This work was funded by NIH grants DA059310, DA058586, AG082539, and GM109091. We appreciate the generous gift of the EcoHIV-NL4-3-EGFP from Dr. Potash of the Icahn School of Medicine at Mount Sinai.

The Animal Care and Use Committee at the University of South Carolina approved all animal protocols (federal assurance number: D16-00028). All experiments strictly followed the guidelines established by the National Institutes of Health in the Guide for the Care and Use of Laboratory Animals. Tmem119-EGFP knock-in mice (30 days old, male, 23-26 g body weight) were obtained from a commercial source and group-housed in AAALAC-accredited facilities. All animals were housed under a 12/12 h light-dark cycle with free access to food and water. The details of the animals, reagents, and equipment used in this study are listed in the Table of Materials.
1. EcoHIV-mScarlet packaging in 293FT cells
<ol>
	<li>Incubate the 293 FT cells in a gelatin-precoated 75 cm2 flask. Keep cells growing to 30% confluent at transfection.</li>
	<li>Perform the transfection of plasmid DNA (15 &#181;g) of EcoHIV-mScarlet (Supplementary File 1) using Lipofectamine 3000 (22.5 &#181;L) following the manufacturer&#39;s instructions (see Table of Materials).</li>
	<li>Culture the cells in DMEM medium with 10% FBS serum for 3 days at 37 &#176;C.</li>
	<li>Collect the conditional medium with viral suspension. Centrifuge at 500 &#215; g for 10 min at 4 &#176;C. Transfer the supernatant with a 10 mL pipette to a sterile 50 mL tube.</li>
	<li>Add a certain amount of Lenti-x concentrator (1:3 ratio) into the viral mixture (for example, 8 mL of concentrator with 24 mL of viral mixture). Gently invert the tube five times.</li>
	<li>Keep the virus-Lenti concentrator mixture at 4 &#176;C for 2 days. Centrifuge at 1,500 &#215; g, 45 min, 4 &#176;C. Carefully remove supernatant as much as possible using a pipette.</li>
	<li>Re-suspend the pellet with prechilled 200 &#181;L of 100 mM PBS. Store the virus at -80 &#176;C. 
	NOTE: The details of EcoHIV-mScarlet packaging in 293FT cells were described in our previous study16. Do not vortex or introduce air bubbles in the viral solution.</li>
</ol>
2. EcoHIV-mScarlet infection in primary rat brain cells
<ol>
	<li>Perform the primary brain cell isolation from rat fetuses (18 days) following the previously published report16.</li>
	<li>Transfer dissociated cells to poly-L-Lysine pre-coated 12-well plates with glass inserts containing 1 mL of DMEM/F12 medium plus 10% FBS. Replace medium the next day for Neurobasal medium with B27 supplement.</li>
	<li>Culture primary fetal brain cells in a 5% CO2 incubator for 3 weeks.</li>
	<li>Add EcoHIV-mScarlet (60 &#181;L, 1.26 &#215; 106 TU/mL) into the culture medium. Incubate the cultured brain cells with EcoHIV-mScarlet for 6 days.</li>
	<li>Fix the cells with 4% PFA and perform immunostaining on infected brain cells using specific primary antibodies (CD11b/c, Iba1).</li>
	<li>Obtain images using 40&#215; objective under a confocal microscopy system.</li>
</ol>
3. EcoHIV-mScarlet virus infection in primary glia cells from adult mice
<ol>
	<li>Anesthetize adult mice with 5% sevoflurane for 5 min (following institutionally approved protocols). Sterilize the skin of the head with 70% EtOH.</li>
	<li>After confirming the mouse is no longer responsive to noxious stimuli, use a sterilized sharpened scissor to perform decapitation. Transfer the head to a new Petri dish filled with 5 mL of HBSS.</li>
	<li>Open the scalp and transfer brain tissue into another Petri dish containing 5 mL of sterilized HBSS. Peel off the meninges and transfer the frontal cortex into 2 mL of HBSS.</li>
	<li>Add 20 &#181;L of 0.25% Trypsin-EDTA into the mixture. Incubate for 15 min at room temperature; gently swirl the tube every few minutes.</li>
	<li>Transfer dissociated cells to a poly-L-Lysine pre-coated 75 cm2 flask containing 10 mL of DMEM/F12 medium and 10% FBS.</li>
	<li>Culture cells at 37 &#176;C, 5% CO2 incubator, until 90% confluency. Digest brain cells with 2 mL of 0.25% Trypsin-EDTA.</li>
	<li>Subculture brain cells into 35 mm glass-bottom dishes containing 5 mL of DMEM/F12 growth medium till 80% confluency.</li>
	<li>Add EcoHIV-mScarlet (8 &#181;L, 1.26 &#215; 106 TU/mL) into the culture medium. Incubate mouse glial cells for 2 days.</li>
	<li>Check red (mScarlet) fluorescence signals daily under a fluorescent microscope.</li>
</ol>
4. EcoHIV-mScarlet virus retroorbital injection into Tmem119-EGFP mice
<ol>
	<li>Use 3% sevoflurane to anesthetize the Tmem119-EGFP mice (both male and female mice at 30 days of age) until they are no longer responsive to noxious stimuli.</li>
	<li>Secure the mice in a lateral position with the injection eye facing up and respiration through the nose cone, which is linked to an anesthesia system. Use an appropriate nose cone size to provide continuous anesthesia.</li>
	<li>Thaw the EcoHIV-mScarlet on ice. Fill the viral solution into an intraocular injector syringe with a 33 G blunt needle.</li>
	<li>Place the mouse&#160;in the right lateral recumbency and keep its head facing to the left. Identify the location of the medial canthus as the injection site.</li>
	<li>After proptosing the eye, slowly and gently insert a needle (45-degree angle) into the medial canthus of the eye. Carefully insert the needle forward into the vessels behind the eyeball (retro-orbital sinus).</li>
	<li>Gently inject 6.5 &#181;L of EcoHIV-mScarlet (1.26 &#215; 106 TU/mL, bilateral-eye inoculation) into the retro-orbital sinus. Carefully remove the needle from the retro-orbital sinus and gently apply pressure to the eyelids to provide hemostasis.</li>
	<li>Apply lubricant to the eye to prevent the cornea from drying or becoming injured.</li>
	<li>Allow the mice to recover in a recovery chamber with a heating pad until it has woken up. 
	NOTE: The needle bevel should not be positioned too deep so that the arteries are not ruptured or the bones fractured. Viral infusion time is dependent upon multiple factors (e.g., injection volume, titer, animal size). For EcoHIV viral infusion, significant expression has been observed one week after retro-orbital injections11,16,17.</li>
</ol>
5. Visualization of brain tissue slices
<ol>
	<li>Deeply anesthetize mice using 5% sevoflurane. Move on to step 5.2 when the mice show no response to noxious stimuli and reflexes are absent.</li>
	<li>Keep the mice in a supine position inside a chemical fume hood.</li>
	<li>Open the skin along the thoracic midline. Separate the diaphragm and open the chest with scissors.</li>
	<li>Insert a 22 G1 1/2 needle into the left ventricle. Open the right atrium with scissors.</li>
	<li>Perfuse 50 mL of pre-chilled 100 mM PBS. Perfuse 100 mL of pre-chilled 4% paraformaldehyde buffer16.</li>
	<li>Remove the entire mouse brain16.</li>
	<li>Fix overnight with 4% paraformaldehyde.</li>
	<li>Secure the brain tissue using tissue adhesive on the metal platform of the Vibratome. Cut 50 &#181;m thickness of coronal sections with carbon steel blades.</li>
	<li>Place the brain slices onto glass slides using a brush. Immediately add 0.1 mL of the antifade mounting medium to each section.</li>
	<li>Place a 22 mm x 50 mm coverslip over brain sections. Dry the super-frost slides in the dark for 1 day.</li>
	<li>Configure the confocal microscope system to a magnification of 60&#215; (A/1.4, oil) and set a Z-plane interval of 0.15 &#181;m, with a pinhole size of 30 &#181;m and a back-projected pinhole radius of 167 nm.</li>
	<li>Use the 488 nm and 594 nm wavelengths to acquire multi-channel images of brain regions of interest.</li>
</ol>

EcoHIV Infection in Tmem119-EGFP Mice for Studying Microglial Alterations

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Neuroimmune Pharmacol. 8 (4), 988-997 (2013).</li><li>Repunte-Canonigo, , 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+expression+changes+consistent+with+neuroAIDS+and+impaired+working+memory+in+HIV-1+transgenic+rats.">Gene expression changes consistent with neuroAIDS and impaired working memory in HIV-1 transgenic rats.</a> Mol Neurodegener. 9, 26 (2014).</li><li>Reid, W., 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=An+HIV-1+transgenic+rat+that+develops+HIV-related+pathology+and+immunologic+dysfunction.">An HIV-1 transgenic rat that develops HIV-related pathology and immunologic dysfunction.</a> Proc Natl Acad Sci USA. 98 (16), 9271-9276 (2001).</li><li>McLaurin, K. A., Li, H., Booze, R. M., Mactutus, C. 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None of the authors have conflicts of interest to declare.

In the present study, it was found that (1) the novel EcoHIV-mScarlet successfully infected primary rat brain cells in vitro; (2) the triple labeling of mScarlet, CD11b/c, and Iba1 identified microglia as the predominant cell type for this EcoHIV expression in rat brain cells in vitro; (3) primary mouse brain cells from adults in vitro further evidence the EcoHIV-mScarlet infection; (4) the EcoHIV-mScarlet distribution in the Tmem119-EGFP knock-in mouse line demonstrated a microglia-specific distribution pattern of EcoHIV infection.
Emerging studies suggested that various types of brain cells identified within the central nervous system (neurons, astrocytes, microglia, oligodendrocytes, etc.) exhibit functional and transcriptomic alteration during HIV and HIV-associated neurocognitive disorders18,19. For example, astrocytes contribute 30%-70% of the brain and perform surveillance to maintain brain homeostasis20. Astrocytes also modulate immune function and regulate the secretion of multi-cytokines and chemokines, especially in the situation of brain inflammation and neurodegeneration in HIV21,22. HIV infection of microglia not only results in a continuous release of viral proteins and proinflammatory cytokines and chemokines, but also provides predominant sources for HIV viral reservoirs23,24,25,26. Furthermore, activated microglia contribute a critical immunological function in the CNS; however, prolonged activation may also exacerbate neurodegeneration in HIV progression10,27. Oligodendrocytes also play an important function, including releasing several neurotrophins (such as nerve growth factor, brain-derived neurotrophic factor, etc.)28. A previous study found that the number of oligodendrocytes significantly decreased in the brains of AIDS patients, which may indicate direct damage to oligodendrocytes from HIV viral proteins29. Therefore, a specific type of cell-manipulated infection model of HIV should provide fundamental means of identifying the differential functions of various brain cells following infection. In the current study, a biological system was developed to mimic features of HIV-1 by chimeric HIV (EcoHIV) inoculation. This HIV inoculation was also combined with the Tmem119-EGFP knock-in mouse line to generate and validate a microglia-manipulated rodent model of HIV.
However, the limitations of the present study must be recognized. There were a few Iba1/CD11b/c negative cells that presented mScarlet fluorescent signals in vitro. Other types of brain cells, such as brain macrophages or pericytes, may be involved in EcoHIV infection, or alternatively, the cell culture environment may promote aberrant infection patterns relative to in vivo inoculation. Future, whole animal studies should further clarify the function of mScarlet+ microglia in the process of EcoHIV infection, and further define the regional distribution of mScarlet+EcoHIV microglia in the brain. Additionally, neurocognitive alterations occurring as a result of microglial infections may also be addressed in this rodent model. Collectively, the EcoHIV-mScarlet inoculation of Tmem119-EGFP knock-in mice provides a novel model and research strategy for investigating the microglial-driven mechanisms of HIV-associated neurocognitive disorders.

Despite the profound benefits derived from antiretroviral therapy, people living with HIV (PLWH) still experience neurocognitive disorders. To better understand the neuronal mechanisms of HIV-associated neurocognitive disorder (HAND), there is a critical need for HIV models to further elucidate the specific cell type involvement in NeuroHIV.
The HIV-1 transgenic rat, which has constitutive exposure to HIV-1 viral proteins, is a popular rodent model used to investigate neurocognitive disorders1,2,3,4and neuroanatomical alterations5,6,7 associated with HAND. The functional deletion of the gag and pol domains prevents viral replication, rendering the HIV-1 transgenic rat noninfectious8,9. Recently, a chimeric HIV (EcoHIV) infection model in mice was initially reported by Potash et al.10 and later extended to rats, which may be advantageous for further studies on HAND and substance use disorders11. In this novel biological system, systemic HIV-1 infection was observed along with many clinical features of HIV-1 in humans, including lymphocyte and macrophage involvement, antiviral immune responses, neuroinvasiveness, and brain inflammation.
Microglia play a critical role as specialized brain-resident macrophages in maintaining brain function and homeostasis. To distinguish microglia from closely related cell types (e.g., blood monocytes, perivascular macrophages, meningeal macrophages), the current study used the Tmem119-EGFP knock-in mouse line. Previous studies have reported that transmembrane protein 119 (Tmem119) exhibits an exclusively microglia-specific expression pattern in rodent and human brain tissue12,13,14,15. The EGFP signal in Tmem119-EGFP knock-in mice was observed throughout the brain and localized specifically to microglial cells.
In the present study, Tmem119-EGFP knock-in mice were inoculated with the EcoHIV-mScarlet virus, and cells positive for EcoHIV in the central nervous system were identified. Here, we present a protocol for EcoHIV-mScarlet inoculation in Tmem119-EGFP knock-in mice, providing a reliable model for therapeutically targeting microglial alterations in HIV.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>293FT cells</td>
			<td>ThermoFisher Scientific</td>
			<td>R70007</td>
			<td></td>
		</tr>
		<tr>
			<td>33 G, Small Hub RN Needle, (Point Style: 3, Needle Length: 19.25 mm)</td>
			<td>Hamilton</td>
			<td>7803-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic solution</td>
			<td>Cellgro</td>
			<td>30004CI</td>
			<td>100x</td>
		</tr>
		<tr>
			<td>C57BL/6-Tmem119em2Gfng/J</td>
			<td>The Jackson Laboratory</td>
			<td>Strain #:031823</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning BioCoat Gelatin 75cm&#178; Rectangular Canted Neck Cell Culture Flask with Vented Cap</td>
			<td>Life Technologies</td>
			<td>354488</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#160;DMEM with L-Glutamine, 4.5 g/L Glucose and Sodium Pyruvate</td>
			<td>Life Technologies</td>
			<td>10013CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>VWR</td>
			<td>637-137</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>World Precision Instruments</td>
			<td>14095</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #7 Forceps</td>
			<td>World Precision Instruments</td>
			<td>14097</td>
			<td></td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit (10)</td>
			<td>Qiagen</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf&#160;Snap-Cap Microcentrifuge Biopur Safe-Lock Tubes</td>
			<td>Life Technologies</td>
			<td>22600028</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather Double Edge Carbon Steel Blades</td>
			<td>Ted Pella, inc.</td>
			<td>121-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisher BioReagents&#160;Microbiology Media: LB Broth, Miller</td>
			<td>Fisher Scientific</td>
			<td>BP1426-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Human recombinant anti-CD11b antibody</td>
			<td>Miltenyi Biotec</td>
			<td>130-120-214</td>
			<td>1:50 dilution</td>
		</tr>
		<tr>
			<td>Intraocular Injector Syringe (6.5 &#181;L), Removable Needle</td>
			<td>Hamilton</td>
			<td>6609071-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen&#160;Lipofectamine 3000 Transfection Reagent&#160;</td>
			<td>Life Technologies</td>
			<td>L3000015</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen&#160;One Shot TOP10 Electrocomp&#160;E. coli</td>
			<td>Fisher Scientific</td>
			<td>C404052</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Forceps</td>
			<td>World Precision Instruments</td>
			<td>15914</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors</td>
			<td>World Precision Instruments</td>
			<td>500216</td>
			<td></td>
		</tr>
		<tr>
			<td>Lentivirus-Associated p24 ELISA Kit</td>
			<td>Cell Biolabs, inc.</td>
			<td>VPK-107-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Lenti-X Concentrator</td>
			<td>Takara</td>
			<td>PT4421-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Life Technologies</td>
			<td>11058021</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160;</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>PELCO easiSlicer&#160;Vibratory Tissue Slicer</td>
			<td>Ted Pella, inc.</td>
			<td>11000</td>
			<td></td>
		</tr>
		<tr>
			<td>PELCO&#160;Pro CA44 Tissue Adhesive</td>
			<td>Ted Pella, inc.</td>
			<td>10033</td>
			<td></td>
		</tr>
		<tr>
			<td>PELCO&#160;Pro Specimen Retrievers</td>
			<td>Ted Pella, inc.</td>
			<td>101-33</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold</td>
			<td>Fisher Scientific</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit monoclonal anti-Iba1 antibody</td>
			<td>Abcam</td>
			<td>ab178847</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>RN Compression Fitting 1 mm</td>
			<td>Hamilton</td>
			<td>55750-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sevoflurane</td>
			<td>Merritt Veterinary Supply</td>
			<td>347075</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague Dawley pregnant rat</td>
			<td>Inotiv</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFrost Plus Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-154%&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>To-Pro-3</td>
			<td>ThermoFisher Scientific</td>
			<td>T3605</td>
			<td>Nucleus staining kit</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>ThermoFisher Scientific</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Scissors</td>
			<td>World Precision Instruments</td>
			<td>500086</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of ecohiv-infected cells in microglia-manipulated transgenic mice

Combined antiretroviral therapy (cART) has dramatically improved the quality of life for people living with HIV (PLWH). However, over 4 million PLWH are over the age of fifty and experience accompanying HIV-associated neurocognitive disorders (HAND). To understand how HIV impacts the central nervous system, a reliable and feasible model of HIV is necessary. Previously, a novel biological system using chimeric HIV (EcoHIV) inoculation was developed in a rat model to investigate neurocognitive impairments and synaptic dysfunction. Nevertheless, a significant challenge remains in clarifying EcoHIV's neuroanatomical distribution, particularly its differential expression in various cell types in the brain. In the current study, EcoHIV with mScarlet fluorescence labeling was modified and retro-orbitally injected into Tmem119-EGFP knock-in mice (which express enhanced green fluorescence protein primarily in microglia) to determine if microglia are the major cell type responsible for viral expression and reservoirs of HIV in the brain. The current data show that: (1) in vitro, EcoHIV-mScarlet fluorescence signals were predominantly localized in microglia-like cells among primary rodent brain cells; (2) in vivo, injection of EcoHIV-mScarlet into Tmem119-EGFP mice induced significant HIV expression in the mouse brain. The co-localization of mScarlet and EGFP signals suggests that microglia are the main cell type harboring HIV in the brain. Overall, EcoHIV in rodents offers a valuable biological system to study microglial alterations, viral reservoirs in the brain, and the neurological mechanisms of HIV-associated neurocognitive disorders.

A fragment of mScarlet (1858 bp) containing enzyme sites of &#34;Cla1&#34; at the 3&#39; end and &#34;Not1&#34; at the 5&#39; end was inserted into pNL4-3-EcoHIV lentiviral vector (Figure 1). To validate the expression of EcoHIV-mScarlet, primary brain cells isolated from the cortex of rat E18 embryos were treated with EcoHIV-mScarlet (60 &#181;L, 1.26 &#215; 106 TU/mL) for 6 days in vitro. The data in Figure 2 showed that red fluorescent signals of mScarlet were mainly localized in glial types of cells based on the different cell morphology. Furthermore, CD11b/c and Iba1 (cell markers for microglia) labeling showed that mScarlet signals were co-localized with CD11b/c + and/or Iba1 + cells. The data indicated that microglia were the major cell type of EcoHIV-mScarlet distribution in vitro. There was no significant neuronal infection in the cultured cells (Supplementary Figure 1). 
Next, the infection of EcoHIV-mScarlet was tested on adult mouse primary mixed glial cells. To do so, mixed glia cells were first isolated and purified from adult mice (2 months) and infected with EcoHIV-mScarlet (8 &#181;L, 1.26 &#215; 106 TU/mL) for 2 days. The images in Figure 3 showed that EcoHIV-mScarlet successfully infected adult mouse glia.
To further address the distribution pattern of EcoHIV-mScarlet in the mouse brain, specifically for identifying the infected cell type, EcoHIV-mScarlet was retro-orbital injected into Tmem119-EGFP knock-in mouse line in which microglia were specifically labeled with EGFP signals without any other macrophage types of conflicts5. The results in Figure 4 (also in Supplementary Figure 2) show that mScarlet red fluorescence signals were observed in EGFP-positive cells, suggesting microglia as the major cell type of EcoHIV expression in mouse brains.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67150/67150fig01.jpg" /> 
Figure 1: The vector map of EcoHIV-NL-4-3-mScarlet. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67150/67150fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67150/67150fig02.jpg" /> 
Figure 2: The EcoHIV-mScarlet infection in primary rat brain cells. (A,E) Representative images of CD11b/c staining at primary brain cells. The brain cells were isolated from E18 rat embryos and infected with the EcoHIV-mScarlet virus for 6 days. (B,F) Representative images of mScarlet fluorescent signals from in vitro brain cells. (C,G) Representative images of Iba1 staining at primary brain cells. (D,H) Merged images of triple labeling of CD11b/c, mScarlet, and Iba1. Scale bars: 50 &#181;m. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67150/67150fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67150/67150fig03.jpg" /> 
Figure 3: EcoHIV-mScarlet infection in primary mouse mixed glial cells. (A,B) Representative confocal images of mScarlet distribution in vitro. The primary mixed glia cells were isolated from adult C57BL6 mice (2-month-old) and cultured for 2 weeks before viral infection. The EcoHIV-mScarlet was added into the culture medium for two days and captured the images under the 60&#215; objective of the Confocal microscope. Scale bar: 10 &#181;m. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67150/67150fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67150/67150fig04.jpg" /> 
Figure 4: EcoHIV-mScarlet distribution in Tmem119-EGFP knock-in mouse line. (A,B) Merged images of mScarlet/EGFP/ToPro3 signals in brain sections. The yellow frame indicates the target area of (B). Scale bar: 300 &#181;m. (C) Representative image of mScarlet distribution in the external plexiform layer of the olfactory region of Tmem119-EGFP knock-in mouse line. (D) Representative image of EGFP distribution. The fluorescent signals were localized in microglia cells in the Tmem119-EGFP knock-in mouse line. (E) Representative image of TO-PRO-3 nucleus staining. Scale bars: 300 &#181;m. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67150/67150fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: MAP2 and MOG staining of primary rat brain cells infected with EcoHIV-mScarlet virus. Scale bars: 50 &#181;m. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67150/Supplementary Figure 1.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Confocal images of EcoHIV-mScarlet infection in Tmem119-EGFP knock-in mouse line. Scale bars: 75 &#181;m. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67150/Supplementary Figure 2.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 1: EcoHIV-mScarlet plasmid DNA sequence. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67150/Supplementary data - EcoHIV-mScarlet plasmid DNA sequence.zip" target="_blank">Please click here to download this File.</a>

Identification of EcoHIV-Infected Cells in Microglia-Manipulated Transgenic Mice

This protocol describes how the combination of EcoHIV infection with Tmem119-EGFP mice offers a valuable biological system for investigating microglial alterations and viral reservoirs in rodent models of HIV-associated neurocognitive disorders.

Combined antiretroviral therapy (cART) has dramatically improved the quality of life for people living with HIV (PLWH). However, over 4 million PLWH are ...

identification-ecohiv-infected-cells-microglia-manipulated-transgenic

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

283 Views.  University of South Carolina. This protocol describes how the combination of EcoHIV infection with Tmem119-EGFP mice offers a valuable biological system for investigating microglial alterations and viral reservoirs in rodent models of HIV-associated neurocognitive disorders.

Video: Identification of EcoHIV-Infected Cells in Microglia-Manipulated Transgenic Mice

Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron precursors (CGNPs) as the cell-of-origin for medulloblastoma1-4. However, the diverse clinical presentations of medulloblastoma subtypes in human patients (nodular, desmoplastic, classical and large cell/anaplastic), and the fact that medulloblastoma is found in a subset of human patients with no ectopic expression of CGNP marker5, suggest that the cellular and molecular origins of medulloblastoma are more complex and far from being completely deciphered. Therefore, it is essential to determine whether there is an alternative medulloblastoma tumor cell-of-origin based on which cell-type specific therapeutic modality can be developed. To this end, intracranial orthotopic allografting of genetically marked tumor cell types followed by subsequent analyses of secondary tumor development in recipients will allow determination of the cellular origin of tumor-initiating cells. Here we describe the experimental protocol for intracranial orthotopic allografting of medulloblastoma cells derived from primary tumor tissue, and this procedure can also be used for transplanting cells from established cell lines.

This protocol describes the isolation and dissociation of mouse medulloblastoma tissue, and subsequent allografting of the tumor cells into immunocompromised recipient mice in order to initiate secondary medulloblastoma.

Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron ...

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein prionoids, which cause neuropathology within the central nervous system (CNS). Here we describe that intraglossal or intraperitoneal injection of alpha-synuclein fibrils into bigenic Tg(M83+/-:Gfap-luc+/-) mice, which overexpress human alpha-synuclein with the A53T mutation from the prion protein promoter and firefly luciferase from the promoter for glial fibrillary acidic protein (Gfap), is sufficient to induce neuropathologic disease. In comparison to homozygous Tg(M83+/+) mice that develop severe neurologic symptoms beginning at an age of 8 months, heterozygous Tg(M83+/-:Gfap-luc+/-) animals remain free of spontaneous disease until they reach an age of 22 months. Interestingly, injection of alpha-synuclein fibrils via the intraperitoneal route induced neurologic disease with paralysis in four of five Tg(M83+/-:Gfap-luc+/-) mice with a median incubation time of 229 &#177;17 days. Diseased animals showed severe deposits of phosphorylated alpha-synuclein in their brains and spinal cords. Accumulations of alpha-synuclein were sarkosyl-insoluble and colocalized with ubiquitin and p62, and were accompanied by an inflammatory response resulting in astrocytic gliosis and microgliosis. Surprisingly, inoculation of alpha-synuclein fibrils into the tongue was less effective in causing disease with only one of five injected animals showing alpha-synuclein pathology after 285 days. Our findings show that inoculation via the intraglossal route and more so via the intraperitoneal route is suitable to induce neurologic illness with relevant hallmarks of synucleinopathies in Tg(M83+/-:Gfap-luc+/-) mice. This provides a new model for studying prion-like pathogenesis induced by alpha-synuclein prionoids in greater detail.

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the identification of the neuronal type in in vivo electrophysiological experiments in broad brain regions. In optogenetics experiments, it is critical to deliver the light to the recording site. However, it is often hard to deliver the stimulation light to the deep brain regions from the brain&#39;s surface. Especially, it is difficult for the stimulation light to reach the deep brain regions when the optical transparency of the brain surface is low, as is often the case with recordings from awake animals. Here, we describe a method to record spike responses to the light from an awake mouse using a custom-made glass optrode. In this method, the light is delivered through the recording glass electrode so that it is possible to reliably stimulate the recorded neuron with light in the deep brain regions. This custom-made optrode system consists of accessible and inexpensive materials and is easy to assemble.

This work introduces a method to perform an optogenetic single-unit recording reliably from an awake mouse using a custom-made glass optrode.

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS) when compared to spinal motor neurons (SMNs). The ability to isolate and culture primary mouse CN3s, CN4s, and SMNs would provide an approach to study mechanisms underlying this selective vulnerability. To date, most protocols use heterogeneous cell cultures, which can confound the interpretation of experimental outcomes. To minimize the problems associated with mixed-cell populations, pure cultures are indispensable. Here, the first protocol describes in detail how to efficiently purify and cultivate CN3s/CN4s alongside SMNs counterparts from the same embryos using embryonic day 11.5 (E11.5) IslMN:GFP transgenic mouse embryos. The protocol provides details on the tissue dissection and dissociation, FACS-based cell isolation, and in vitro cultivation of cells from CN3/CN4 and SMN nuclei. This protocol adds a novel in vitro CN3/CN4 culture system to existing protocols and simultaneously provides a pure species- and age-matched SMN culture for comparison. Analyses focusing on the morphological, cellular, molecular, and electrophysiological characteristics of motor neurons are feasible in this culture system. This protocol will enable research into the mechanisms that define motor neuron development, selective vulnerability, and disease.

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons.

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral ...

Identification of EcoHIV-Infected Cells in Microglia-Manipulated Transgenic Mice

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Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Isl^mn:GFP Transgenic Mice