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. ...

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 border="0" cellpadding="0" cellspacing="0" style="width:1120px;" width="1119">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:339px;">ddY mice</td>
			<td style="width:133px;">SLC</td>
			<td style="width:109px;"></td>
			<td style="width:539px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Eight-well culture slide</td>
			<td>Falcon</td>
			<td>354108</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">poly D lysine</td>
			<td>Wako</td>
			<td>168-19041</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Culture medium, Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">house serum</td>
			<td>Gibco</td>
			<td>26050-088</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">glucose</td>
			<td>Wako</td>
			<td>049-31165</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-glutamine</td>
			<td>Wako</td>
			<td>074-00522</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.05% trypsin</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DNase I</td>
			<td>Worthington</td>
			<td>DP</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">soybean trypsin inhibitor</td>
			<td>Gibco</td>
			<td>17075-029</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Filter with 70 &#181;m mesh size, cell strainer</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">B-27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CO2 incubator</td>
			<td>Astec</td>
			<td>SCA-165DS</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Amyloid &#946;1-42</td>
			<td>Sigma-Aldrich</td>
			<td>A9810</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">paraformaldehyde</td>
			<td>Wako</td>
			<td>162-16065</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">sucrose&#160;</td>
			<td>Wako</td>
			<td>196-00015</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Aqueous mounting medium, Aqua-Poly/Mount</td>
			<td>polysciences</td>
			<td>18606-20</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Inverted microscope A</td>
			<td>Carl Zeiss</td>
			<td>Axio Observer Z1&#160;</td>
			<td>Connected with AxioCam MRm, Heating Unit XL S, CO2 Module S1, and TempModule S1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Objective Plan-Apochromat 20x</td>
			<td>Carl Zeiss</td>
			<td>420650-9901</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Objective Plan-Apochromat 63x</td>
			<td>Carl Zeiss</td>
			<td>440762-9904</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Objective, CFI Plan Apo Lambda 40X</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">anti-MAP2 IgG</td>
			<td>Abcam</td>
			<td>ab32454</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">anti-tau-1 IgG</td>
			<td>Chemicon</td>
			<td>MAB3420</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">anti-amyloid &#946; antibody</td>
			<td>IBL</td>
			<td>10379</td>
			<td>clone 11A1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">normal goat serum</td>
			<td>Wako</td>
			<td>143-06561</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">bovine serum albumin</td>
			<td>Wako</td>
			<td>010-25783</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">t-octylphenoxypolyethoxyethanol</td>
			<td>Wako</td>
			<td>169-21105</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">goat anti-mouse IgG conjugated with AlexaFluor 594</td>
			<td>Invitrogen</td>
			<td>A11032</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">goat anti-rabbit IgG conjugated with AlexaFluor 488</td>
			<td>Invitrogen</td>
			<td>A11029</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">hot plate</td>
			<td>NISSIN</td>
			<td>NHP-M30N</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">cover glass</td>
			<td>Fisher Scientific</td>
			<td>12-545-85</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">35 mm dish</td>
			<td>IWAKI</td>
			<td>1000-035</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Silicone RTV</td>
			<td>Shin-Etsu</td>
			<td>KE42T</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:339px;">hand punch</td>
			<td style="width:133px;">Roper Whitney</td>
			<td style="width:109px;">No. XX</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fluorescence membrane probe, FM1-43FX</td>
			<td>Invitrogen</td>
			<td>F35355</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ca2+- and Mg2+-free Hanks&#39; balanced salt solution</td>
			<td>Gibco</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Transfection solution, Nucleofector solution</td>
			<td>Lonza</td>
			<td>VPG-1001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electroporator, Nucleofector I</td>
			<td>Amaxa</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Inverted microscope B</td>
			<td>Keyence</td>
			<td>BZ-X710</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:339px;">Image software, ImageJ</td>
			<td style="width:133px;">NIH</td>
			<td style="width:109px;"></td>
			<td style="width:539px;">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&#9...

Watch this Scientific Journal Video about Visualizing Axonal Growth Cone Collapse and Early Amyloid ÃŽÂ² Effects in Cultured Mouse Neurons at JoVE.com

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 ...

visualizing-axonal-growth-cone-collapse-early-amyloid-effects

Research

JoVE Journal

Neuroscience

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

Mechanical Manipulation of Neurons to Control Axonal Development

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the 10-1000 &mu;dyne range1,2. Force measurements are obtained through observation of the Hookean bending of the glass needles, which are calibrated by a direct and empirical method3. Equipment requirements and procedures for fabricating, calibrating, treating, and using the needles on cells are fully described. The force regimes previously used and different cell types to which these techniques have been applied demonstrate the flexibility of the methodology and are given as examples for future investigation4-6. The technical advantages are the continuous 'visualization' of the forces produced by the manipulations and the ability to directly intervene in a variety of cellular events. These include direct stimulation and regulation of axonal growth and retraction7; as well as detachment and mechanical measurements on any type of cultured cell8.

Application and direct measurements of forces on neurons in the 2-1000 microdyne range are achieved with high precision using calibrated glass needles. This methodology can be used to control and measure several aspects of axonal development, including axonal initiation, axonal tension, velocity of axonal elongation, and force vectors.

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light whereas cones operate continuously under rapidly changing bright light conditions. Absorption of light by rod- and cone-specific visual pigments in the outer segments of photoreceptors triggers a phototransduction cascade that eventually leads to closure of cyclic nucleotide-gated channels on the plasma membrane and cell hyperpolarization. This light-induced change in membrane current and potential can be registered as a photoresponse, by either classical suction electrode recording technique1,2 or by transretinal electroretinogram recordings (ERG) from isolated retinas with pharmacologically blocked postsynaptic response components3-5. The latter method allows drug-accessible long-lasting recordings from mouse photoreceptors and is particularly useful for obtaining stable photoresponses from the scarce and fragile mouse cones. In the case of cones, such experiments can be performed both in dark-adapted conditions and following intense illumination that bleaches essentially all visual pigment, to monitor the process of cone photosensitivity recovery during dark adaptation6,7. In this video, we will show how to perform rod- and M/L-cone-driven transretinal recordings from dark-adapted mouse retina. Rod recordings will be carried out using retina of wild type (C57Bl/6) mice. For simplicity, cone recordings will be obtained from genetically modified rod transducin &alpha;-subunit knockout (T&alpha;-/-) mice which lack rod signaling8.

We describe a relatively simple method of transretinal electroretinogram (ERG) recordings for obtaining rod and cone photoresponses from intact mouse retina. This approach takes advantage of the block of synaptic transmission from photoreceptors to isolate their light responses and record them using field electrodes placed across the isolated flat-mounted retina.

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile stimulation is a positive early experience that mimics maternal licking and grooming in the rat. Exposing rat pups to this positive experience can be completed easily and cost-effectively by using highly accessible materials such as a household duster. Using a cross-litter design, pups are either stroked or left undisturbed, for 15 min, three times per day throughout the perinatal period. To measure the neuroplastic changes related to this positive early experience, Golgi-Cox staining of brain tissue is utilized. Owing to the fact that Golgi-Cox impregnation stains a discrete number of neurons rather than all of the cells, staining of the rodent brain with Golgi-Cox solution permits the visualization of entire neuronal elements, including the cell body, dendrites, axons, and dendritic spines. The staining procedure is carried out over several days and requires that the researcher pay close attention to detail. However, once staining is completed, the entire brain has been impregnated and can be preserved indefinitely for ongoing analysis. Therefore, Golgi-Cox staining is a valuable resource for studying experience-dependent plasticity.

This paper describes the procedures for tactile stimulation of rat pups and subsequent Golgi-Cox staining of neuronal morphology. Tactile stimulation is a positive experience that is administered in the perinatal period by stroking pups with a household duster. Golgi-cox staining is a reliable procedure permitting the visualization of entire neurons.

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile ...

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are increasingly viewed as an early feature of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD) and Huntington&#8217;s disease (HD). As yet unclear is the mechanism(s) by which axonal injury is induced. We reported the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. Quantum dot-labeled BDNF (QD-BDNF) was produced by conjugating quantum dot 655 to mBtBDNF. A microfluidic device was used to isolate axons from neuron cell bodies. Addition of QD-BDNF to the axonal compartment allowed live imaging of BDNF transport in axons. We demonstrated that QD-BDNF moved essentially exclusively retrogradely, with very few pauses, at a moving velocity of around 1.06 &#956;m/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders.

Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function. We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons.

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

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 ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...