inhibitor. We also present several variations on the general protocol including two unique stereotactic variations, retrograde neuronal tracing, as well as fresh tissue labeling and dissection that greatly expand the potential applications of this technique. These variations allow for multiple approaches to analyze the neurobiologic effects of this common and understudied form of stroke.

Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and ...

Medicine

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific genes. Multiple means of assessing non-coding DNA and various chromatin states have proven useful in predicting the presence of enhancer sequences in the genome, but validating the activity of these sequences and finding the organs and developmental stages they are active in is a labor-intensive process. Recent advances in adeno-associated virus (AAV) vectors have enabled the widespread delivery of transgenes to mouse tissues, enabling in vivo enhancer testing without necessitating a transgenic animal. This protocol shows how a reporter construct that expresses EGFP under the control of a minimal promoter, which does not drive significant expression on its own, can be used to study the activity patterns of candidate enhancer sequences in the mouse brain. An AAV-packaged reporter construct is delivered to the mouse brain and incubated for 1-4 weeks, after which the animal is sacrificed, and brain sections are observed under a microscope. EGFP appears in cells in which the tested enhancer is sufficient to initiate gene expression, pinpointing the location and developmental stage in which the enhancer is active in the brain. Standard cloning methods, low-cost AAV packaging, and expanding AAV serotypes and methods for in vivo delivery and standard imaging readout make this an accessible approach for the study of how gene expression is regulated in the brain.

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

This protocol describes a novel rAAV-based transient enhancer-reporter assay. This assay can be used to induce enhancer-driven expression in vivo in the mouse brain.

Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

Selective Gene Silencing in the Choroid Plexus

Targeted Knockdown of Genes in the Choroid Plexus

The choroid plexus (ChP) serves as a critical gateway for immune cell infiltration into the central nervous system (CNS) under both physiological and pathological conditions. Recent research has shown that regulating ChP activity may offer protection against CNS disorders. However, studying the biological function of the ChP without affecting other brain regions is challenging due to its delicate structure. This study presents a novel method for gene knockdown in ChP tissue using adeno-associated viruses (AAVs) or cyclization recombination enzyme (Cre) recombinase protein consisting of TAT sequence (CRE-TAT). The results demonstrate that after injecting AAV or CRE-TAT into the lateral ventricle, the fluorescence was exclusively concentrated in the ChP. Using this approach, the study successfully knocked down the adenosine A2A receptor (A2AR) in the ChP using RNA interference (RNAi) or Cre/locus of X-overP1 (Cre/LoxP) systems, and showed that this knockdown could alleviate the pathology of experimental autoimmune encephalomyelitis (EAE). This technique may have important implications for future research on the ChP's role in CNS disorders.

Author Spotlight: Insights and Innovations in Gene Expression Manipulation Techniques for Choroid Plexus Research 

Here, we describe a method to selectively alter gene expressions in the choroid plexus while avoiding any impact in other brain areas.

The choroid plexus (ChP) serves as a critical gateway for immune cell infiltration into the central nervous system (CNS) under both physiological and ...

AAV Systems and Mouse Models for Investigating Ectopic Expression of Neurod1 in Transduced Cells at Subacute and Chronic Times Post-Ischemic Stroke

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