This work was partially supported by research grants from JSPS (KAKENHI 18K07389), Japan, Takeda Science Foundation, Japan, and Kobayashi Pharmaceutical Co., Ltd., Japan.

All experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals at the Sugitani Campus of the University of Toyama and were approved by the Committee for Animal Care and Use of Laboratory Animals at the Sugitani Campus of the University of Toyama (A2014INM-1, A2017INM-1).
1. Collapse Assay
<ol>
	<li>Poly-D-lysine coating
	<ol>
		<li>Coat 8-well culture slides with 400 &#956;L of 5 &#956;g/mL poly-D-lysine (PDL) in phosphate-buffered saline (PBS) and incubate them at 37 &#176;C overnight.</li>
		<li>Remove the PDL solution and wash the wells 3 times with distilled water.</li>
	</ol>
	</li>
	<li>Neuron culture24
	<ol>
		<li>Mince freshly isolated cerebral cortices from embryonic day 14 (E14) ddY mice with microscissors in neuron culture medium containing 12% horse serum, 0.6% glucose, and 2 mM L-glutamine (medium A). Do not add antibiotics. 
		NOTE: In this protocol, the ddY mouse is used. This is an outbred strain commonly used in Japan. This neuron culture protocol can be also applied for rat cortical neurons7,25.</li>
		<li>Centrifuge the tissues at 87 x g for 3 min.</li>
		<li>Remove the supernatant. Then to the pellet, add 2 mL of 0.05% trypsin and incubate for 15 min at 37&#176;C. Mix by tapping every 5 min.</li>
		<li>Add 4 mL of medium A and mix by tapping.</li>
		<li>Centrifuge the tissues at 178 x g for 3 min.</li>
		<li>Remove the supernatant, and incubate the tissues with 600 U/mL DNase I and 0.3 mg/mL soybean trypsin inhibitor dissolved in PBS for 15 min at 37 &#176;C. Mix by tapping every 5 min.</li>
		<li>After incubation, add 4 mL of medium A and mix by tapping.</li>
		<li>Centrifuge the tissues at 178 x g for 3 min.</li>
		<li>After removing the supernatant, add 4 mL of medium A and triturate the tissues with a polished Pasteur pipette.</li>
		<li>Filter the triturated tissues with a 70 &#181;m pore-size mesh. After filtration, calculate the density of cells with a hemocytometer.</li>
		<li>Culture the cells in the 8-well culture slide at 0.8 x 104 cells/well with medium A and maintain them in a CO2 incubator with a humidified atmosphere of 10% CO2 at 37 &#176;C.</li>
		<li>After 4 h of culturing, replace the culture medium to one containing 2% supplement for neuronal culture, 0.6% glucose, and 2 mM L-glutamine (medium B). 
		NOTE: The purity of neurons was approximately 75%, as described previously26.</li>
	</ol>
	</li>
	<li>Collapse assay27
	<ol>
		<li>Dissolve commercially obtained full-length amyloid &#946;1-42 (A&#946;1-42) in distilled water at a concentration of 0.5 mM and incubate at 37 &#176;C for 7 days. After the incubation, store the aggregated A&#946;1-42 solution in a -30 &#176;C freezer until use. 
		NOTE: This incubation is necessary for aggregation and toxicity of A&#946;27,28,29,30.</li>
		<li>After 4 days of neuronal culture, treat the wells with 100 &#956;L of new medium B, containing 0.5 &#956;M aggregated A&#946;1-42 or vehicle solution (distilled water) for 1 h. 
		NOTE: Effects of A&#946;1-42 were dose-dependently increased from 0.1 to 5 &#956;M, and peaked at 0.5 &#956;M as described previously27. Similar results can be observed when by A&#946;1-42 treatment for 1 h after 3 days of neuronal culture31.</li>
		<li>Remove the culture medium and immediately fix the neurons with 4% paraformaldehyde containing 4% sucrose in PBS for 1 h at 37 &#176;C on a hot plate.</li>
		<li>After fixation, wash the neurons 3 times with PBS and mount them with an aqueous mounting medium. Dry the mounting medium at 4 &#176;C for 2&#8211;4 days.</li>
		<li>Capture the entire area (7.8 x 9 mm2) of each well with a 20X dry objective lens on an inverted microscope.</li>
		<li>Classify the longest neurites of each neuron in stage 3 or 4 as axons, as previously described32,33.</li>
		<li>Classify growth cones according to the following criteria: 1) axonal growth cones lacking lamellipodia or 2) possessing fewer than three filopodia are considered collapsed growth cones, as described previously17. 
		NOTE: Healthy growth cones are scored as 0 point; collapsed growth cones are scored as 1 point. Mean collapse scores are calculated for each treatment.</li>
	</ol>
	</li>
</ol>
2. Amyloid &#946; Immunostaining
<ol>
	<li>Culture mouse cortical neurons for 3 days, as described in step 1.2.</li>
	<li>Treat with aggregated A&#946;1-42 (5 &#956;M) or vehicle for 4 h at 37 &#176;C in a CO2 incubator.</li>
	<li>Without removing the medium, add an equal volume of 4% paraformaldehyde containing 4% sucrose in PBS to each well, and maintain the culture at 37 &#176;C on a hot plate for 5 min.</li>
	<li>Replace the solution with 400 &#956;L of 4% paraformaldehyde containing 4% sucrose in PBS, and maintain at 37 &#176;C on the hot plate for 1 h. This fixation protocol was modified from a previous report34.</li>
	<li>Wash the neurons 3 times with PBS.</li>
	<li>Block with 5% normal goat serum in PBS.</li>
	<li>Incubate the neurons with mouse anti-amyloid &#946; immunoglobulin G (IgG) (1:50) and 1% bovine serum albumin in PBS at 4 &#176;C overnight.</li>
	<li>Wash the neurons 3 times with PBS.</li>
	<li>Incubate the neurons with a fluorescence-conjugated secondary antibody (1:400) and 1% bovine serum albumin in PBS at room temperature for 2 h.</li>
	<li>Wash the neurons 3 times with PBS and mount them with an aqueous mounting medium.</li>
	<li>Capture fluorescence images and bright field images with oblique illumination by using a 40X dry objective lens on inverted microscope B.</li>
</ol>
3. Axonal Immunostaining27
<ol>
	<li>Wash the neurons 3 times with PBS after cultured neuron fixation, as described in step 1.3.3.</li>
	<li>Incubate the neurons with mouse anti-tau-1 IgG (1:500), rabbit anti-microtubule associated protein 2 (MAP2) IgG (1:500) in 5% normal goat serum, and 0.3% t-octylphenoxypoly-ethoxyethanol in PBS at 4 &#176;C overnight.</li>
	<li>Wash the neurons 3 times with PBS.</li>
	<li>Incubate the neurons with fluorescence-conjugated secondary antibodies (1:400) and 0.3% t-octylphenoxypolyethoxyethanol in PBS at room temperature for 2 h.</li>
	<li>Wash the neurons 3 times with PBS, and mount using an aqueous mounting medium.</li>
	<li>Capture fluorescence and differential interference contrast (DIC) images by using a 20&#215; dry objective lens on inverted microscope A.</li>
</ol>
4. Live Cell Imaging27
<ol>
	<li>Coat glass-based dishes with 500 &#956;L of PDL (5 &#956;g/mL), as described in step 1.1.1. 
	NOTE: In this protocol, homemade glass-based dishes were used. Commercially available glass-based dishes can also be used for live imaging. Homemade glass-based dishes were prepared as follows: 1) make a hole approximately 1.4 mm in diameter in the center of a 35-mm dish with a hand punch, and 2) attach a glass coverslip (diameter of 22 mm) to the back of the dish with silicone.</li>
	<li>Wash the plates with distilled water, as described in step 1.1.2, and culture the cortical neurons in the glass-based dish at 3 x 104 cells/dish with medium A, as described in step 1.2.</li>
	<li>After 4 days of cell culture, replace the medium with 2 mL of new medium B, and transfer the dish to inverted microscope A. Maintain the culture in a humidified atmosphere of 10% CO2 at 37 &#176;C.</li>
</ol>
5. Endocytosis Experiment
<ol>
	<li>Culture the mouse cortical neurons as described in step 1.2.</li>
	<li>Four days later, replace the medium with 100 &#956;L of new medium B containing 20 &#956;M fluorescence membrane probe for 1 min.</li>
	<li>Add 1 &#956;L of 0.05 mM aggregated A&#946;1-42 (final 0.5 &#956;M) or vehicle (distilled water) solution and mix by pipetting. Incubate for 20 min.</li>
	<li>Remove the medium and wash the wells twice with medium B that has been pre-warmed to 37 &#176;C.</li>
	<li>Fix, wash, and mount the neurons as described in steps 1.3.3 and 1.3.4.</li>
	<li>Capture fluorescent and DIC images with a 63X oil objective lens on inverted microscope A.</li>
	<li>Quantify the density of the fluorescence membrane probe-positive area in each healthy growth cone by using an image software.</li>
</ol>
6. Gene Transfection
<ol>
	<li>Prepare cortical neurons as described in step 1.2. After completing steps 1.2.1 to 1.2.10, centrifuge the neurons at 178 x g for 3 min.</li>
	<li>Remove the supernatant, add 4 mL of Ca2+-free and Mg2+-free Hanks&#39; balanced salt solution (CMF-HBSS), and mix by pipetting.</li>
	<li>Centrifuge the cells at 178 x g for 3 min.</li>
	<li>Remove the supernatant, add 4 mL of CMF-HBSS, and mix by pipetting. Next, calculate the cell density, as described in step 1.2.10.</li>
	<li>Transfer 5 x 106 cells to a 1.5 mL tube and centrifuge at 1,677 x g for 1 min.</li>
	<li>Remove the supernatant, add 100 &#956;L of transfection solution with supplement and 3 &#956;g of DNA plasmid encoding EGFP or EGFP-AP180 C-terminus, and mix by pipetting.</li>
	<li>Transfer the above solution (step 6.6) to a certified cuvette and transfect with an electroporator, according to the manufacturer&#39;s protocol.</li>
	<li>Immediately after transfection, add 500 &#956;L of medium A into the cuvette and transfer the solution to a 1.5-mL tube with a certified pipette. Next, calculate the cell density, as described in step 1.2.10.</li>
	<li>Culture the cells in an 8-well culture slide at 0.8 x 104 cells/well, as described in steps 1.2.11 and 1.2.12.</li>
	<li>After 4 days of cell culture, perform a collapse assay as described in step 1.3.</li>
</ol>

The author has nothing to disclose.

The protocol described in this study enabled the observation of early phenomena in axonal growth cones after A&#946;1-42 treatment. A&#946;1-42 induced endocytosis in axonal growth cones within 20 min, and growth cone collapse was observed within 1 h of treatment. This endocytosis was probably mediated by clathrin. By using this protocol, the inhibition of clathrin-mediated endocytosis was confirmed to prevent A&#946;1-42-induced growth cone collapse and axonal degeneration in cultured neurons27. Additionally, the inhibition of clathrin-mediated endocytosis attenuated A&#946;1-42-induced axonal degeneration and memory deficits in vivo27. These results indicate that clathrin-mediated endocytosis is a promising therapeutic avenue for AD prevention.
This protocol was developed from collapse assays for axonal growth repellents, such as semaphorin 3A and ephrin-A516,17,18,19,20. Collapse assays have been used in studies assessing the development of neuronal networks. I have shown that this protocol can be applied to pathological analyses, particularly those involving mechanisms of AD; however, a limitation may be that approximately 40% of growth cones collapsed in the healthy condition. This percentage is higher than results from cultured dorsal root ganglion neurons, which are more commonly used in collapse assays16,17,18,19,20. Therefore, the difference in cell types might be linked to differences in collapse ratios. The collapse ratios found in this study were consistent with those found in previous studies with normal cultured cortical neurons40,41. Furthermore, A&#946;1-42 induced similar levels of growth cone collapse when compared with other collapse factors, such as semaphorin 3A and ephrin-A527. Therefore, this protocol is valid for the quantification of A&#946;1-42-induced growth cone collapse. This fixation protocol is important to maintain the shape of growth cones. If the cells were conventionally fixed with 4% paraformaldehyde at room temperature, more growth cones may have collapsed due to the fixation procedure (data not shown). Alternatively, glutaraldehyde and fixation buffers are available for rigid fixation, as previously described42; however, glutaraldehyde exhibits autofluorescence, which is a significant impediment for fluorescence imaging.
A recent study with the same protocol showed that the water extract from Radix Polygalae (roots of Polygala tenuifolia) inhibited A&#946;1-42-induced endocytosis in cultured neurons, prevented axonal degeneration, and reduced memory deficits in a transgenic mouse model of AD31. A novel candidate for AD prevention has been found with this protocol. A combination of gene transfection and live cell imaging in this protocol might show the other cellular events found in axons and their terminals before and after A&#946; treatment, such as Ca2+ imaging, microtubule dynamics, and cell adhesion dynamics, which are reportedly related to axonal growth43,44,45. This protocol may help reveal more detailed mechanisms of A&#946; toxicity and may help lead to the prevention and/or treatment of AD.

Amyloid-&#946; (A&#946;) deposits are found in the brain of patients with Alzheimer's disease (AD) and are considered a critical cause of AD1 that disrupt neuronal networks, leading to memory impairments2,3,4. Many clinical drug candidates have been shown to effectively prevent amyloid-&#946; (A&#946;) production or remove A&#946; deposits. However, none have succeeded in improving memory function in AD patients5. A&#946; is already deposited in the brain prior to the onset of memory impairments6; therefore, decreasing A&#946; levels in the brains of patients exhibiting memory impairments may be ineffective. A&#946; deposition is present in preclinical AD patients; however, these patients rarely present with neuronal degeneration and memory deficits6. There is a time lag between A&#946; deposition and memory impairments. Therefore, a critical strategy for the prevention of AD is blocking A&#946; toxicity signaling during the early stages of AD, prior to the development of memory deficits. A&#946; deposition induces axon degeneration7,8,9,10,11,12,13, which may lead to a disruption of neural networks and permanent impairment of memory function. Many studies have investigated the mechanisms of A&#946; toxicity; for example, the degenerated axons of AD mice brains have been shown to have increased autophagy14. Calcineurin activation has been reported as a possible mechanism of A&#946;-induced axonal degeneration15; however, the direct trigger of axonal degeneration remains unknown. 
This study focuses on the collapse of axonal endings called growth cones. The collapse of axonal growth cones can be caused by axonal growth repellents, such as semaphorin-3A and ephrin-A516,17,18,19,20. Collapse-like dystrophic axonal endings have been observed in the brains of AD patients21,22. Additionally, a failure of growth cone functioning can provoke axonal degeneration23. However, it is unknown whether A&#946; induces growth cone collapse. Therefore, this study presents a novel protocol to observe the early effects of A&#946; in cultured neurons and investigate A&#946;-induced growth cone collapse.

<table><tbody><tr><td>ddY mice</td><td>SLC</td><td></td><td></td></tr><tr><td>Eight-well culture slide</td><td>Falcon</td><td>354108</td><td></td></tr><tr><td>poly D lysine</td><td>Wako</td><td>168-19041</td><td></td></tr><tr><td>Culture medium, Neurobasal medium</td><td>Gibco</td><td>21103-049</td><td></td></tr><tr><td>house serum</td><td>Gibco</td><td>26050-088</td><td></td></tr><tr><td>glucose</td><td>Wako</td><td>049-31165</td><td></td></tr><tr><td>L-glutamine</td><td>Wako</td><td>074-00522</td><td></td></tr><tr><td>0.05% trypsin</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>DNase I</td><td>Worthington</td><td>DP</td><td></td></tr><tr><td>soybean trypsin inhibitor</td><td>Gibco</td><td>17075-029</td><td></td></tr><tr><td>Filter with 70 &amp;micro;m mesh size, cell strainer</td><td>Falcon</td><td>352350</td><td></td></tr><tr><td>B-27 supplement</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Astec</td><td>SCA-165DS</td><td></td></tr><tr><td>Amyloid &amp;beta;1-42</td><td>Sigma-Aldrich</td><td>A9810</td><td></td></tr><tr><td>paraformaldehyde</td><td>Wako</td><td>162-16065</td><td></td></tr><tr><td>sucrose&amp;nbsp;</td><td>Wako</td><td>196-00015</td><td></td></tr><tr><td>Aqueous mounting medium, Aqua-Poly/Mount</td><td>polysciences</td><td>18606-20</td><td></td></tr><tr><td>Inverted microscope A</td><td>Carl Zeiss</td><td>Axio Observer Z1&amp;nbsp;</td><td>Connected with AxioCam MRm, Heating Unit XL S, CO2 Module S1, and TempModule S1</td></tr><tr><td>Objective Plan-Apochromat 20x</td><td>Carl Zeiss</td><td>420650-9901</td><td></td></tr><tr><td>Objective Plan-Apochromat 63x</td><td>Carl Zeiss</td><td>440762-9904</td><td></td></tr><tr><td>Objective, CFI Plan Apo Lambda 40X</td><td>Nikon</td><td></td><td></td></tr><tr><td>anti-MAP2 IgG</td><td>Abcam</td><td>ab32454</td><td></td></tr><tr><td>anti-tau-1 IgG</td><td>Chemicon</td><td>MAB3420</td><td></td></tr><tr><td>anti-amyloid &amp;beta; antibody</td><td>IBL</td><td>10379</td><td>clone 11A1</td></tr><tr><td>normal goat serum</td><td>Wako</td><td>143-06561</td><td></td></tr><tr><td>bovine serum albumin</td><td>Wako</td><td>010-25783</td><td></td></tr><tr><td>t-octylphenoxypolyethoxyethanol</td><td>Wako</td><td>169-21105</td><td></td></tr><tr><td>goat anti-mouse IgG conjugated with AlexaFluor 594</td><td>Invitrogen</td><td>A11032</td><td></td></tr><tr><td>goat anti-rabbit IgG conjugated with AlexaFluor 488</td><td>Invitrogen</td><td>A11029</td><td></td></tr><tr><td>hot plate</td><td>NISSIN</td><td>NHP-M30N</td><td></td></tr><tr><td>cover glass</td><td>Fisher Scientific</td><td>12-545-85</td><td></td></tr><tr><td>35 mm dish</td><td>IWAKI</td><td>1000-035</td><td></td></tr><tr><td>Silicone RTV</td><td>Shin-Etsu</td><td>KE42T</td><td></td></tr><tr><td>hand punch</td><td>Roper Whitney</td><td>No. XX</td><td></td></tr><tr><td>Fluorescence membrane probe, FM1-43FX</td><td>Invitrogen</td><td>F35355</td><td></td></tr><tr><td>Ca&lt;sup&gt;2+&lt;/sup&gt;- and Mg&lt;sup&gt;2+&lt;/sup&gt;-free Hanks&#039; balanced salt solution</td><td>Gibco</td><td>14175-095</td><td></td></tr><tr><td>Transfection solution, Nucleofector solution</td><td>Lonza</td><td>VPG-1001</td><td></td></tr><tr><td>Electroporator, Nucleofector I</td><td>Amaxa</td><td></td><td></td></tr><tr><td>Inverted microscope B</td><td>Keyence</td><td>BZ-X710</td><td></td></tr><tr><td>Image software, ImageJ</td><td>NIH</td><td></td><td>https://imagej.nih.gov/ij/</td></tr></tbody></table>

visualizing axonal growth cone collapse and early amyloid &#946; effects in cultured mouse neurons

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the brains of AD patients, these do not improve memory functions. Since A&#946; aggregates in the brain before the appearance of memory impairments, targeting A&#946; may be inefficient for treating AD patients who already exhibit memory deficits. Therefore, downstream signaling due to A&#946; deposition should be blocked before AD development. A&#946; induces axonal degeneration, leading to the disruption of neuronal networks and memory impairments. Although there are many studies on the mechanisms of A&#946; toxicity, the source of A&#946; toxicity remains unknown. To help identify the source, we propose a novel protocol that uses microscopy, gene transfection, and live cell imaging to investigate early changes caused by A&#946; in axonal growth cones of cultured neurons. This protocol revealed that A&#946; induced clathrin-mediated endocytosis in axonal growth cones followed by growth cone collapse, demonstrating that inhibition of endocytosis prevents A&#946; toxicity. This protocol will be useful in studying the early effects of A&#946; and may lead to more efficient and preventative AD treatment.

In this protocol, A&#946;1-42 was incubated at 37 &#176;C for 7 days before use, because incubation of A&#946;1-42 was needed for producing toxic forms27,28,30,35. After this incubation, aggregated forms of A&#946; were observed (Figure 1A). It has been reported that similar incubation of A&#946;1-42 produced the fibril form of A&#946;36. After treatment with this aggregated A&#946;1-42, immunostaining with an antibody for the toxic oligomer of A&#946;35,37 was performed, and positive staining was detected on cultured neurons (Figure 1B). Considering the above, this incubation protocol produces the toxic forms of A&#946;.
Several days were required for the induction of axonal degeneration after A&#946; exposure. The events prior to axonal degeneration remain unclear. Therefore, this protocol has been developed to further understand the mechanisms involved. Using this protocol, the early phenomena induced by A&#946; treatment were analyzed. Cortical neurons were cultured for 4 days. The longest neurites in the cultured neurons were identified as axons; these were confirmed by positive immunostaining for the axonal marker, tau-1, and negative immunostaining for the dendritic marker, MAP2 (Figure 2). After 1 h of vehicle treatment, growth cones had spread lamellipodia and processed several filopodia. These were identified as healthy growth cones. Conversely, 1 h of A&#946;1-42 treatment led to shrunken growth cones, which developed no lamellipodia or filopodia. These were identified as collapsed growth cones. Collapse scores were calculated as described in step 1.3.7. When shapes of growth cones were unclear, they were eliminated from the analysis. A&#946;1-42 treatment led to a significant increase in collapse score, corresponding to increased axonal growth collapse, when compared to the collapse score of vehicle-treated growth cones27.
Axonal growth cones were observed before and after treatment with A&#946;1-42 (Figure 3). Cells were maintained in the inverted microscope with a humidified atmosphere of 10% CO2 at 37 &#176;C. Images were captured every 5 min. As shown in Figure 3, growth cones collapsed between 21 and 26 min after A&#946;1-42 treatment. Growth cones were excluded from live cell imaging if they did not retain their healthy shape for 1 h prior to any treatment.
To visualize the early effects of A&#946;1-42-treatment, endocytosis was used as the focus of this analysis, because endocytosis inhibitors can block A&#946;1-42-induced growth-cone collapse27. Endocytosis was visualized with a fluorescence membrane probe (i.e., a fluorescent dye that binds to plasma membranes and is spontaneously endocytosed). A previous study showed that growth cones do not collapse at 20 min after A&#946;1-42-treatment27; therefore, healthy growth cones were selected by DIC imaging in vehicle- or A&#946;1-42-treated cells after 20 min. Following A&#946;1-42-treatment, numerous fluorescent membrane probe-positive puncta were observed in the growth cone (Figure 4). The density of fluorescence membrane probe-positive puncta in growth cones was significantly increased27. This suggests that A&#946;1-42-induced growth cone endocytosis occurs prior to collapse.
To confirm the role of endocytosis, a DNA plasmid encoding EGFP-AP180 C-terminus was transfected into cultured cortical neurons. Cells expressing the AP180 C-terminus selectively inhibited clathrin-mediated endocytosis38,39. If EGFP expression was observed at the cell body in the neuron, the AP180 C-terminus was considered to be expressed at the axonal growth cone of the neuron. Transfection of AP180 C-terminus blocked A&#946;1-42-induced growth cone collapse (Figure 5)27.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58229/58229fig1.jpg" /> 
Figure 1: Incubation of A&#946;1-42 aggregates A&#946;. (A) A&#946;1-42 was dissolved in distilled water at a concentration of 0.5 mM and incubated at 37 &#176;C for 7 days (after incubation), or stored at -30 &#176;C without incubation (no incubation). Each A&#946; solution was diluted to 0.1 mM; then, 10 &#956;L of each diluted solution was dropped on glass slides and covered with coverslips. Bright-field images with oblique illumination were captured by using inverted microscope B. Scale bar = 20 &#956;m. (B) Aggregated A&#946;1-42 or vehicle treatment on cultured neurons for 4 h. Following treatment, the neurons were fixed and immunostained for toxic A&#946; oligomers. Fluorescence images (red) and bright-field images with oblique illumination (gray) are shown. Scale bar = 20 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58229/58229fig1large.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/58229/58229fig2.jpg" /> 
Figure 2: A&#946;1-42-induced axonal growth cone collapse. After A&#946;1-42- or vehicle-treatment, neurons were fixed and immunostained for tau-1 (red) and microtubule associated protein 2 (MAP2, green). Fluorescence and differential interference contrast (DIC) images are shown. Magnified views of the regions of interest (ROI, rectangles) are shown below their corresponding images. White scale bars = 50 &#956;m; black scale bars = 10 &#956;m. This figure has been modified from Kuboyama et al, 201527. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58229/58229fig2large.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/58229/58229fig3.jpg" /> 
Figure 3: Live cell imaging before and after A&#946;1-42 treatment. After 4 days of culture, cells were transferred to an inverted microscope and DIC images were captured every 5 min. Time-lapse images are shown. The digits represent minutes:seconds after the application of aggregated A&#946;1-42 (final concentration, 0.5 &#956;M). Scale bar = 10 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58229/58229fig3large.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/58229/58229fig4.jpg" /> 
Figure 4: Twenty minutes of A&#946;1-42 treatment induced endocytosis. Cortical neurons were cultured for 4 days and treated with a fluorescence membrane probe. Then, neurons were treated for 20 min with A&#946;1-42 or vehicle. Fluorescence images of the growth cones are shown. The yellow dotted lines represent the outlines of the growth cones. Scale bar = 10 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58229/58229fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58229/58229fig5.jpg" /> 
Figure 5: Expression of AP180-C terminus blocks A&#946;1-42-induced collapse. Four days after transfection of EGFP (A, B) or EGFP-AP180 C-terminus (C, D); A&#946;1-42 (B, D) or vehicle (A, C) was added to cortical neurons for 1 h. DIC (upper panels) and fluorescence (bottom panels) images are shown. Arrows indicate growth cones. Scale bars = 10 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58229/58229fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Visualizing Axonal Growth Cone Collapse and Early Amyloid &#946; Effects in Cultured Mouse Neurons

Here a protocol to investigate the early effects of amyloid-&#946; (A&#946;) in the brain is presented. This shows that A&#946; induces clathrin-mediated endocytosis and collapse of axonal growth cones. The protocol is useful in studying early effects of A&#946; on axonal growth cones and may facilitate prevention of Alzheimer's disease.

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the ...

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7.9K Views.  University of Toyama. Here a protocol to investigate the early effects of amyloid-β (Aβ) in the brain is presented. This shows that Aβ induces clathrin-mediated endocytosis and collapse of axonal growth cones. The protocol is useful in studying early effects of Aβ on axonal growth cones and may facilitate prevention of Alzheimer's disease.

Video: Visualizing Axonal Growth Cone Collapse and Early Amyloid β Effects in Cultured Mouse Neurons

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

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

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

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

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

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

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

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

Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding immunostaining (specifically for ionized calcium-binding adapter molecule 1 (IBA1), a calcium binding protein expressed by microglia) and tissue processing for electron microscopy (EM). Methoxy-X04 is a fluorescent dye that crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. Injection of the animals with methoxy-X04 prior to sacrifice and brain fixation allows pre-screening and selection of the plaque-containing brain sections for further processing with time-consuming manipulations. This is particularly helpful when studying early AD pathology within specific brain regions or layers that may contain very few plaques, present in only a small fraction of the sections. Post-mortem processing of tissue sections with Congo Red, Thioflavin S, and Thioflavin T (or even with methoxy-X04) can label &#946;-pleated sheets, but requires extensive clearing with ethanol to remove excess dye and these procedures are incompatible with ultrastructural preservation. It would also be inefficient to perform labeling for A&#946; (and other cellular markers such as IBA1) on all brain sections from the regions of interest, only to yield a small fraction containing A&#946; plaques at the right location. Importantly, A&#946; plaques are still visible after tissue processing for EM, allowing for a precise identification of the areas (generally down to a few square millimeters) to examine with the electron microscope.

This article describes a protocol for visualizing amyloid A&#946; plaques in Alzheimer&#39;s disease mouse models using methoxy-X04, which crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. It allows pre-screening of plaque-containing tissue sections prior to immunostaining and processing for electron microscopy.

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Medicine

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 ...

Detecting Amyloid-&#946; Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies on accumulation of amyloid-&#946; (A&#946;) in the brain. A&#946; is produced from the proteolytic cleavage of the beta-amyloid precursor protein (APP) by &#946;-secretase and &#947;-secretase. In pathological circumstances, the increased &#946;-cleavage of APP leads to overproduction of A&#946;, which aggregates into A&#946; plaques. Since A&#946; plaques are a characteristic of AD pathology, detecting the amount of A&#946; is very important in AD research. In this protocol, we introduce the immunofluorescent staining method to visualize A&#946; deposition. The mouse model used in our experiments is 5&#215;FAD, which carries five mutations found in human familial AD. The neuropathological and behavioral deficits of 5xFAD mice are well-documented, which makes it a good animal model to study A&#946; pathology. We will introduce the procedure including transcardial perfusion, cryosectioning, immunofluorescent staining and quantification to detect A&#946; accumulation in 5&#215;FAD mice. With this protocol, researchers can investigate A&#946; pathology in an AD mouse model.

In the neuropathology of Alzheimer's disease, one of the most crucial characteristics is the deposition of amyloid-&#946;. In this protocol, we describe the method of immunofluorescent staining in 5&#215;FAD transgenic mouse to detect amyloid-&#946; accumulation in plaques. The process of perfusion, cryosectioning, staining and quantification will be described in detail.

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies ...

Visualizing Neuronal and Glial Cell Dynamics in the Developing Mouse Brain

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

Author Spotlight: Exploring Cell Migration and Gene Roles in the Developing Brain

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...

In Vitro Assessment of Aggregated Amyloid-&beta; on Neuronal Growth Cone Collapse

Source: Kuboyama, T. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58229">Visualizing Axonal Growth Cone Collapse and Early Amyloid &#946; Effects in Cultured Mouse Neurons.</a> J. Vis. Exp. (2018)
This video demonstrates an in vitro assessment of neuronal growth cone collapse by amyloid-&#946; (A&#946;) aggregates. Treating the neurons with A&#946; aggregates causes cytoskeletal destabilization, leading to the collapse of growth cones at the tip of the axons. The treated cells are compared with control cells to assess the extent of growth cone collapse.

1. Collapse Assay

	Poly-D-lysine coating 
	
		Coat 8-well culture slides with 400 &#956;L of 5 &#956;g/mL poly-D-lysine (PDL) in phosphate-buffered ...

Visualizing Axonal Growth Cone Collapse and Early Amyloid β Effects in Cultured Mouse Neurons

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Chapters in this video

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Intracerebroventricular Injection of Amyloid-β Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Correlative Light and Electron Microscopy to Study Microglial Interactions with β-Amyloid Plaques

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

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

Detecting Amyloid-β Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

In Vitro Assessment of Aggregated Amyloid-β on Neuronal Growth Cone Collapse