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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of Matrices
<ol>
	<li>Boil 4-8 silicone matrices in a microwave or on a hot plate for 5 min and allow them to dry completely for 1 h under laminar flow (striped side up). 
	NOTE: The following procedures should be performed under laminar flow.</li>
	<li>Blow compressed air or use transparent sticky tape to remove any dust from the striped side of the matrices. Keep the striped side clean so it can firmly adhere to the plate surface.</li>
	<li>Carefully place the matrices onto 6-cm plastic dishes by pressing with a finger (one matrix per dish; stripe side down). Avoid getting air bubbles between the matrix and the dish. Mark the location of the stripes on the bottom side of the culture dish.</li>
	<li>If the matrix fails to attach, repeat from step 1.2.</li>
</ol>
2. Stripe Generation and Laminin Coating for Neuron Culture
<ol>
	<li>Prepare fluorescently labeled recombinant protein (25 &#181;l each for injection [step 2.2] or 100 &#181;l each for the placement method [step 2.2.1]) by mixing the Fc-tagged protein (10-50 &#181;g/ml) and a fluorescent dye-conjugated anti-human Fc antibody (1 to 3 ratio: 30-150 &#181;g/ml) in phosphate buffered saline (PBS). Then, incubate the mixture for 30 min at RT.</li>
	<li>Using a 22-G syringe, inject 25 &#181;l of the recombinant protein prepared in the previous step (2.1) through the small hole on the side of the matrix (arrows in Figure 1A, 1C, and 1E).
	<ol>
		<li>Alternatively, place 100 &#181;l of the recombinant protein into the top slit of the matrix and aspirate approximately half of the solution from the small hole (arrows in Figure 1A, 1C, and 1E), so that the recombinant protein remains in the striped regions.</li>
	</ol>
	</li>
	<li>Incubate the dish for 30 min at 37 &#176;C in a cell culture incubator.</li>
	<li>Place 300 &#181;l of PBS into the top slit (Figure 1B) and aspirate from the small hole located on the side of the matrix (arrows in Figure 1A, C and E) to remove unattached recombinant proteins. Do not aspirate the PBS completely, because the proteins will dry. Repeat this step twice. (3 times total)</li>
	<li>Carefully remove the matrix and immediately place ~100 &#181;l of control protein (10-50 &#181;g/ml; Fc) to cover the entire striped area, creating an alternate coating. Then, incubate the dish for 30 min at 37 &#176;C in the cell culture incubator.</li>
	<li>After washing the dish surface three times with PBS, coat it with ~100 &#181;l of 20 &#181;g/ml laminin in PBS. 
	NOTE: Laminin should be thawed on ice to prevent solidification.</li>
	<li>Incubate the dish for 1 h at 37 &#176;C in the cell culture incubator.</li>
	<li>Wash the dish surface three times with PBS and add culture medium. Place the coated dish with culture medium in a 5% CO2 incubator at 37 &#176;C until use.</li>
</ol>
3. Culturing Hippocampal Neurons from E15.5 Mouse Embryos
<ol>
	<li>Euthanize the mother via cervical dislocation and isolate three E15.5 embryos.</li>
	<li>Cut the skin and skull sagittally at the midline of the head with scissors, and remove the brain using a small spoon. Transfer the brain carefully to a 60-mm Petri dish containing 3 ml of Hank&#39;s Balanced Salt Solution (HBSS).</li>
	<li>Using a scalpel, cut out the hemispheres including cortex, hippocampus and striatum (Figure 2A). Then, dissect the hippocampal region by carefully removing the meninges, and transfer the dissected hippocampal tissues to a 15-ml centrifuge tube containing 2 ml of HBSS solution, on ice (Figure 2B-C). Collect 6 hippocampi from 3 embryos in the tube, which are sufficient for culturing neurons in 8 dishes.</li>
	<li>Replace the HBSS solution with trypsin/ethylenediamine tetraacetic acid (trypsin/EDTA) solution (2 ml) and incubate the tube at 37 &#176;C for 15 min in a water bath. 
	NOTE: Aspirate HBSS without centrifugation and do not decant the HBSS solution by tilting the tube.</li>
	<li>Neutralize the trypsin activity by adding 500 &#181;l of fetal bovine serum (FBS).</li>
	<li>Centrifuge the mixture at 100 x g for 5 min. Remove the supernatant and wash the pellet with 2 ml of culture medium. Repeat this step once more.</li>
	<li>Pipet the hippocampi up and down 10 times in a culture medium using a 1,000-&#181;l tip with a large hole (cut-tip) to dissociate the tissue.</li>
	<li>Change to a regular (uncut) 1,000-&#181;l tip and pipet the dissociated tissue up and down to obtain a single-cell suspension.</li>
	<li>Separate the single cells by passing them through an 80-100-&#181;m mesh cell strainer.</li>
	<li>Centrifuge the filtered single-cell suspension at 150 x g for 5 min.</li>
	<li>Remove the supernatant by aspirating, and resuspend the pellet (neurons) in 2 ml of culture medium.</li>
	<li>Count the cells using a hemocytometer with trypan blue staining to determine the density and viability. Then, plate 10,000 neurons in 150 &#181;l of culture medium for each set of stripes. The suspension should be carefully placed to cover the entire stripe region.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL centrifuge tube</td>
			<td>Violamo</td>
			<td>1-3500-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat anti-human IgG antibody</td>
			<td>Thermo Scientific</td>
			<td>A11013</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Donkey anti-mouse IgG antibody</td>
			<td>Thermo Scientific</td>
			<td>A-21203</td>
			<td>Dilution 1/500</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Thermo Scientific</td>
			<td>17504-044</td>
			<td>Dilution 1/50</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>01-2030-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 100 um</td>
			<td>BD Falcon</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugation machine</td>
			<td>Kubota</td>
			<td>2410</td>
			<td></td>
		</tr>
		<tr>
			<td>DAKO pen</td>
			<td>DAKO</td>
			<td>S2002</td>
			<td>Alternative water-repellent pen may be used</td>
		</tr>
		<tr>
			<td>Disposable scalpel</td>
			<td>Feather</td>
			<td>2975#11</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Scientific</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps No. 5</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Thermo Scientific</td>
			<td>35050-061</td>
			<td>Dilution 1/200</td>
		</tr>
		<tr>
			<td>Hamilton Syringe</td>
			<td>Hamilton</td>
			<td>805N</td>
			<td>22 gauge, 50 uL</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Thermo Scientific</td>
			<td>14170-112</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IgG, Fc Fragment</td>
			<td>Jackson</td>
			<td>009-000-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Thermo Scientific</td>
			<td>23017-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Thermo Scientific</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Nacalai</td>
			<td>14249-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Scientific</td>
			<td>15070-063</td>
			<td>Dilution 1/100</td>
		</tr>
		<tr>
			<td>Plastic culture dish, 60 mm</td>
			<td>Thermo Scientific</td>
			<td>150288</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone Matrices</td>
			<td></td>
			<td></td>
			<td>Available and purchasable from Prof. Martin Bastmeyer (bastmeyer@kit.edu)</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Tip, 1000 uL</td>
			<td>Watson</td>
			<td>125-1000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent sticky tape</td>
			<td>Tesa</td>
			<td>57315</td>
			<td>Alternative sticky tape may be used</td>
		</tr>
		<tr>
			<td>Trypan blue, 0.4%</td>
			<td>Bio-Rad</td>
			<td>145-0013</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Thermo Scientific</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture medium</td>
			<td></td>
			<td></td>
			<td>Neurobasal supplemented with B27, GlutaMAX and Penicillin-Streptomycin.</td>
		</tr>
	</tbody>
</table>

assessing attractive or repulsive activity of proteins using hippocampal neurons

Source: Yamagishi, S. et al., <a href="https://app.jove.com/v/54096">Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons.</a> J. Vis. Exp. (2016)
This video demonstrates a method to assess protein activity using hippocampal neurons over patterned protein zones. Neurons exhibit repulsion in test protein zones and attachment in control protein zones, highlighting the effects of attractive or repulsive protein activity.

<img alt="Figure 1" src="/files/ftp_upload/22966/22966_Figure1.jpg" /> 
Figure 1. Silicon Matrix used to create the striped protein stamp. (A, B) Top view of the silicon matrix. The size of matrix is 30 mm x 25 mm x 5mm. White arrow in A indicates a small hole where the recombinant protein is injected (step 2.2). Arrowhead in B indicates a slit where the recombinant protein is alternatively applied (step 2.2.1). (C, D) Bottom view of the silicon matr...

Assessing Attractive or Repulsive Activity of Proteins Using Hippocampal Neurons

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Begin with a dish containing a silicon matrix that has multiple small channels connected to inlet and outlet ports.
Inject the test protein with repulsive activity through the inlet to fill the channels.
Incubate to allow protein adherence in the channel area, forming test protein zones, then wash.
Remove the matrix, overlay the coated area with a control protein possessing attractive activity, and incubate.
This facilitates the control protein&#39;s adherence to the empty areas between the test protein zones, creating control protein zones with an alternating striped pattern,then wash.
Add laminin to coat the striped area, then wash.
Seed the coated area with a single-cell suspension of hippocampal neurons and incubate.
In the test protein zone, the neurons interact with the proteins, triggering signals that cause the neurons to repel, inhibiting their attachment.
In contrast, neuron-protein interactions in the control protein zone promote neuronal attachment, supporting their growth and axon extension.

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26 Views. Source: Yamagishi, S. et al., Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons. J. Vis. Exp. (2016) This video demonstrates a method to assess protein activity using hippocampal neurons over patterned protein zones. Neurons exhibit repulsion in test protein zones and attachment in control protein zones, highlighting the effects of attractive or repulsive protein activity. 

Video: Assessing Attractive or Repulsive Activity of Proteins Using Hippocampal Neurons - Concept

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

JoVE Journal

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.

Here, we described a protocol to quantitatively study the assembly and structure of the axon initial segments (AIS) of hippocampal neurons that lack pre-assembled AIS due to the absence of a giant ankyrin-G.

Assessing Attractive or Repulsive Activity of Proteins Using Hippocampal Neurons

Transcript

Assessing Attractive or Repulsive Activity of Proteins Using Hippocampal Neurons

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments