The authors thank Olga Sharma, Theresa Hundt, and the staff of the DZNE microscopy and animal facilities for technical support.

All procedures including animals were performed with approval of the animal protection committee of North Rhine-Westphalia State Environment Agency (LANUV). Animals were housed and cared for according to standard conditions with a 12 h light/dark cycle and free access to food and water.
1. Animal Model
<ol>
	<li>Intercross hemizygous Tg(Gfap-luc+/-) mice with hemizygous Tg(M83+/-) mice to generate hemizygous bigenic Tg(M83+/-:Gfap-luc+/-) mice15,16.</li>
	<li>Genotype the progeny with real-time PCR for the presence of the transgene encoding human alpha-synuclein and with standard PCR for firefly luciferase12,17.</li>
	<li>Inoculate the animals at an age of six-to-eight weeks.</li>
</ol>
2. Inoculum Preparation
<ol>
	<li>Prepare monomeric recombinant human alpha-synuclein with the A53T mutation in Tris-buffered saline (TBS) buffer containing 150 mM NaCl in 20 mM Tris-HCl (pH 7.2) as has been previously described14.</li>
	<li>Prepare fibrillar alpha-synuclein for inoculations by agitating 3 &#181;g/&#181;L of monomeric recombinant human alpha-synuclein in an orbital shaker at 800 rpm and 37 &#176;C for 5 days.</li>
	<li>Dilute fibril assemblies in phosphate-buffered saline (PBS) to reach a final concentration of 1 &#181;g/&#181;L for intraperitoneal or 2 &#181;g/&#181;L for intraglossal inoculations.</li>
	<li>Fragment the alpha-synuclein fibrils with a rod sonicator for 1 min (40 pulses of 0.5 s duration with a one-second pause between each pulse and an amplitude set to 50%) on ice. To avoid contamination by cross-seeding. Use clean sonication probes to prepare different inoculum.</li>
</ol>
3. Intraglossal Injections
<ol>
	<li>Anesthetize animals with an intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine. Pinch the animal&#39;s toe and confirm that it does not withdraw its hindlimb to ensure proper anesthesia. Use vet ointment on the animal&#39;s eyes to prevent dryness while under anesthesia.</li>
	<li>Fix narcotized animals carefully with adhesive tape onto a heating plate in a dorsal recumbent position with their heads facing towards the investigator.</li>
	<li>Fill a 27 G disposable hypodermic syringe with 5 &#181;L of sonicated alpha-synuclein fibrils or PBS.</li>
	<li>Use one blunt-nosed thumb forceps with serrated tips to hold the animal&#8217;s mouth open, and a second smaller pair of forceps to carefully pull out the tongue to make the bottom side of the tongue accessible for injection.</li>
	<li>Inject the needle of the syringe into the right or left bottom side of the tongue in proximity to the hypoglossal nerve. Slowly inject the inoculum after 5 s. Slowly retract the needle after 5 s to ensure that the inoculum has penetrated the tissue and is not lost while retracting the needle.</li>
	<li>Release the animal from its fixations and leave it on the heating plate under constant monitoring until complete recovery.</li>
</ol>
4. Intraperitoneal Injections
<ol>
	<li>For intraperitoneal injections, narcotize the animals shortly in an anesthesia chamber by inhalation with isoflurane/oxygen using a flow rate of 2 L/min and the vaporizer set to 2%. Pinch the animal&#39;s toe and confirm that it does not withdraw its hindlimb to ensure proper anesthesia.</li>
	<li>Fill a 27 G disposable hypodermic syringe with 50 &#181;L of sonicated alpha-synuclein fibrils or PBS.</li>
	<li>Directly inject into the peritoneum of the mouse and avoid penetrating the small intestine or cecum located behind the abdominal wall by holding the animal in a dorsal position with the head facing away from the investigator and downward at approximately 45&#176;. Monitor animals until they have recovered from the anesthesia.</li>
</ol>
5. Bioluminescence Imaging
<ol>
	<li>Image Tg(M83+/-:Gfap-luc+/-) mice every 2-4 weeks using a bioluminescence imaging system.</li>
	<li>Prior to imaging shortly anesthetize mice in an isoflurane/oxygen chamber with a flow rate of 2 L/min and the vaporizer set to 2%. Pinch the animal&#39;s toe and confirm that it does not withdraw its hindlimb to ensure proper anesthesia.</li>
	<li>Shave and depilate the heads of the mice with a depilatory cream. Color the ears in black with a non-irritating marker to block unspecific bioluminescence.</li>
	<li>Dilute D-luciferin potassium salt, the substrate of luciferase, in PBS to a 30 mg/mL stock solution. Store this in 1 mL aliquots at -20 &#176;C. Protect the dissolved D-luciferin from exposure to light.</li>
	<li>Weigh the animals prior to injection. Calculate the exact volume of D-luciferin for injection of 150 mg/kg body weight. Intraperitoneally inject D-luciferin and return the animal to the anesthesia chamber.</li>
	<li>Start the imaging software. After the imaging system has reached its operating temperature initialize the system by clicking on the &#39;Initialize&#39; button in the control panel.</li>
	<li>Select the buttons &#39;Luminescent&#39; and &#39;Photograph&#39;. Set the exposure time to 60 s, the binning to &#39;Medium&#39;, the F/Stop to &#39;1&#39;, and the EM gain to &#39;Off&#39;.</li>
	<li>Confirm that the excitation filter is set to &#39;Block&#39; and the emission filter to &#39;Open&#39;, and set the subject height to 1.50 cm.</li>
	<li>Ten min after injection with D-luciferin, place the animals onto the heating plate in the imaging chamber and ensure that their muzzles are correctly placed in the anesthesia outlet. Close the isoflurane/oxygen flow from the inhalation chamber and open the flow for the imaging chamber with a flow rate of 0.25 L/min and an evaporation of 2%. Close the door of the imaging chamber properly.</li>
	<li>Click on the &#39;Acquire&#39; button to measure the bioluminescence, which takes 60 s. Stop the isoflurane flow and monitor the animals until they have completely recovered after returning them back to their cages.</li>
	<li>Use the imaging software to quantify the bioluminescence. Within the &#39;Tool Palette&#39; select &#39;ROI Tools&#39;. Under &#39;Type&#39; within the &#39;ROI Tools&#39; select &#39;Measurement ROI&#39;.</li>
	<li>Within the &#39;ROI Tools&#39; click the &#34;circle&#34; tool and select the number of ROIs to draw from the pull down menu &#8211; ideally &#39;3&#39; for three mice.</li>
	<li>In the image, right-click each ROI and under &#39;Properties&#39; adjust the width and height to 1.25 cm. Position each ROI over the brain area that is quantified.</li>
	<li>In the upper left of the image, set the units panel to &#39;radiance (photons)&#39;.</li>
	<li>Within the &#39;Tool Palette&#39;, select &#39;Image Adjust&#39; and adjust the minimum and the maximum of the &#39;Color Scale&#39;, for instance, from 0.20e6 to 1.00e6.</li>
	<li>Under &#39;File&#39; save the data with &#39;Save&#39;.</li>
</ol>
6. Biochemical Analysis
<ol>
	<li>Isolate the brains and spinal cords according to established protocols18,19.</li>
	<li>Homogenize whole brain and whole spinal cord samples separately in PBS in the presence of protease and phosphatase inhibitors (diluted to 1x in PBS) in two 30 s cycles with 6,000 rpm in a tissue homogenizer. Ensure that the brain and spinal cord samples are homogenized to a final concentration of 20% (wt/vol).</li>
	<li>Sonicate the samples twice while keeping them on ice with a constant pulse of 10 s and an amplitude set to 50%.</li>
	<li>Adjust the homogenates to 10% in PBS and 750 mM NaCl by diluting them 1:1 with 1.5 M NaCl in PBS.</li>
	<li>To separate the cellular debris, centrifuge the homogenates at 1,000 x g for 5 min at 4 &#176;C. Keep the supernatants for the next steps and discard the pellets.</li>
	<li>Measure the protein concentration in the homogenates using the bicinchoninic acid (BCA) assay according to the manufacturer&#39;s instructions. Incubate 1.0 mg of total protein from brain homogenates or 0.8 mg from spinal cord homogenates in N-laurylsarcosyl at a final concentration of 10% (wt/vol) for 15 min on ice.</li>
	<li>Overlay the homogenates onto a 3 mL sucrose cushion of 10% (wt/vol) in distilled water and ultracentrifuge them at 465,000 x g for 1 h at 4 &#176;C.</li>
	<li>Resuspend the pellets in a buffer containing 4% sodium dodecyl sulfate (SDS), 2% &#946;-mercaptoethanol, 192 mM glycine, 25 mM Tris, and 5% (wt/vol) sucrose.</li>
	<li>Boil the samples at 100 &#176;C for 5 min and load them onto SDS-polyacrylamide gels for electrophoresis in a morpholineethanesulfonic acid (MES) buffer system.</li>
	<li>Transfer the separated proteins to polyvinylidene difluoride (PVDF) membranes in a semidry blotting system.</li>
	<li>Incubate the membranes in 0.4% (vol/vol) formalin solution in PBS for 30 min at room temperature on a rotor with 35 rpm to cross-link the proteins with the membrane.</li>
	<li>Block the membranes in TBS buffer containing 0.05% (vol/vol) Tween 20 and 5% (wt/vol) milk for 1 h at room temperature.</li>
	<li>Incubate the blots with the primary antibody in blocking buffer overnight at 4 &#176;C.</li>
	<li>Wash the membranes three times in TBS buffer and incubate them with peroxidase- or fluorophore-conjugated secondary antibodies in TBS for 1 h at room temperature.</li>
	<li>Visualize the bound secondary antibodies by chemiluminescence or fluorescence according to established protocols20.</li>
</ol>
7. Immunofluorescence Analysis
<ol>
	<li>Dehydrate the dissected brains and spinal cords in a tissue processing station and embed them into paraffin using a paraffin station.</li>
	<li>Cut the paraffin-embedded tissues with a microtome in 8 &#181;m thick coronal sections and mount the sections on glass slides.</li>
	<li>Deparaffinize the tissue sections by incubating them in two separate xylol baths for 5 to 10 min and rehydrate them through a series of graded ethanol baths (100%, 90%, 70%, 50%) and finally with H2O.</li>
	<li>Incubate the sections in 0.01 M citrate buffer (pH 6.0) for 5 min at room temperature and additionally boil them for 10 min in a microwave oven.</li>
	<li>Let the sections cool down to room temperature and incubate them with a 3% hydrogen peroxide solution for 30 min to inhibit endogenous peroxidases.</li>
	<li>Block the tissue sections by incubation with 20% (vol/vol) normal goat serum, 1% (vol/vol) bovine serum albumin (BSA), and 0.5% Triton X-100 in PBS for 1 h at room temperature.</li>
	<li>Incubate the sections with the primary antibody diluted in 1% (vol/vol) normal goat serum, 1% (vol/vol) BSA, and 0.25% Triton X-100 in PBS overnight at 4 &#176;C.</li>
	<li>Wash the sections twice with 0.25% (vol/vol) Triton X-100 in PBS and once with PBS.</li>
	<li>Stain the sections with the corresponding fluorophore-conjugated secondary antibodies and the nuclear dye DAPI diluted in 1% (vol/vol) normal goat serum, 1% (vol/vol) BSA, and PBS for 1 h at room temperature.</li>
	<li>After washing twice with 0.25% (vol/vol) Triton X-100 in PBS and once with PBS, coverslip the slides with embedding media and visualize the staining with a confocal laser scanning microscope.</li>
</ol>

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The authors have nothing to disclose.

Peripheral injection of alpha-synuclein fibrils into the peritoneum of Tg(M83+/-:Gfap-luc+/-) mice represents a facile method to induce neurologic disease accompanied by neuroinflammation to recapitulate important characteristics of synucleinopathies. Similarly, tongue injection represents another route for neuroinvasion by alpha-synuclein prionoids in transgenic mice but is less efficient. We chose to terminate our experiments at 420 days after injection and we cannot exclude the possibility that more or all of the fibril injected mice may have developed disease at a later time point. Additionally, the use of Tg(M83+/-:Gfap-luc+/-) mice enables non-invasive visualization of astrogliosis and represents a simple method for monitoring inflammatory responses caused by the neuroinvasion of alpha-synuclein over the entire lifespan of the mouse.
A critical step is the preparation of the inoculum. Seemingly trivial factors like aggregation time, sonication time, the abundance of endotoxins, or the amount of fibrils used for injection could influence the outcome of an experiment by affecting the seeding propensity of misfolded alpha-synuclein21. Thus it is important to always use the same conditions when preparing inocula for injection. For intraglossal injections it has to be considered that not only the hypoglossal nerve but also other cranial nerves like the glossopharyngeal nerve innervate the tongue and could be involved in the interneuronal transport of alpha-synuclein prionoids to the brain. Thus targeting different areas of the tongue could result in different spreading kinetics to the brain. We did not directly inject into the hypoglossal nerve and it is possible that targeting the hypoglossal nerve could improve CNS transmission of alpha-synuclein prionoids. For intraperitoneal injections with alpha-synuclein fibrils it is critical that the inoculum is correctly injected into the peritoneum and not accidentally into internal organs like the intestine or cecum, which could negatively affect neuroinvasion. This is also important for bioluminescence imaging when injecting D-luciferin. Mistargeting of D-luciferin to internal organs could slow down its distribution to the brain and result in lower bioluminescence. To obtain uniform results during imaging it is also critical to always freshly prepare the D-luciferin solution prior to injection and to let it exactly incubate for 10 min after intraperitoneal injection before imaging.
Bioluminescence imaging is a useful method to non-invasively measure changes in astrogliosis over time and can be applied not only after intraperitoneal or intraglossal injections but also after every type of treatment including, for example, intracerebral or intravenous injections. To monitor spreading of alpha-synuclein prionoids from the periphery to the CNS and the ensuing neuroinflammation we used bigenic Tg(M83+/-:Gfap-luc+/-) mice. Because hemizygous expression of alpha-synuclein in these animals is 40% lower relative to homozygous animals they remain free of spontaneous disease for up to 22 months of age and have a suitable genetic background for alpha-synuclein transmission studies13. However, modifications regarding the choice of the mouse model for bioluminescence imaging can be desirable. An interesting option is the use of monogenic Tg(Gfap-luc+/-) mice expressing only luciferase as a transgene, which allows an assessment of the prionoid behavior of alpha-synuclein fibrils on a background that is essentially wild-type as far as alpha-synuclein is concerned14. Moreover, crossing Tg(Gfap-luc+/-) mice to other transgenic mice, such as Tg(SOD1G93A) mice for amyotrophic lateral sclerosis or to Tg(APP23) mice for Alzheimer&#39;s disease, enables imaging of neuroinflammation in other disease models22,23.
A limitation of this protocol is that based on the site of injection the injected inoculum cannot exceed a certain volume. Thus injections into the tongue can only be performed with smaller volumes than those into the peritoneum, which may affect transmission times to the CNS. Another limitation is that the Gfap-luc transgene only allows detection of astrogliosis but not of microgliosis, which is equally important in neuroinflammation. Lastly, this model does not provide insights as to how alpha-synuclein prionoids reach the CNS after intraperitoneal injection.
The application of exogenous alpha-synuclein prionoids via injection has been shown to be a useful method to study transmission pathways that play a critical role in recapitulating characteristics of synucleinopathies. Intracerebral injections of recombinant alpha-synuclein fibrils or disease-associated mouse or human alpha-synuclein species into animal models have been used in several studies to analyze their seeding properties and transmission efficiency over time5,7,17,24,25,26. Since stereotactic injections into the brain always induce a local inflammatory response shortly after surgery, this transmission route might influence the spreading and infectivity of the inoculum caused by an alteration of brain homeostasis. More recent studies have changed the focus to peripheral or systemic applications, not primarily to avoid the surgical procedure but rather to analyze the periphery, the intestinal wall, skeletal muscle, or blood, as an initial point from where neuroinvasion to the CNS could commence6,9,14,27. For prions it has been shown that transmission through the peritoneum or the tongue leads to neuroinvasion and neurologic disease; after tongue infection, prions spread within only two weeks to the hypoglossal nucleus in the brain stem11. Since alpha-synuclein has been discussed to have neuroinvasive and seeding properties like prions, we showed that transmission via the tongue and the peritoneum represent further entrance points for alpha-synuclein prionoids to invade the CNS14. Quantification of astrogliosis by bioluminescence imaging facilitates the tracking of the neuroinflammatory process over time and represents a non-invasive alternative to histological analysis.
To obtain a deeper insight into how the CNS responds to neuroinvasion of alpha-synuclein prionoids and to other treatments that cause neuroinflammation, it would be beneficial to generate mouse models that also allow non-invasive monitoring of microgliosis, e.g. with a transgene that drives luciferase expression from a promoter specific for protein expression in microglia.

There is growing evidence that alpha-synuclein has characteristics that are similar to those of the prion protein, particularly in its capacity to self-seed and propagate misfolding between cells and along neuronal pathways. This property of alpha-synuclein is also referred to as &#39;prion-like&#39; or &#39;prionoid&#39;, and is supported by observations in transplantation experiments, which suggest the transmissibility of misfolded alpha-synuclein from diseased neurons to newly transplanted healthy neurons1,2,3,4. Also direct injection of misfolded alpha-synuclein into the brain or the periphery, e.g. the hindlimb muscle or intestinal wall, results in a spread of alpha-synuclein pathology to distal parts of the CNS5,6,7,8,9,10. We analyzed transmission of alpha-synuclein prionoids via peripheral routes in more detail and addressed the question whether misfolded alpha-synuclein may neuroinvade the CNS after a single intraglossal or intraperitoneal injection, a feature that previously had been shown for prions but not for misfolded alpha-synuclein. After injection of prions into the tongue, neuroinvasion of the CNS is achieved via propagation along the hypoglossal nerve of the tongue that leads to the nucleus of the hypoglossal nerve, which is located in the brain stem11. As a mouse model we chose Tg(M83+/-:Gfap-luc+/-) mice that overexpress the A53T mutant of human alpha-synuclein from the prion promoter, and firefly luciferase under control of a Gfap promoter to monitor astrocytic activation by bioluminescence, as previously shown in the brain of prion-infected mice12. In our hands bigenic Tg(M83+/-:Gfap-luc+/-) mice did not develop disease until 23 months of age as has been shown by others13. A single injection of human alpha-synuclein fibrils via the intraglossal or intraperitoneal route induced neurologic disease with pathology in the brains and spinal cords of Tg(M83+/-:Gfap-luc+/-) mice supporting the hypothesis that alpha-synuclein prionoids share important characteristics with prions14.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-actin antibody</td>
			<td>Merck Millipore</td>
			<td>MAB1501</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-alpha-synuclein, phospho S129 antibody [pSyn#64]</td>
			<td>Wako</td>
			<td>015-25191</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-alpha-synuclein, phospho S129 antibody [EP1536Y]</td>
			<td>Abcam</td>
			<td>ab51253</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-GFAP antibody</td>
			<td>Dako</td>
			<td>Z0334 01</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-IBA-1 antibody</td>
			<td>Wako</td>
			<td>019-19741</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Sequestosome-1 (p62) antibody</td>
			<td>Proteintech</td>
			<td>18420-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-ubiquitin antibody [Ubi-1]</td>
			<td>Merck Millipore</td>
			<td>MAB1510</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Invitrogen</td>
			<td>14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine&#160;</td>
			<td>Ratiopharm</td>
			<td></td>
			<td>100 mg/kg</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Ratiopharm</td>
			<td></td>
			<td>10 mg/kg</td>
		</tr>
		<tr>
			<td>27-gauge syringe</td>
			<td>VWR</td>
			<td>613-4900</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane&#160;</td>
			<td>Piramal Healthcare</td>
			<td>PZN&#160; 4831850</td>
			<td></td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Veet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Secureline lab marker&#160;</td>
			<td>Neolab</td>
			<td>25040</td>
			<td></td>
		</tr>
		<tr>
			<td>D-luciferin potassium salt</td>
			<td>Acris</td>
			<td>LK10000</td>
			<td>30 mg/mL stock solution</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>5776671</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonopuls Mini20 sonicator</td>
			<td>Bandelin</td>
			<td>3648</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina II imaging system</td>
			<td>PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Living Image 3.0 Software</td>
			<td>PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tg(M83+/-) mice or B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J mice</td>
			<td>The Jackson Laboratory</td>
			<td>004479</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard pattern forceps</td>
			<td>Fine Science Tools</td>
			<td>11000-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow pattern forceps</td>
			<td>Fine Science Tools</td>
			<td>11002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>N-laurylasarcosyl</td>
			<td>Sigma</td>
			<td>L5125-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima Max-XP ultracentrifuge&#160;</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>TLA-110 rotor&#160;</td>
		</tr>
		<tr>
			<td>Thickwall polycarbonate tubes</td>
			<td>Beckman Coulter</td>
			<td>362305</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAG Novex 4-12% Bis-Tris Midi Protein Gels</td>
			<td>Thermo Fisher Scientific</td>
			<td>WG1401BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP conjugated antibody</td>
			<td>Cayman</td>
			<td>Cay10004301-1</td>
			<td></td>
		</tr>
		<tr>
			<td>IR Dye 680 conjugated antibody&#160;</td>
			<td>LI-COR Biosciences</td>
			<td>926-68070</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal West Dura Extended Duration Substrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>34075</td>
			<td></td>
		</tr>
		<tr>
			<td>Stella 3200 imaging system</td>
			<td>Raytest</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey infrared imaging system&#160;</td>
			<td>LI-COR Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20&#160;</td>
			<td>MP Biomedicals</td>
			<td>TWEEN201</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>SA/T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-FL PVDF membrane</td>
			<td>Merck Millipore</td>
			<td>IPFL00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylol</td>
			<td>Sigma</td>
			<td>Roth</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>Sigma</td>
			<td>SA/00216763/000500</td>
			<td>working solution 3%</td>
		</tr>
		<tr>
			<td>Bovine serum albumine (BSA)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A3294-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>PCN5000</td>
			<td></td>
		</tr>
		<tr>
			<td>4,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D1306</td>
			<td>working dilution 1:50,000</td>
		</tr>
		<tr>
			<td>Fluoromount media&#160;</td>
			<td>Omnilab</td>
			<td>SA/F4680/000025</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM700 confocal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HALT protease and phosphatase inhibitors</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>10516495</td>
			<td></td>
		</tr>
		<tr>
			<td>Precellys 24-Dual homogenizer&#160;</td>
			<td>Peqlab</td>
			<td>91-PCS24D</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 conjugated antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A31619</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 conjugated antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11005</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce&#160;BCA&#160;Protein&#160;Assay&#160;Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>10741395</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome RM2255</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSM700 confocal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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 prionoids via the tongue or the peritoneum induced neuropathology in the CNS of bigenic Tg(M83+/-:Gfap-luc+/-) mice (Table 1 and Figure 1). After a single intraperitoneal injection with alpha-synuclein fibrils, four of five mice developed neurologic disease with a median incubation time of 229 &#177;17 days. Surprisingly, only one of five mice developed CNS disease after intraglossal injection with alpha-synuclein fibrils after 285 days. Inoculation with PBS did not cause disease within the 420 day period of the experiment.
Biochemical analysis by Western blotting and probing with the phospho-specific EP1536Y antibody against Ser129 of alpha-synuclein revealed that for both transmission routes diseased animals had accumulated aggregates of phosphorylated and sarkosyl-insoluble alpha-synuclein in their brains and spinal cords (Table 2 and Figure 2). In contrast, brains and spinal cords of PBS-injected and non-diseased animals only contained monomeric species of phosphorylated alpha-synuclein.
Immunofluorescent staining of brain slices of intraperitoneally injected and diseased mice with the phospho-specific pSyn#64 antibody revealed a wide distribution of phosphorylated alpha-synuclein deposits throughout the CNS (Table 2 and Figure 3). Moreover, phosphorylated alpha-synuclein deposits colocalized with ubiquitin and p62 indicating an aberrant protein homeostasis that might contribute to the formation of deposits.
The accumulation of alpha-synuclein aggregates in diseased animals was accompanied by neuroinflammatory changes, which was detected by immunofluorescence staining for GFAP, a marker of astrocytes that can reveal reactive astrogliosis (Figure 4). Additionally, activated microglia were also found in regions with abundant alpha-synuclein pathology by immunofluorescence staining for IBA-1 (ionized calcium-binding adapter molecule 1). In accordance, bioluminescence imaging revealed increased radiance (&#62;2 &#215; 106 p/s/cm2/sr) emitted from the brains of Tg(M83+/-:Gfap-luc+/-) mice injected with alpha-synuclein fibrils, shortly before they developed signs of neurologic disease. In contrast, brains of mice injected with PBS did not show any increase in radiance. Increased radiance is indicative of reactive astrogliosis, a hallmark of neuroinflammation, since luciferase expression is driven from the Gfap promoter.
<img alt="Figure 1" src="/files/ftp_upload/55503/55503fig1.jpg" /> 
Figure 1: Injection with alpha-synuclein fibrils via the intraperitoneal or intraglossal route causes disease in bigenic Tg(M83+/-:Gfap-luc+/-) mice. (A) Electron micrograph of sonicated recombinant human alpha-synuclein fibrils that were intraperitoneally and intraglossally injected into mice. Negative staining with uranyl acetate revealed multiple clusters of short, rod-shaped aggregates. Scale bars = 100 nm. (B) Kaplan-Meier survival curves show that after intraperitoneal injection with alpha-synuclein fibrils four of five Tg(M83+/-:Gfap-luc+/-) mice developed neurologic disease in 229 &#177;17 days (purple closed squares), whereas none of the PBS-injected control mice developed signs of neurologic dysfunction within 420 days (purple open squares). After intraglossal inoculation with alpha-synuclein fibrils one mouse of five died after 285 days (green closed circles). In contrast, none of the PBS-injected control mice developed neurologic disease or spontaneously died (green open circles). Modified from Breid et al., 201614. <a href="http://cloudflare.jove.com/files/ftp_upload/55503/55503fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55503/55503fig2.jpg" /> 
Figure 2: Biochemical analysis of phosphorylated alpha-synuclein in the CNS of peripherally injected Tg(M83+/-:Gfap-luc+/-) mice. (A) Immunoblotting with the EP1536Y antibody, recognizing phosphorylation at Ser129 of alpha-synuclein, showed that intraperitoneally injected and diseased mice accumulated high-molecular-weight species of sarkosyl-insoluble aggregates of phosphorylated alpha-synuclein in their brains and spinal cords. Control mice challenged with PBS did not accumulate aggregates of phosphorylated alpha-synuclein and showed bands only for its monomeric form. (B) After intraglossal challenge with alpha-synuclein fibrils only one mouse, animal 121, showed sarkosyl-insoluble aggregates of phosphorylated alpha-synuclein in its brain and spinal cord, whereas all other intraglossally inoculated mice remained healthy without deposits of alpha-synuclein. Molecular masses are shown in kilodaltons. Sample loading in each lane is shown by detection of actin. Modified from Breid et al., 201614. <a href="http://cloudflare.jove.com/files/ftp_upload/55503/55503fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55503/55503fig3.jpg" /> 
Figure 3: Immunofluorescence analysis shows that deposits of phosphorylated alpha-synuclein colocalize with ubiquitin and p62 in the brains and spinal cords of diseased Tg(M83+/-:Gfap-luc+/-) mice. Co-staining of phosphorylated alpha-synuclein, detected with the EP1536Y antibody, and ubiquitin revealed a distinct colocalization in the brains, as observed in the thalamic nucleus (A), and spinal cords, as detected in the grey matter (B), of diseased mice after intraperitoneal challenge with alpha-synuclein fibrils. Similarly, phosphorylated alpha-synuclein, detected with the pSyn#64 antibody, also colocalized with p62 in brains (C) and spinal cords (D) of diseased animals. (A to D) PBS-injected healthy control animals did not show deposits or colocalization for any of these proteins. Nuclear staining with DAPI is shown in blue. Scale bars = 20 &#181;m. Modified from Breid et al., 201614. <a href="http://cloudflare.jove.com/files/ftp_upload/55503/55503fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55503/55503fig4.jpg" /> 
Figure 4: Tg(M83+/-:Gfap-luc+/-) mice developed gliosis after peripheral challenge with alpha-synuclein fibrils. (A) Immunofluorescence analysis of brain sections of diseased animals with an antibody against GFAP, a marker of astrocytes, revealed reactive astrogliosis in areas with deposits of phosphorylated alpha-synuclein, which were detected in the brain stem with the pSyn#64 antibody. In contrast, there was no reactive astrogliosis in brains of PBS-injected control mice. (B) Similarly, staining with an antibody to IBA-1, a marker of microglia, demonstrated microgliosis in areas with deposits of phosphorylated alpha-synuclein in diseased animals. Brains of healthy, PBS-injected control mice did not show signs of microgliosis. (C) Bioluminescence imaging revealed elevated radiance from the brains and spinal cords of Tg(M83+/-:Gfap-luc+/-) mice intraperitoneally injected with alpha-synuclein fibrils (left panel), which was caused by the activation of astrocytes, shortly before the mice developed neurologic symptoms. In the brains and spinal cords of PBS-injected control mice the basal radiance did not increase with time (right panel). After intraglossal inoculation with alpha-synuclein fibrils, one Tg(M83+/-:Gfap-luc+/-) mouse, animal 121, showed signs of reactive astrogliosis shortly before it died at 285 days (center panel). (D) After intraperitoneal challenge of Tg(M83+/-:Gfap-luc+/-) mice with alpha-synuclein fibrils, increased levels of bioluminescence (&#62;2 &#215; 106 p/s/cm2/sr) were measured from the brains of four mice (blue, green, brown, and orange circles) some weeks before they developed neurologic signs of disease. One of five animals also showed elevated levels of bioluminescence shortly before it died 285 days after intraglossal injection with alpha-synuclein fibrils (magenta circles). In contrast, for healthy, PBS-injected control mice (black circles; error bars show SD [n = 4]) and one animal that did not develop disease after intraperitoneal injection with alpha-synuclein fibrils (red circles), the bioluminescence signal remained below a threshold of 2 x 106 p/s/cm2/sr within the period of observation. Scale bars = 20 &#181;m. Modified from Breid et al., 201614. <a href="http://cloudflare.jove.com/files/ftp_upload/55503/55503fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mouse line</td>
			<td>Inoculum (&#956;g)</td>
			<td>Inoculation route</td>
			<td>No. of mice with disease/no. of mice inoculated</td>
			<td>Mean survival time &#177;SD (days)</td>
		</tr>
		<tr>
			<td>Tg(M83+/-:Gfap-luc+/-)</td>
			<td>human &#945;-synuclein fibrils (50)</td>
			<td>intraperitoneal</td>
			<td>4/5</td>
			<td>229 &#177;17/420</td>
		</tr>
		<tr>
			<td>Tg(M83+/-:Gfap-luc+/-)</td>
			<td>PBS</td>
			<td>intraperitoneal</td>
			<td>0/5</td>
			<td>0/420</td>
		</tr>
		<tr>
			<td>Tg(M83+/-:Gfap-luc+/-)</td>
			<td>human &#945;-synuclein fibrils (10)</td>
			<td>intraglossal</td>
			<td>1/5</td>
			<td>285/420</td>
		</tr>
		<tr>
			<td>Tg(M83+/-:Gfap-luc+/-)</td>
			<td>PBS</td>
			<td>intraglossal</td>
			<td>0/4</td>
			<td>0/420</td>
		</tr>
	</tbody>
</table>
Table 1: Inoculation experiments. *Modified from Breid et al., 201614
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Target [antibody clone]</td>
			<td>Host</td>
			<td>Immunogen</td>
			<td>Dilution in IF</td>
			<td>Dilution in WB</td>
		</tr>
		<tr>
			<td>Actin [C4]</td>
			<td>Mouse</td>
			<td>-</td>
			<td>-</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>&#945;-synuclein, phospho S129 [pSyn#64]</td>
			<td>Mouse</td>
			<td>pSer129</td>
			<td>1:1200</td>
			<td>-</td>
		</tr>
		<tr>
			<td>&#945;-synuclein, phospho S129 [EP1536Y]</td>
			<td>Rabbit</td>
			<td>pSer129</td>
			<td>1:100</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Glial fibrillary acidic protein (GFAP)</td>
			<td>Rabbit</td>
			<td>-</td>
			<td>1:200</td>
			<td>-</td>
		</tr>
		<tr>
			<td>IBA-1</td>
			<td>Rabbit</td>
			<td>-</td>
			<td>1:500</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sequestosome-1 (p62)</td>
			<td>Rabbit</td>
			<td>-</td>
			<td>1:100</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ubiquitin [Ubi-1]</td>
			<td>Mouse</td>
			<td>-</td>
			<td>1:500</td>
			<td>-</td>
		</tr>
	</tbody>
</table>
Table 2: Antibodies used for immunofluorescence (IF) and Western blotting (WB). *Modified from Breid et al., 201614

Watch this Scientific Journal Video about Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils at JoVE.com

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

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 ...

bioluminescence-imaging-neuroinflammation-transgenic-mice-after

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.5K Views.  German Center for Neurodegenerative Diseases (DZNE). 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.

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

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, the olfactory bulb (OB), the dorsal motor nucleus of the vagus (DMV)3, the intermediolateral nucleus of the spinal cord 4 and the substantia nigra, providing the basis for the neuropathological staging of the disease4,5. The ENS and the OB are the most exposed nervous structures and the first ones to be affected. Interestingly, PD has been related to pesticide exposure6-8. Here we show in detail two methods used in our previous study 9. In order to analyze the effects of rotenone acting locally on the ENS, we administered rotenone using a gavage to one-year old C57/BL6 mice. Rotenone is a widely used pesticide that strongly inhibits mitochondrial Complex I 10. It is highly lipophylic and poorly absorbed in the gastrointestinal tract 11. Our results showed that the administration of 5 mg/kg of rotenone did not inhibit mitochondrial Complex I activity in the muscle or the brain. Thus, suggesting that using our administration method rotenone did not cross the hepatoportal system and was acting solely on the ENS. Here we show a method to administer pesticides using a gavage and the image analysis protocol used to analyze the effects of the pesticide in alpha-synuclein accumulation in the ENS. The first part shows a method that allows intragastric administration of pesticides (rotenone) at a desired precise concentration. The second method shows a semi-automatic image analysis protocol to analyze alpha-synuclein accumulation in the ENS using an image analysis software.

Parkinson's disease has been related to the exposure to pesticides. Here we show a method to deliver pesticides using a gastric tube at the desired concentration and a method to analyze their effect in alpha-synuclein accumulation in the enteric nervous system.

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

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 ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the diagnosis still relies only on clinical signs of motor involvement that appear later on in the disease course, when most of the dopaminergic neurons are already lost. Hence, there is a strong need for a biomarker that can identify patients at the beginning of disease or at the risk of developing it. Over the last few years, skin biopsy has proved to be an excellent research and diagnostic tool for peripheral nerve diseases such as small fiber neuropathy. Interestingly, a small fiber neuropathy and alpha synuclein (&#945;Syn) neural deposits have been shown by skin biopsy in PD patients. Indeed, skin biopsy has the great advantage of being an easily accessible, minimally invasive and painless procedure that allows the analysis of peripheral nervous tissue prone to the pathology. Moreover, the possibility of repeating the skin biopsy in the course of the follow-up of the same patient allows studying the longitudinal correlation with the disease progression. We set up a standardized reliable protocol to investigate the presence of &#945;Syn aggregates in skin nerve fibers of the PD patient. This protocol involves few short fixation steps, a cryotome sectioning and then a free-floating immunofluorescence double-staining with two specific antibodies: anti Protein Gene Product 9.5 (PGP9.5) to mark the cutaneous nerve fibers and anti 5G4 for detecting &#945;Syn aggregates. It is a versatile, sensitive and easy to perform protocol that can also be applied for targeting other proteins of interest in skin nerves. The ability to mark &#945;Syn aggregates is another step forward to the use of skin biopsy as a tool for establishing a pre-mortem histopathological diagnosis of PD.

Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...