eoe sub articles

EoE Sub Articles

1. Adaptation of embryonic stem cells (ESCs) to Feeder Cell-free Suspension Culture
<ol>
	<li>Thaw ESCs at 37 &#176;C until a sliver of ice remains.</li>
	<li>Using a P1000 pipet, gently transfer 1 - 2.5 x 106&#160;dissociated ESCs to a non-tissue culture-treated dish containing 10 ml of pre-warmed ESC medium. Incubate in a humidified tissue culture incubator for 20 hr at 37 &#176;C and 5% carbon dioxide (CO2).</li>
	<li>After 20 hr, wash cells by gently transferring the suspension culture to a 15 ml conical tube using a 10 ml serological pipet. Pellet ESCs for 3 min at 200 x g. During spin, add 10 ml of fresh ESC medium to low-adhesion dish.</li>
	<li>Aspirate supernatant, taking care to avoid dislodging the cell pellet, and add 1 ml fresh ESC medium to the cell pellet. Transfer cells to the bacterial dish using a P1000 pipet and return to the incubator.</li>
	<li>Observe cells daily until aggregates become visible to the naked eye (typically 4 - 8 days (d); aggregates will be 0.2 - 0.5 mm). If aggregates are not apparent after 4 d, repeat step 1.3. Once aggregates are visible, dissociate and maintain ESCs as described in Section 2.</li>
</ol>
2. ESC Passaging and Maintenance
<ol>
	<li>Transfer ESC aggregates to a 15 ml conical tube using a sterile 10 ml pipet and allow aggregates to settle to a compact pellet (typically 3 - 5 min). Aspirate the supernatant, being careful to avoid disrupting the cell pellet, and wash aggregates with 5 ml phosphate-buffered saline (PBS). Although gravity settling is recommended, alternatively, pellet cells at 100 x g for 2.5 min instead to save time.</li>
	<li>Aspirate PBS and add 500 &#181;l trypsin. Incubate aggregates in trypsin for 3 min in a water bath at 37 &#176;C. Add 500 &#181;l ESC medium to dilute the trypsin and gently triturate 10 times with a P1000 pipet to break up aggregates. Count cells using a hemocytometer.</li>
	<li>Pellet dissociated cells for 3 min at 200 x g and resuspend cells to a final concentration of 1.0 x 107&#160;cells/ml in ESC medium. Transfer 150 &#181;l (1.5 x 106&#160;cells) to 10 ml of fresh ESC medium in a 10 cm bacterial dish and return to tissue culture incubator for 48 hr. Excess ESCs can be differentiated, cryopreserved at 1 - 5 x 106&#160;cells/ml in 90% ESC medium supplemented with 10% dimethyl sulfoxide (DMSO), or discarded.</li>
	<li>Passage aggregates every 48 hr. Aggregates ready for passage will be clearly visible, with diameters exceeding 0.5 mm.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Thawing and culture of cryopreserved, suspension-adapted cells are accomplished per steps 1.2 - 1.4. Aggregates will be ready for passaging within 48 - 72 hr.</li>
</ol>
3. Neuronal Differentiation
NOTE: Conduct steps 3.1 - 3.5 before noon and step 3.7 after noon. An overview of the differentiation procedures is presented in&#160;Figure 1A.
<ol>
	<li>Dissociate ESCs as in steps 2.1 - 2.5. Transfer 350 &#181;l (3.5 x 106) of dissociated ESCs to a 10 cm low-attachment dish containing 25 ml differentiation medium. Place on an orbital shaker set to 30 - 45 rpm inside a tissue culture incubator at 37 &#176;C with 5% CO2. This is the first day of differentiation, termed day&#160;in vitro&#160;(DIV) -8. 
	NOTE: Using an ultra-low attachment dish increases the cost of the method but produces slightly larger yields than bacterial Petri dishes since aggregates can occasionally adhere to Petri dishes. Medium volumes and cell numbers can be scaled accordingly if different dish sizes are preferred.</li>
	<li>After 48 hr (DIV -6), use a 25 ml pipet to transfer the differentiating cell aggregates to a 50 ml conical tube. Immediately add a fresh 25 ml of differentiation medium to the Petri dish.</li>
	<li>Allow aggregates to settle over 2 - 5 min, producing a visible pellet that is 1 - 2 mm deep. Ignore single cells or small aggregates remaining in suspension. Carefully aspirate the medium and transfer the cell pellet back to the petri dish using a P1000. Place on rotary shaker in a tissue culture incubator.</li>
	<li>At DIV -4, repeat step 3.2. The pellet will be 2 - 4 mm deep. Replace 30 ml differentiation medium supplemented with 6 &#181;M all-trans retinoic acid (RA) to the petri dish. Return to the rotary shaker in the tissue culture incubator for an additional 48 hr.</li>
	<li>At DIV -2, repeat step 3.3. The pellet will be 4 - 8 mm deep at this point.</li>
	<li>At DIV -1, prepare plating surfaces as in Section 4.</li>
	<li>At DIV 0, thaw 5 ml of pre-aliquoted and frozen neural progenitor cells (NPC) trypsinization medium at 37 &#176;C for 5 - 10 min and place in a tissue culture hood. Using a 25 ml pipet, transfer differentiating aggregates to a 50 ml conical tube. Allow aggregates to settle and carefully aspirate the medium. Wash the pellet twice with 10 ml PBS, allowing the aggregates to settle between washes.</li>
	<li>After the second PBS wash, add 5 ml of NPC trypsinization medium to the pellet and incubate at 37 &#176;C for 5 min. Gently flick the tube after 2.5 min.</li>
	<li>Add 5 ml of 0.1% soybean trypsin inhibitor (STI) to inactivate trypsin and mix by inverting. Gently triturate 10 - 15 times with a 10 ml serological pipet until a relatively homogenous cell suspension is produced.</li>
	<li>Slowly transfer the cell suspension to a 40 &#181;m or 70 &#181;m cell strainer placed on the top of a 50 ml conical tube. Once all the suspension has been filtered, add 1 ml of N2 medium to wash the remaining cells through the filter and pellet the dissociated cell suspension for 6 min at 200 x g.</li>
	<li>Aspirate medium without disturbing the pellet. Wash cells twice with 10 ml N2 medium, pellet cells for 5 min at 200 x g, and triturate between washes with a P1000. Before the second wash, count cells using a hemocytometer.</li>
	<li>Resuspend cells in N2 medium at 1 x 107&#160;cells per ml and plate embryonic stem cell-derived neurons (ESNs) at a cell density of 150,000 - 200,000 cells/cm2.</li>
	<li>Transfer newly plated ESNs to a humidified tissue culture incubator at 37 &#176;C and 5% CO2&#160;and maintain as in Section 5.</li>
</ol>
4. Preparing Culture Surfaces for Plating Neural Precursors at DIV 0
<ol>
	<li>Prepare tissue culture-treated dishes at least 1 day before plating. Add sufficient polyethyleneimine (PEI; 25 &#181;g/ml in sterile H2O) or poly-D-lysine (PDL; 100 &#181;g/ml in sterile H2O) to cover tissue-culture-treated plastic dishes and incubate O/N at 37 &#176;C.</li>
	<li>The morning of plating, wash dishes twice with double-distilled H2O and once with PBS. After the final wash, add sufficient N2 medium to cover the dish (e.g.,&#160;1 ml per well of a 12-well dish or 4 ml per 6 cm dish).</li>
	<li>Prepare 18 mm glass coverslips at least one day before neuron plating. Clean coverslips by plasma-cleaning for 4 min.</li>
	<li>Immediately transfer cleaned coverslips to an ethanol-washed parafilm in the bottom of a large sterile dish and add 400 &#181;l of PEI or PDL solution, prepared as in step 4.1. Incubate overnight (O/N) at 37 &#176;C in a tissue culture incubator.</li>
	<li>In the morning, wash coverslips three times with water and add 5 &#181;g/ml laminin in PBS for 1 - 3 hr at 37 &#176;C. Before dissociating neurons, aspirate the laminin and immediately transfer the coverslip to a well of a 12-well dish containing 1 ml of NPC medium, being sure to keep the treated side facing up.</li>
	<li>Store dishes and coverslips at 37 &#176;C until NPCs are ready to plate.</li>
</ol>
5. Maintenance of Neurons
<ol>
	<li>At DIV 1, aspirate medium and replace with N2 culture medium.</li>
	<li>At DIV 2 and 4, aspirate medium and replace with B27 culture medium.</li>
	<li>At DIV 8, aspirate medium and replace with B27 culture medium containing mitotic inhibitors to eliminate contaminating non-neuronal cells.</li>
	<li>At DIV 12, replace with B27 culture medium.</li>
	<li>Do not remove DIV 12+&#160;ESNs from 5% CO2&#160;until ready.</li>
</ol>
6. Measured Inhibition of Synaptic Transmission (MIST) Assay for Quantifying Miniature Excitatory Post-synaptic Currents
CAUTION: Clostridial neurotoxins are the most poisonous substances known, with estimated human LD50&#160;values as low as 0.1 - 1 ng/kg. Obtain necessary approvals before using these toxins and use appropriate precautions.
<ol>
	<li>Carefully dilute BoNT/A to 100x, the desired final concentration in ESN culture medium, and warm to 37 &#186;C. Add the appropriate volume of toxin to DIV 21+&#160;ESN cultures, swirl the culture dish, and return to the incubator.</li>
	<li>If cells will be analyzed more than 4 hours after intoxication, add a toxin to the dish and swirl without removing from 5% CO2, such as directly in the incubator or in a constant CO2&#160;chamber.</li>
	<li>At the appropriate time point, aspirate the ESN culture medium and wash twice with extracellular recording buffer (ERB). Add 4 ml of ERB supplemented with 5.0 &#181;M tetrodotoxin and 10 &#181;M bicuculline, blocking action potentials and antagonizing GABAA&#160;receptor activity, respectively.</li>
	<li>Transfer the dish to the electrophysiology rig. Neither perfusion nor temperature control is required for the MIST assay.</li>
	<li>Pull borosilicate glass using a micropipette puller to produce a recording pipette with 5-10 m&#937; of resistance and fill with intracellular recording buffer. Gently dip filled recording pipette in siliconizing reagent before recording.</li>
	<li>Using an air-filled syringe, provide positive pressure as the recording pipette is lowered into the ERB. After gently landing the recording pipette on the neuron&#39;s soma to be recorded, remove positive pressure and form a gigaseal. Decrease the holding voltage to -70 mV. Carefully apply negative pressure to break into whole-cell configuration.</li>
	<li>Switch to the current clamp to monitor and record resting membrane potential. Without adjustment for liquid junction potentials, the resting membrane potential will be between -67 and -82 mV.</li>
	<li>Perform a continuous -70 mV voltage-clamp recording for 4 - 5 min to detect miniature excitatory post-synaptic currents (mEPSCs).</li>
	<li>Analyze 4 min of recorded data for mEPSC detection using spike detection software with the following settings: Threshold, 5; Period to search a local maximum, 10,000 &#181;sec; Time before a peak for baseline, 5,000 &#181;sec; Period to search a decay time, 20,000 &#181;sec; Fraction of peak to find a decay time, 0.37; Period to average a baseline, 1,000 &#181;sec; Area threshold, 20; Number of points to average peak, 1; Direction of peak, negative.</li>
	<li>Collect and save information on detected events. Divide the number of detected events in 4 min by 240 to determine the mEPSC frequency in Hz.</li>
	<li>Collect mEPSC frequencies for 8 - 12 controls and 8 - 12 BoNT-treated samples for each exposure condition. Analyze frequency against age- and lot-matched controls. Determine the statistical significance of % inhibition of synaptic activity using one-way ANOVA and Dunnett&#39;s&#160;post-hoc&#160;test.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Table 1. ESC and ESN</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Life Technologies</td>
			<td>10829-018</td>
			<td>Media: ESC culture medium</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Formulation and notes: 500 mL</td>
		</tr>
		<tr>
			<td>100x MEM NEAA</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td>Media: store at 4 &#176;C for up to 1 month</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Formulation and notes: 6 mL</td>
		</tr>
		<tr>
			<td>200 mM L-Alanyl-L-Glutamine</td>
			<td>ATCC</td>
			<td>30-2115</td>
			<td>6 mL</td>
		</tr>
		<tr>
			<td>ES qualified FBS</td>
			<td>Applied Stem Cell</td>
			<td>ASM-5007</td>
			<td>90 mL</td>
		</tr>
		<tr>
			<td>100x antibiotics</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td>3 mL</td>
		</tr>
		<tr>
			<td>55 mM 2-mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>21985-023</td>
			<td>1.1 mL</td>
		</tr>
		<tr>
			<td>107&#160;Units/mL LIF</td>
			<td>Millipore</td>
			<td>ESG1107</td>
			<td>60 &#956;L</td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Life Technologies</td>
			<td>10829-018</td>
			<td>Media: ESC differentiation medium</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Formulation and notes: 436.6 mL</td>
		</tr>
		<tr>
			<td>100x MEM NEAA</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td>Media: store at 4 &#176;C for up to 1 month</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Formulation and notes: 5 mL</td>
		</tr>
		<tr>
			<td>200 mM L-Alanyl-L-Glutamine</td>
			<td>ATCC</td>
			<td>30-2115</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>ESC-qualified serum</td>
			<td>Applied Stem Cell</td>
			<td>ASM-5007</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>100x antibiotics</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td>2.5 mL</td>
		</tr>
		<tr>
			<td>55 mM 2-mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>21985-023</td>
			<td>0.9 mL</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td>Media: NPC trypsinization medium</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Formulation and notes: 100 mL</td>
		</tr>
		<tr>
			<td>0.5 M EDTA (18.3%)</td>
			<td>Sigma-Aldrich</td>
			<td>3690</td>
			<td>Media: freeze in 5 mL aliquote</td>
		</tr>
		<tr>
			<td>&#160; &#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Formulation and notes: 0.266 mL</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T8802</td>
			<td>50 mg</td>
		</tr>
		<tr>
			<td>Polyethyleneimine (PEI)</td>
			<td>Sigma-Aldrich</td>
			<td>P3143</td>
			<td>Media: Surface coating solutions</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Formulation and notes: 2.5 &#181;g/mL in H20</td>
		</tr>
		<tr>
			<td>Poly-D-lysine (PDL)</td>
			<td>Sigma-Aldrich</td>
			<td>P7280</td>
			<td>Media: use within 1 wk</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Formulation and notes: 100 &#181;g/mL in H20</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td>5 &#181;g/mL in H20</td>
		</tr>
		<tr>
			<td>DMEM/F12 + GlutaMAX</td>
			<td>Life Technologies</td>
			<td>10565-018</td>
			<td>Media: NPC culture medium</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Formulation and notes: 492.5 mL</td>
		</tr>
		<tr>
			<td>100x N2 vitamins</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td>Media: store at 4 &#176;C for up to 1 month</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Formulation and notes: 5 mL</td>
		</tr>
		<tr>
			<td>100x antibiotics</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td>2.5 mL</td>
		</tr>
		<tr>
			<td>Neurobasal A</td>
			<td>Life Technologies</td>
			<td>10888-022</td>
			<td>Media: ESN culture medium</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Formulation and notes: 482.5 mL</td>
		</tr>
		<tr>
			<td>50x B27 vitamins</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td>Media: store at 4 &#176;C for up 1 month</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Formulation and notes: 10 mL</td>
		</tr>
		<tr>
			<td>200 mM L-Alanyl-L-Glutamine</td>
			<td>ATCC</td>
			<td>30-2115</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>100x antibiotics</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td>2.5 mL</td>
		</tr>
		<tr>
			<td>5-fluoro-2&#39;-deoxyuridine (5FDU)</td>
			<td>Sigma-Aldrich</td>
			<td>F0503</td>
			<td>Media: 2000x Mitotic inhibitors aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Formulation and notes: dd 150 mg 5FDU and 350 mg uridine to 10 mL Neurobasal A. Sterile filter, aliquot and freeze. Use 2000x in ESN culture medium.</td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma-Aldrich</td>
			<td>U3003</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Trypsin</td>
			<td>Life Technologies</td>
			<td>12605-010</td>
			<td>Media: Miscellaneous</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Formulation and notes: store at RT</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor (STI)</td>
			<td>Sigma-Aldrich</td>
			<td>T6414</td>
			<td>store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4403</td>
			<td>Prepare 100 mM stock by dissolving 100 mg ascorbic acid in 5.7 mL of 50:50 DMSO/ethanol mix.</td>
		</tr>
		<tr>
			<td>Retinoic acid (RA)</td>
			<td>Sigma-Aldrich</td>
			<td>R2625</td>
			<td>Resuspend 15 mg RA in 9 mL 50:50 DMSO/ethanol. Supplement with 1 mL of 100 mM ascorbic acid stock. Aliquot and stored at -80 &#176;C. Stable for 6 mos.</td>
		</tr>
		<tr>
			<td>Table 2. MIST</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>71386</td>
			<td>Media: Extracellular Recording Buffer</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Final Concentration (mM): 58.44</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>MW: 140</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>60135</td>
			<td>Media: (ERB; pH = 7.3, 315 mO)</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Final Concentration (mM): 74.56</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>MW: 3.5</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>S3139</td>
			<td>Media: Stable at 4 &#176;C for at least 6 mo.</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Final Concentration (mM): 119.98</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>MW: 1.25</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>21115</td>
			<td>Final Concentration (mM): 110.98</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>MW: 2</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>63069</td>
			<td>Final Concentration (mM): 95.21</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>MW: 1</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8644</td>
			<td>Final Concentration (mM): 180.16</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>MW: 10</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H0887</td>
			<td>Final Concentration (mM): 238.3</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>MW: 10</td>
		</tr>
		<tr>
			<td>K-gluconate</td>
			<td>Sigma-Aldrich</td>
			<td>P1847</td>
			<td>Media: Intracellular Recording Buffer</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Final Concentration (mM): 234.5</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>MW: 140</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>71386</td>
			<td>Media: (IRB; pH = 7.3, 320 mO)</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Final Concentration (mM): 58.44</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>MW: 5</td>
		</tr>
		<tr>
			<td>Mg-ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A9187</td>
			<td>Media: Stable at 4 &#176;C for at least 6 mo.</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Final Concentration (mM): 507.18</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>MW: 2</td>
		</tr>
		<tr>
			<td>Li-GTP</td>
			<td>Sigma-Aldrich</td>
			<td>G5884</td>
			<td>Final Concentration (mM): 523.18</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>MW: 0.5</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>21115</td>
			<td>Media: Stable at 4 &#176;C for at least 6 mo.</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Final Concentration (mM): 110.98</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>MW: 0.1</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>63069</td>
			<td>Media: Stable at 4 &#176;C for at least 6 mo.</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Final Concentration (mM): 95.21</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>MW: 1</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td>380.35 
			Media: Stable at 4 &#176;C for at least 6 mo. 
			Final Concentration (mM): 380.35 
			MW: 1</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H0887</td>
			<td>Media: Stable at 4 &#176;C for at least 6 mo.</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Final Concentration (mM): 238.3</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>MW: 10</td>
		</tr>
		<tr>
			<td>Bicuculline</td>
			<td>Tocris</td>
			<td>131</td>
			<td>Media: Miscellaneous</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Final Concentration (mM): 0.01</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>MW: 417.85</td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Sigma-Aldrich</td>
			<td>C239</td>
			<td>Final Concentration (mM): 0.01</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>MW: 276.12</td>
		</tr>
		<tr>
			<td>Tetrodotoxin</td>
			<td>Sigma-Aldrich</td>
			<td>A8001</td>
			<td>Final Concentration (mM): 0.005</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>MW: 645.74</td>
		</tr>
		<tr>
			<td>BoNT/A-/G</td>
			<td>Metabiologics</td>
			<td>N/A</td>
			<td>Media:</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Final Concentration (mM): TBD</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>MW: 150,000</td>
		</tr>
		<tr>
			<td>Tetanus toxin</td>
			<td>Sigma-Aldrich</td>
			<td>T3194</td>
			<td>Final Concentration (mM): TBD</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>MW: 150,000</td>
		</tr>
		<tr>
			<td>Sigmacote</td>
			<td>Sigma-Aldrich</td>
			<td>SL-2</td>
			<td>Final Concentration (mM): N/A</td>
		</tr>
		<tr>
			<td>&#160; &#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>MW: N/A</td>
		</tr>
		<tr>
			<td>Table 3. Equipment and Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MiniAnalysis (version 6.0.7)</td>
			<td>Synaptosoft, Inc.</td>
			<td>N/A</td>
			<td>Event detection software</td>
		</tr>
		<tr>
			<td>Igor Pro (version 6.22A)</td>
			<td>WaveMetrics</td>
			<td>N/A</td>
			<td>Software for visualization of recordings</td>
		</tr>
		<tr>
			<td>Patchmaster</td>
			<td>Heka</td>
			<td>N/A</td>
			<td>Data acquisition software</td>
		</tr>
		<tr>
			<td>Olympus IX51 inverted microscope</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instrument</td>
			<td>MPC-365</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch-clamp amplifier</td>
			<td>Heka</td>
			<td>ECB10USB</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate capillary tubes</td>
			<td>Sutter Instrument</td>
			<td>B150-86-10</td>
			<td>Corning 7740</td>
		</tr>
		<tr>
			<td>18 mm glass coverslips</td>
			<td>Fisher</td>
			<td>12-545-84</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-32G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer (40 &#181;m)</td>
			<td>Fisher</td>
			<td>08-771-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Stovall Belly Dancer Shaker</td>
			<td>Fisher</td>
			<td>15-453-211</td>
			<td></td>
		</tr>
		<tr>
			<td>Low adhesion dishes</td>
			<td>Fisher</td>
			<td>05-539-101</td>
			<td>Corning 3262</td>
		</tr>
		<tr>
			<td>Bacterial dishes</td>
			<td>VWR</td>
			<td>25384-302</td>
			<td>100 x 15 mm</td>
		</tr>
	</tbody>
</table>

a technique to assess the inhibitory effect of toxin exposure on miniature excitatory postsynaptic currents (mepscs)

Source: Hubbard, K. et. al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/52361">Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons.</a>&#160;J. Vis. Exp. (2015)
The video demonstrates the electrophysiological analysis of embryonic stem cell-derived neurons after exposure to neurotoxins. Treating these neurons with botulinum neurotoxins reduces the frequency of spontaneous miniature excitatory post-synaptic currents, confirming inhibition of synaptic transmission.

<img alt="Figure 1" src="/files/ftp_upload/22071/22071_Figure1.jpg" /> 
Figure 1. Suspension-adapted ESNs remain mitotically stable and express markers of pluripotency.&#160;(A) Schematic of ESC maintenance and differentiation. The presence or absence of retinoic acid (RA) or leukemia inhibitory factor (LIF) is marked by a + or &#8211;. A comparison between days&#160;in vitro&#160;(DIV) and classical developmental stages (DS) for primary neu...

A Technique to Assess the Inhibitory Effect of Toxin Exposure on Miniature Excitatory Postsynaptic Currents (mEPSCs)

1. Adaptation of embryonic stem cells (ESCs) to Feeder Cell-free Suspension Culture

	Thaw ESCs at 37 &#176;C until a sliver of ice remains.
	Using a ...

Treat neuronal cultures with increasing concentrations of a test toxin.
The toxin enters the cells and is released into the cytosol.
Depending on its potential, the toxin may prevent vesicular fusion, inhibiting neurotransmitter release.
Replace the medium with a buffer containing specific neuropharmacological compounds to block neuronal action potentials.
Place the culture dish on an electrophysiology stage. To start recording, connect a neuron soma with a pipette containing a recording electrode.
Apply steady negative pressure to aspirate a small membrane portion, forming a gigaseal, a tight seal that ensures minimal interference during recording.
Maintain a constant membrane voltage to measure neuronal currents.
Apply negative pressure pulses to rupture the membrane, establishing direct contact between the neuron and the pipette.
Record the currents produced in the neuron due to the uptake of neurotransmitters, termed mEPSCs.
A decrease in mEPSC frequency with increased toxin exposure indicates the inhibition of neurotransmitter release.

a-technique-to-assess-inhibitory-effect-toxin-exposure-on-miniature

Universidad San Sebastian

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

117 Views. Source: Hubbard, K. et. al., Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons. J. Vis. Exp. (2015) The video demonstrates the electrophysiological analysis of embryonic stem cell-derived neurons after exposure to neurotoxins. Treating these neurons with botulinum neurotoxins reduces the frequency of spontaneous miniature excitatory post-synaptic currents, confirming inhibition of synaptic transmission. 

Video: A Technique to Assess the Inhibitory Effect of Toxin Exposure on Miniature Excitatory Postsynaptic Currents (mEPSCs) - Concept

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

JoVE Journal

Neuroscience

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

Immunology and Infection

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) pathophysiology in vitro. Multi-electrode array (MEA) recordings are a means to record electrical field potentials from large populations of neurons and analyze network activity over time. It was previously demonstrated that the presence of hiPSC-A that are differentiated using techniques to promote a spinal cord astrocyte phenotype improved maturation and electrophysiological activity of regionally specific spinal cord hiPSC-motor neurons (MN) when compared to those cultured without hiPSC-A or in the presence of rodent astrocytes. Described here is a method to co-culture spinal cord hiPSC-A with hiPSC-MN and record electrophysiological activity using MEA recordings. While the differentiation protocols described here are particular to astrocytes and neurons that are regionally specific to the spinal cord, the co-culturing platform can be applied to astrocytes and neurons differentiated with techniques specific to other fates, including cortical hiPSC-A and hiPSC-N. These protocols aim to provide an electrophysiological assay to inform about glia-neuron interactions and provide a platform for testing drugs with therapeutic potential in ALS.

We describe a method for differentiating spinal cord human induced pluripotent-derived astrocytes and neurons and their co-culture for electrophysiological recording.

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) ...

A Technique to Assess the Inhibitory Effect of Toxin Exposure on Miniature Excitatory Postsynaptic Currents (mEPSCs)

Transcript

A Technique to Assess the Inhibitory Effect of Toxin Exposure on Miniature Excitatory Postsynaptic Currents (mEPSCs)

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons