The authors thank Mr. Masaaki Kaneta, Ms. Hiromi Suzuki, and Ms. Mitsue Kawashima (Department of Regenerative and Infectious Pathology, Hamamatsu University School of Medicine) for their excellent technical assistance. This work was supported by the Japan Society for the Promotion of Science, KAKENHI Grant Number 23590445.

All the experimental protocols were approved by the Animal Care Committee of Hamamatsu University of School of Medicine.
1. Preparation of MCMV (Smith strain) and Recombinant M32-enhanced Green Fluorescent Protein (EGFP)-MCMV
<ol>
	<li>Generate recombinant M32-EGFP-MCMV according to the method as follows (1.2 - 1.9) and as previously described8.</li>
	<li>Use recombinant viruses derived from the Smith strain of wild-type MCMV (accession number: U68299). Insert EGFP (4,361 base pairs; bp) between 37,089 and 41,450 bp (M32 - M31 locus) in the MCMV genome by homologous recombination.</li>
	<li>Amplify by polymerase chain reaction the MCMV M32 (41,450 - 39,286 bp, left flanking sequence and M31 (37,089 - 39,246 bp, right flanking sequence gene loci using the following primers: M32, 5&#8242;-CTACTAGCTAGCCTTCCGCGAGTCGCTGTATT-3&#8242; (forward primer), 5&#8242;-CTACTAGCTAGCCTTCCGCGAGTCGCTGTATT-3&#8242; (reverse primer); M31, 5&#8242;-CTAAATTAACTTAAGTCGTCCTCTTCGTCTCACAA-3&#8242; (forward primer), 5&#8242;-AACTAGTCTAGATCGCTCCTGGTTGGTTTTTA-3&#8242; (reverse primer).</li>
	<li>Insert the M32 locus into the EGFP-expressing vector using NheI and BamHI restriction sites. Insert the M31 locus into the M32-EGFP-recombinant plasmid using AflII and XbaI sites. Cleave the M32-EGFP-M31 sequence with NheI and AflII from the M32-EGFP-M31 recombination plasmid and dissolve in H2O.</li>
	<li>Transfect the cleaved construct into the nuclei of MCMV (Smith strain)-infected NIH3T3 cells 24 hr post-infection (hpi) using an electroporation system to induce homologous recombination.</li>
	<li>At 3 days post-infection, co-culture the cells with uninfected mouse embryonic fibroblasts (MEFs) at a ratio of 1/1,000 in six-well plates and screen for EGFP-expressing foci of infected cells.</li>
	<li>Harvest the virus-containing supernatants from the wells containing green fluorescent foci and dilute tenfold. For purification, choose wells that display single green fluorescent foci.</li>
	<li>When all foci resulting from the limiting-dilution infection display EGFP expression, the virus preparation is pure, indicating that no wild-type virus remains. Quantify the virus by the plaque-assay method as previously described17.</li>
	<li>Treat MCMV in certified biosafety cabinets at biosafety level 2 wearing gloves and mask.</li>
	<li>Passage MCMV (Smith strain) and recombinant MCMV in MEFs prepared from 12-day-old ICR mouse embryos as previously described17.</li>
	<li>Remove cells from the supernatants of infected MEF cultures by centrifugation at 3,000 &#215; g for 20 min at 16 &#176;C.</li>
	<li>Ultracentrifuge the supernatants for 40 min at 70,000 &#215; g. Resuspend the pellets containing virions in 1 ml of Tris-buffered saline and transfer onto a preformed linear sorbitol gradient (25% - 70%). Ultracentrifuge again at 70,000 x g for 60 min18.</li>
	<li>Harvest the virion-containing band with a syringe. Pellet the harvested virions by an additional ultracentrifugation step at 70,000 &#215; g for 40 min.</li>
	<li>Resuspend the pellet in 1 ml of phosphate-buffered saline (PBS) and store at -80 &#176;C until the infection experiments.</li>
	<li>Quantify the virus titer by the plaque-assay method as previously described19.</li>
	<li>Visualize the EGFP expression of the recombinant MCMV particles (excitation at 489 nm, emission at 508 nm) by fluorescent microscopy (Figure 1A) and the particle structure by transmission electron microscopy (TEM)8 (Figure 1B).</li>
</ol>
2. Preparation of Nile Red Fluorescent Microbeads
<ol>
	<li>Purchase carboxyl fluorescent Nile red microbeads and place 500 &#181;l of microbeads into tubes according to diameter (0.04 - 0.06 &#181;m, approximately 3.63 &#215; 1012 particles; 0.1 - 0.3 &#181;m, approximately 5.75 &#215; 1010 particles; and 1.7 - 2.2 &#181;m, approximately 6.85 &#215; 107 particles).</li>
	<li>Treat beads with 500 &#181;l of 0.1 M NaOH for approximately 1 day to remove any endotoxins and resuspend the beads in sterile water at RT.</li>
	<li>Adsorb the beads O/N with 10% mouse serum obtained from C57BL/6 mice at RT prior to use.</li>
	<li>To separate aggregates, vortex the beads and sonicate thoroughly before use.</li>
</ol>
3. ICV Injection of MCMV and Fluorescent Microbeads into Neonatal Mice
<ol>
	<li>Maintain normal pregnant ICR mice in a temperature-controlled facility under a 12 hr light/dark cycle. The neonates are designated as P 0.5 on the day of birth.</li>
	<li>Sterilize a 10-&#181;l syringe and a 27 G&#160;needle with 70% alcohol.</li>
	<li>Load the injection solution (5 &#181;l) containing MCMV or fluorescent microbeads into the needle by carefully pulling the plunger of the syringe.</li>
	<li>Restrain neonatal mouse (P 0.5) by putting the mouse on ice for 3 - 4 min. Once the animal is under anesthesia, use the toe-pinch response method to determine the depth of anesthesia.</li>
	<li>Mark the injection site with a non-toxic laboratory pen at a location approximately 0.7 - 1.0 mm lateral to the sagittal suture and 0.7 - 1.0 mm caudal from the neonatal bregma (Figure 2A).</li>
	<li>Insert the needle 2 mm deep, perpendicular to the skull surface at the marked injection site. For reference, mark 2 mm from the tip of needle with a non-toxic maker.</li>
	<li>Slowly inject 5 &#181;l of MCMV (approximately 5 &#215; 105 PFU) into the lateral ventricle without opening the scalp (Figure 2B).</li>
	<li>In another group of mice, inject a 5 &#181;l solution containing fluorescent microbeads (0.1 - 0.3 &#181;m, approximately 5.75 &#215; 108 particles) by the same method.</li>
	<li>Slowly remove the needle 10 - 20 sec after discontinuing the plunger movement to prevent backflow.</li>
	<li>To recover, keep the mice for 5 - 10 min in a warm container until movement and general responsiveness are restored.</li>
	<li>Harvest the brains as described in section 5 at a range of time points (3, 12, 24, 48, and 72 hr) post-injection.</li>
</ol>
4. IV Injection of MCMV or Fluorescent Microbeads into Neonatal Mice
<ol>
	<li>Restrain neonatal mouse (P 0.5) by putting the mouse on ice for 3 - 4 min.</li>
	<li>Use a 1-ml syringe with a 35 G needle to perform the intravenous injection of MCMV in P 0.5 neonates.</li>
	<li>Use a transilluminator (vein finder) to visualize the superficial temporal (facial) vein. Before injection, secure the neonate to the transilluminator using surgical tape (Figure 2C).</li>
	<li>While wearing magnifying glasses (1.5X), slowly infuse 50 &#181;l of MCMV (approximately 5.45 &#215; 109 particles) or fluorescent microbeads (0.04 - 0.06 &#181;m, approximately 3.63 &#215; 1011 particles; 0.1 - 0.3 &#181;m, approximately 5.75 &#215; 109 particles; and 1.7 - 2.2 &#181;m, approximately 6.85 &#215; 106 particles) into the superficial temporal vein (Figure 2D).</li>
	<li>After removing the needle, use gauze containing 70% alcohol to apply pressure to the injection site until the bleeding ceases.</li>
	<li>Give the neonate approximately 5 min in a warm container to recover before returning it to the cage.</li>
	<li>Harvest the brains as described in section 5 at a range of time points (3, 12, 24, and 72 hr) post-injection.</li>
</ol>
5. Brain Tissue Sample Preparation for Paraffin Sections
<ol>
	<li>Place the injected neonates in a small plastic dish&#160;on crushed ice.</li>
	<li>Once the animal is under anesthesia, use the toe-pinch response method to determine the depth of anesthesia.</li>
	<li>Make one central or two end horizontal end cuts through the rib cage to open up the thoracic cavity.</li>
	<li>Make a cut in the atrium with sharp scissors. Infuse 4% paraformaldehyde (PFA) solution into the atrium. Stop perfusion when spontaneous movement (PFA dance) and lightened-color of the liver are observed. Do not exsanguinate.</li>
	<li>Dissect the neonate (as previously described20) by first removing the head using a pair of scissors.</li>
	<li>Make a midline incision along the integument from the neck to the nose to expose the skull.</li>
	<li>Place the sharp end of a pair of iris scissors into the foramen magnum on one side, carefully sliding along the inner surface of the skull.</li>
	<li>Make a cut extending to the distal edge of the posterior skull surface, and make an identical cut on the contralateral side. Clear away the skull around the cerebellum.</li>
	<li>Carefully peel back the skull on one side to prevent damage to the brain. Repeat this procedure on the other side of the brain.</li>
	<li>Using a spatula, sever the olfactory bulbs and nervous connections along the ventral surface of the brain.</li>
	<li>Gently separate the brain from the head, trimming any dura that still connect the brain to the skull using scissors, and remove the brain.</li>
	<li>Place the brain in a vial of fixative containing 4% PFA at least 10 times the volume of the brain for 24 hr at 4&#176;C.</li>
	<li>Dehydrate the tissue through a series of graded ethanol baths to displace the water, and then infiltrate with paraffin wax, forming a block. Cut the paraffin block with a microtome into slices 4 &#181;m thick21.</li>
	<li>Deparaffinize and rehydrate the slides of the infected brains22. Proceed to in situ hybridization for paraffin embedded sections.</li>
</ol>
6. Brain Tissue Sample Preparation for Frozen Sections
<ol>
	<li>Remove the brain following the steps outlined in 5.1 - 5.11.</li>
	<li>To observe the fluorescent microbeads and M32-EGFP-MCMV, place the resected brain on flat bottom cryomolds. Add embedding medium to completely cover the brain sample. Snap freeze the cryomolds in precooled n-hexane (-80 &#186;C), using forceps to hold the edge of the cryomolds prior to attaching it to the chuck.</li>
	<li>For sectioning, attach the frozen tissue block on the precooled cryostat chuck. Transfer the frozen tissue with the chuck into a cryostat chamber and lower the temperature to between -10 and -20 &#176;C, and cut the slices approximately 8 - 10 &#181;m thick23.</li>
	<li>Fix the sections with cytofixative (mixture of isopropyl alcohol and polyethylene glycol) by spraying. Air dry the sections for 30 min at RT immediately after spraying, and store them at - 80 &#186;C until further use.</li>
	<li>Equilibrate the sections back to RT and wash three times with PBS.</li>
	<li>Stain the sections with fluorescein isothiocyanate (FITC)-conjugated Griffonia simplicifolia isolectin B4 at a concentration of 1:100 for 10 min in PBS at RT (Figure 5) or with PE-conjugated CD31 antibody at a concentration of 1:100 for 30 min in PBS at RT (Figure 6).</li>
	<li>Wash the sections with PBS three times.</li>
	<li>After the washing, stain the sections with 4&#8242;,6-diamidino-2-phenylindole (DAPI) to visualize cell nuclei. Mount the sections in anti-fade reagent and image DAPI (excitation at 345 nm, emission at 455 nm), EGFP (excitation at 489 nm, emission at 508 nm), and Nile red (excitation at 553 nm, emission at 637 nm) with a fluorescent microscope (Figures 3, 5, 6).</li>
</ol>
7. Fluorescent in situ Hybridization (FISH) for Paraffin Embedded Sections
<ol>
	<li>Prepare the FISH probe labeled directly with fluorophore for DNA in situ hybridization by nick translation using a bacterial artificial chromosome containing the whole MCMV DNA genome (pSM3fr)19. The concentration of the FISH probe is 0.1 &#181;g/&#181;l.</li>
	<li>Deparaffinize and rehydrate the slides of the infected brains22.</li>
	<li>Treat the tissue sections with RNase (100 &#181;g/ml in PBS) to detect viral DNA.</li>
	<li>Perform antigen retrieval with a 0.05% NP40, 0.01 M citrate buffer (pH 6.0) at 95 - 98&#176;C for 20 min. Cool the slides down to RT for 20 min.</li>
	<li>In a glass Coplin staining jar, wash the slides in pure water three times for 2 min each time. Perform an additional antigen retrieval step with a 0.06% pepsin, 0.01 N HCl solution for 5 min at 37 &#176;C.</li>
	<li>Rinse slides three times in pure water for 2 min each time in a glass Coplin staining jar.</li>
	<li>Dehydrate the tissue sections again by transferring the slides from 70% ethanol, to 85% ethanol, and then to 100% ethanol.</li>
	<li>For preparing 10 ml of the hybridization buffer, mix 1.25 ml in situ hybridization salts (3 M NaCl, 100 mM Tris-HCl pH 8.0, 100 mM sodium phosphate pH 6.8, 50 mM EDTA) with 5 ml deionized formamide, 2.5 ml 50% Dextran sulfate, 250 &#181;l 50x Denhardt&#39;s solution, 125 &#181;l 100 mg/ml tRNA, and 875 &#181;l H2O.</li>
	<li>Dilute the DNA probe directly labeled with fluorophores that recognize the whole MCMV genome randomly in the hybridization buffer. The final volume should be 10 &#181;l (7 &#181;l hybridization buffer, 1 &#181;l probe (0.1 &#181;g/&#181;l), and 2 &#181;l distilled water).</li>
	<li>Add the probe mix (10 &#181;l) to each slide and cover with a coverslip (15 &#215; 15 mm). Seal the coverslip with rubber cement. Denature the probe mix for 5 min at 85 &#176;C, and complete the hybridization step O/N at 42 &#176;C.</li>
	<li>Wash the slides with 0.3% NP40, 0.4x SSC at 73&#176;C for 2 min; with 0.1% NP40, 0.4x SSC at 73 &#176;C for 1 min; and with 2x SSC twice.</li>
	<li>Counterstain the nuclei with DAPI (10 ng/ml) and cover the slide with a coverslip.</li>
	<li>Mount the sections in anti-fade reagent and image DAPI (excitation at 345 nm, emission at 455 nm) and EGFP (excitation at 489 nm, emission at 508 nm) with a fluorescent microscope (Figure 4).</li>
</ol>

<ol>
	<li>Philippe, N., 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=Pandoraviruses:+amoeba+viruses+with+genomes+up+to+2.5+Mb+reaching+that+of+parasitic+eukaryotes.">Pandoraviruses: amoeba viruses with genomes up to 2.5 Mb reaching that of parasitic eukaryotes.</a> Science. 341 (6143), 281-286 (2013).</li><li>Passini, M. 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=Intraventricular+brain+injection+of+adeno-associated+virus+type+1+(AAV1)+in+neonatal+mice+results+in+complementary+patterns+of+neuronal+transduction+to+AAV2+and+total+long-term+correction+of+storage+lesions+in+the+brains+of+beta-glucuronidase-deficient+mice.">Intraventricular brain injection of adeno-associated virus type 1 (AAV1) in neonatal mice results in complementary patterns of neuronal transduction to AAV2 and total long-term correction of storage lesions in the brains of beta-glucuronidase-deficient mice.</a> J Virol. 77 (12), 7034-7040 (2003).</li><li>Kim, J. 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=Viral+transduction+of+the+neonatal+brain+delivers+controllable+genetic+mosaicism+for+visualising+and+manipulating+neuronal+circuits+in+vivo.">Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualising and manipulating neuronal circuits in vivo.</a> Eur J Neurosci. 37 (8), 1203-1220 (2013).</li><li>McLean, J. 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=Widespread+neuron-specific+transgene+expression+in+brain+and+spinal+cord+following+synapsin+promoter-driven+AAV9+neonatal+intracerebroventricular+injection.">Widespread neuron-specific transgene expression in brain and spinal cord following synapsin promoter-driven AAV9 neonatal intracerebroventricular injection.</a> Neurosci Lett. 576, 73-78 (2014).</li><li>Hsu, K. M., Pratt, J. R., Akers, W. J., Achilefu, S. I., Yokoyama, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+cytomegalovirus+displays+selective+infection+of+cells+within+hours+after+systemic+administration.">Murine cytomegalovirus displays selective infection of cells within hours after systemic administration.</a> J Gen Virol. 90. 90 (Pt 1), 33-43 (2009).</li><li>Sakao-Suzuki, 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=Aberrant+fetal+macrophage/microglial+reactions+to+cytomegalovirus+infection.">Aberrant fetal macrophage/microglial reactions to cytomegalovirus infection.</a> Annals of Clinical and Translational Neruology. 1 (8), 570-588 (2014).</li><li>Gombash Lampe, S. E., Kaspar, B. K., Foust, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+injections+in+neonatal+mice.">Intravenous injections in neonatal mice.</a> J Vis Exp. (93), e52037 (2014).</li><li>Kawasaki, 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=Cytomegalovirus+initiates+infection+selectively+from+high-level+beta1+integrin-expressing+cells+in+the+brain.">Cytomegalovirus initiates infection selectively from high-level beta1 integrin-expressing cells in the brain.</a> Am J Pathol. 185 (5), 1304-1323 (2015).</li><li>Rahim, A. 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=Intravenous+administration+of+AAV2/9+to+the+fetal+and+neonatal+mouse+leads+to+differential+targeting+of+CNS+cell+types+and+extensive+transduction+of+the+nervous+system.">Intravenous administration of AAV2/9 to the fetal and neonatal mouse leads to differential targeting of CNS cell types and extensive transduction of the nervous system.</a> FASEB J. 25 (10), 3505-3518 (2011).</li><li>Cannon, M. J., Davis, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Washing+our+hands+of+the+congenital+cytomegalovirus+disease+epidemic.">Washing our hands of the congenital cytomegalovirus disease epidemic.</a> Bmc Public Health. 5, (2005).</li><li>Frenkel, L. D., Keys, M. P., Hefferen, S. J., Rola-Pleszczynski, M., Bellanti, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unusual+eye+abnormalities+associated+with+congenital+cytomegalovirus+infection.">Unusual eye abnormalities associated with congenital cytomegalovirus infection.</a> Pediatrics. 66 (5), 763-766 (1980).</li><li>Becroft, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatal+cytomegalovirus+infection:+epidemiology,+pathology+and+pathogenesis.">Prenatal cytomegalovirus infection: epidemiology, pathology and pathogenesis.</a> Perspect Pediatr Pathol. 6, 203-241 (1981).</li><li>Conboy, T. 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=Intellectual+development+in+school-aged+children+with+asymptomatic+congenital+cytomegalovirus+infection.">Intellectual development in school-aged children with asymptomatic congenital cytomegalovirus infection.</a> Pediatrics. 77 (6), 801-806 (1986).</li><li>Fowler, K. 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=The+outcome+of+congenital+cytomegalovirus+infection+in+relation+to+maternal+antibody+status.">The outcome of congenital cytomegalovirus infection in relation to maternal antibody status.</a> N Engl J Med. 326 (10), 663-667 (1992).</li><li>Cannon, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Congenital+cytomegalovirus+(CMV)+epidemiology+and+awareness.">Congenital cytomegalovirus (CMV) epidemiology and awareness.</a> J Clin Virol. 46 Suppl 4, S6-S10 (2009).</li><li>Grassi, M. 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=Microglial+nodular+encephalitis+and+ventriculoencephalitis+due+to+cytomegalovirus+infection+in+patients+with+AIDS:+two+distinct+clinical+patterns.">Microglial nodular encephalitis and ventriculoencephalitis due to cytomegalovirus infection in patients with AIDS: two distinct clinical patterns.</a> Clin Infect Dis. 27 (3), 504-508 (1998).</li><li>Kawasaki, H., Mocarski, E. S., Kosugi, I., Tsutsui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclosporine+inhibits+mouse+cytomegalovirus+infection+via+a+cyclophilin-dependent+pathway+specifically+in+neural+stem/progenitor+cells.">Cyclosporine inhibits mouse cytomegalovirus infection via a cyclophilin-dependent pathway specifically in neural stem/progenitor cells.</a> J Virol. 81 (17), 9013-9023 (2007).</li><li>Britt, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cytomegalovirus:+propagation,+quantification,+and+storage.">Human cytomegalovirus: propagation, quantification, and storage.</a> Curr Protoc Microbiol. Chapter 14, Unit 14E 13 (2010).</li><li>Kawasaki, H., Kosugi, I., Arai, Y., Iwashita, T., Tsutsui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+embryonic+stem+cells+inhibit+murine+cytomegalovirus+infection+through+a+multi-step+process.">Mouse embryonic stem cells inhibit murine cytomegalovirus infection through a multi-step process.</a> PLoS One. 6 (3), e17492 (2011).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> J Vis Exp. (65), (2012).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+sections+of+paraffin-embedded+tissues.">Cutting sections of paraffin-embedded tissues.</a> CSH Protoc. , (2008).</li><li>Chi, V., Chandy, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry:+paraffin+sections+using+the+Vectastain+ABC+kit+from+vector+labs.">Immunohistochemistry: paraffin sections using the Vectastain ABC kit from vector labs.</a> J Vis Exp. (8), e308 (2007).</li><li>Wilsbacher, L. D., Coughlin, S. R. <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+cardiomyocyte+development+using+immunofluorescence+in+embryonic+mouse+heart.">Analysis of cardiomyocyte development using immunofluorescence in embryonic mouse heart.</a> J Vis Exp. (97), (2015).</li><li>Ohshima, 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=Intraperitoneal+and+intravenous+deliveries+are+not+comparable+in+terms+of+drug+efficacy+and+cell+distribution+in+neonatal+mice+with+hypoxia-ischemia.">Intraperitoneal and intravenous deliveries are not comparable in terms of drug efficacy and cell distribution in neonatal mice with hypoxia-ischemia.</a> Brain Dev. 37 (4), 376-386 (2015).</li><li>Kim, J. Y., Grunke, S. D., Levites, Y., Golde, T. E., Jankowsky, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebroventricular+viral+injection+of+the+neonatal+mouse+brain+for+persistent+and+widespread+neuronal+transduction.">Intracerebroventricular viral injection of the neonatal mouse brain for persistent and widespread neuronal transduction.</a> J Vis Exp. (91), e51863 (2014).</li><li>Glascock, 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=Delivery+of+therapeutic+agents+through+intracerebroventricular+(ICV)+and+intravenous+(IV)+injection+in+mice.">Delivery of therapeutic agents through intracerebroventricular (ICV) and intravenous (IV) injection in mice.</a> J Vis Exp. (56), (2011).</li></ol>

The authors have nothing to disclose.

In animal models, ICV, intraperitoneal, direct placental, and IV infections have been used to study the pathogenesis of viral encephalitis. We focused on the ICV and IV injection models of neonatal mice for the simplicity of the procedures and the benefit of direct injection of particles into the target region. Although intraperitoneal infection is an easy method, viral particles spread systemically via an indirect process5,24. Direct placental infection is a good method to study embryonic systemic infection. ...

When studying viral encephalitis, the initial distribution of viral particles is very important to understand disease pathogenesis and to identify viral targets in the brain. Most viruses range in size from 20 to 300 nm, although the Pandoravirus is more than 700 nm in size1. The distribution of the viral particles in the acute phase of infection may depend on the size of the particles, the distribution of cellular receptors, or the affinity of the cellular receptors for viruses. In animal models, intracerebroventricular (ICV), intraperitoneal, direct placental, and intravenous (IV) infections have been used to study the pathogenesis of viral encephalitis. ICV inoculation with virus is often used to establish central nervous system (CNS) infections in mice. Studies using this technique report widespread infection, particularly of cells in the periventricular zones and in regions of the brain in direct contact with the cerebrospinal fluid (CSF), similar to the effects of viral ventriculoencephalitis. The small size of adeno-associated virus (AAV) particles (20 - 25 nm in diameter) facilitates their dissemination throughout the brain in ICV infections2-4. Intraperitoneal5, direct placental6, and IV injections7 represent hematogenic systemic administration. The penetration of viral particles through the blood-brain barrier (BBB) allows them to reach the parenchyma of the neonatal brain, representing diffuse microglial nodules8,9.
Cytomegalovirus (CMV) is a common virus that belongs to the herpes virus family. In the United States, 50% - 80% of the people have had CMV infection by age 40. CMV infections are rarely harmful but can cause diseases in immunocompromised patients and fetuses. Of all deliveries, 0.2% - 2% are born with CMV10, resulting in severe symptoms such as microcephaly, periventricular calcification, cerebellar hypoplasia, microphthalmia, and optic nerve atrophy11,12. Furthermore, mental retardation, sensorineural hearing loss, visual defects, seizure, and epilepsy occur in about 10% of non-fatally CMV-infected infants13,14. CNS dysfunction is the most common characteristic symptom of CMV congenital anomaly. More children are permanently disabled each year by congenital CMV than by Down syndrome, fetal alcohol syndrome, or spina bifida15. There are no vaccinations against CMV available at the present, calling for a need of a safe and effective vaccine. Studying the interaction of CMV particles with their receptors in the earliest phase of infection is important to understand the effect of vaccination.
Ventriculoencephalitis and diffuse microglial nodules are the two main pathological characteristics of CMV encephalitis16. It has been uncertain how the CMV particles (150 - 300 nm) spread through the brain in the acute phase of infection and how the distribution of cellular receptors and their affinity for viruses contribute to the viral spread. Kawasaki et al. have evaluated ICV and IV infections from the perspective of the distribution of particles and their receptors (&#946;1 integrin) in the earliest phase of infection. We have found that the dissemination of CMV particles and the expression of &#946;1 integrin are well correlated in the earliest phase of infection in both ICV and IV infections8. ICV infection is a model of ventriculoencephalitis and IV infection is a model of diffuse microglial nodules. Studying the dynamics of viral or fluorescent particles would give useful information on the effect of particle size, viral interactions with cellular receptors, and the mechanism of BBB penetration in the brain. The following protocol could be used to investigate any viral infection and viral vector in the CNS.

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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Tris; tris(hydroxymethyl)- aminomethane</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:80px;">T-6791</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCl</td>
			<td>Sigma-Aldrich</td>
			<td>H-1758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pEGFP-N1 vector&#160;</td>
			<td>Clontech</td>
			<td>#6085-1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">D-sorbitol</td>
			<td>Sigma-Aldrich</td>
			<td>S-1876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SPHERO TM Fluorescent Polystyrene Nile Red 0.04-0.06</td>
			<td>Spherotech, Inc.</td>
			<td>FP-00556-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SPHERO TM Fluorescent Polystyrene Nile Red 0.1-0.3</td>
			<td>Spherotech, Inc.</td>
			<td>FP-0256-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SPHERO TM Fluorescent Polystyrene Nile Red 1.7-2.2</td>
			<td>Spherotech, inc.&#160;</td>
			<td>FP-2056-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% mouse serum</td>
			<td>DAKO&#160;</td>
			<td>X0910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL/6 mouse</td>
			<td>SLC, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ICR mouse</td>
			<td>SLC, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Modified Microliter Syringes (7000 Series)</td>
			<td>Hamilton company</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35-gauge needle</td>
			<td>Saito Medical</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A Wee Sight Transilluminator</td>
			<td>Phillips Healthcare</td>
			<td>1017920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O.C.T.Compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase A</td>
			<td>Sigma-Aldrich</td>
			<td>R4642</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nonidet(R) P-40</td>
			<td>Nacalai</td>
			<td>25223-04</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">citrate buffer (pH6) x10</td>
			<td>Sigma-Aldrich</td>
			<td colspan="2">C9999-100ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pepsin</td>
			<td>Sigma-Aldrich</td>
			<td>P6887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>dojindo</td>
			<td>N001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formamide</td>
			<td>TCI</td>
			<td>F0045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran sulfate sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>42867-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Denhardt&#39;s Solution (50X)</td>
			<td>ThermoFishcer sceintific</td>
			<td>750018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast tRNA (10 mg/mL)</td>
			<td>ThermoFishcer sceintific</td>
			<td>AM7119</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SSC x20</td>
			<td>Sigma-Aldrich</td>
			<td>S6639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>ThermoFishcer sceintific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">n-Hexane</td>
			<td>Sigma-Aldrich</td>
			<td>296090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">superfrost plus glass</td>
			<td>ThermoFishcer sceintific</td>
			<td>12-55-18</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytokeep II</td>
			<td>Nippon Shoji Co.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC-conjugated Griffonia simplicifolia isolectin B4</td>
			<td>Vector laboratories, Inc.</td>
			<td>L1104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Mouse CD31 (PECAM-1) PE</td>
			<td>ebioscience</td>
			<td>12-0311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong&#160; Gold</td>
			<td>ThermoFishcer sceintific</td>
			<td>P36934</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BIOREVO</td>
			<td>KEYENCE</td>
			<td style="width:80px;">BZ-9000E</td>
			<td></td>
		</tr>
	</tbody>
</table>

intracerebroventricular and intravascular injection of viral particles and fluorescent microbeads into the neonatal brain

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 &#181;l of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-&#181;l syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 &#181;l of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

In studies on the pathogenesis of viral encephalitis, the infection method is important. The hematogenous route represents an acute infection of the endothelial cells and pericytes of the brain, while the ICV route represents an acute infection spreading via the CSF through the subarachnoid space, reaching to the meninges and choroid plexus. To analyze the first distribution of particles in acute encephalitis, in situ hybridization detecting the MCMV genomes and direct observatio...

Watch this Scientific Journal Video about Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain at JoVE.com

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

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19.2K Views.  Hamamatsu University School of Medicine. Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization. 

Video: Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

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 ...

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost oligodendrocytes, myelin, axons, and neurons. Remyelination is an endogenous repair mechanism whereby new myelin is produced subsequent to proliferation, recruitment, and differentiation of oligodendrocyte precursor cells into myelin-forming oligodendrocytes, and is necessary to protect axons from further damage. Currently, all therapeutics for the treatment of multiple sclerosis target the aberrant immune component of the disease, which reduce inflammatory relapses but do not prevent progression to irreversible neurological decline. It is therefore imperative that remyelination-promoting strategies be developed which may delay disease progression and perhaps reverse neurological symptoms. Several animal models of demyelination exist, including experimental autoimmune encephalomyelitis and curprizone; however, there are limitations in their use for studying remyelination. A more robust approach is the focal injection of toxins into the central nervous system, including the detergent lysolecithin into the spinal cord white matter of rodents. In this protocol, we demonstrate that the surgical procedure involved in injecting lysolecithin into the ventral white matter of mice is fast, cost-effective, and requires no additional materials than those commercially available. This procedure is important not only for studying the normal events involved in the remyelination process, but also as a pre-clinical tool for screening candidate remyelination-promoting therapeutics.

Demyelinating diseases can be modeled in animals by focal application of lysolecithin into the CNS. A single injection of lysolecithin into mouse spinal cord produces a lesion that spontaneously repairs over time. The goal is to study factors involved in de- and remyelination, and to test agents for enhancing repair.

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

Intracerebroventricular Treatment with Resiniferatoxin and Pain Tests in Mice

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In addition to its peripheral function, its involvement in brain functions has also been suggested. Resiniferatoxin (RTX), an ultrapotent TRPV1 agonist, has been known to induce long-term desensitization of TRPV1, and this desensitization has been an alternative approach for investigating the physiological relevance of TRPV1-expressing cells. Here we describe a protocol for intracerebroventricular (i.c.v.) treatment with RTX in mice. Procedures are described for testing nociception to peripheral TRPV1 stimulation (RTX test) and mechanical stimulation (tail pressure test) then follow. Although the nociceptive responses of mice that had been administered RTX i.c.v. were comparable to those of the control groups, RTX-i.c.v.-administered mice were insensitive to the analgesic effect of acetaminophen, suggesting that i.c.v. RTX treatment can induce supraspinal-selective TRPV1 desensitization. This mouse model can be used as a convenient experimental system for studying the role of TRPV1 in brain/supraspinal function. These techniques can also be applied to studies of the central actions of other drugs.

The transient receptor potential vanilloid type 1 (TRPV1) in the supraspinal region has been suggested to play some roles in the brain function. Described here is a protocol for intracerebroventricular injection of resiniferatoxin for supraspinal TRPV1 desensitization in mice. Procedures for some pain tests are also presented.

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In ...

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

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

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

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

Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain consists of an outer covering of grey matter over the hemispheres known as the cerebral cortex. Underneath this layer reside many other specialized structures that are essential for multiple phenomenon important for existence. Acquiring samples of specific gross brain regions requires quick and precise dissection steps.&#160;It is understood that at the microscopic level, many sub-regions exist and likely cross the arbitrary regional boundaries that we impose for the purpose of this dissection.
Mouse models are routinely used to study human brain functions and diseases. Changes in gene expression patterns may be confined to specific brain areas targeting a particular phenotype depending on the diseased state. Thus, it is of great importance to study regulation of transcription with respect to its well-defined structural organization. A complete understanding of the brain requires studying distinct brain regions, defining connections, and identifying key differences in the activities of each of these brain regions. A more comprehensive understanding of each of these distinct regions may pave the way for new and improved treatments in the field of neuroscience. Herein, we discuss a step-by-step methodology for dissecting the mouse brain into sixteen distinct regions. In this procedure, we have focused on male mouse C57Bl/6J (6-8 week old) brain removal and dissection into multiple regions using neuroanatomical landmarks to identify and sample discrete functionally-relevant and behaviorally-relevant&#160;brain regions. This work will help lay a strong foundation in the field of neuroscience, leading to more focused approaches in the deeper understanding of brain function.

We present a hands-on, step-by-step, rapid protocol for mouse brain removal and dissection of discrete regions from fresh brain tissue. Obtaining brain regions for molecular analysis has become routine in many neuroscience labs. These brain regions are immediately frozen to obtain high quality transcriptomic data for system level analysis.

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain ...