eoe sub articles

EoE Sub Articles

1. Assessing the Role of Presynaptic Proteins in Intact Neuronal Circuits by Targeted Stimulation of Knocked-down Neurons with Optogenetics
NOTE: The following protocol requires previous experience with electrophysiological recordings in acute brain slices and access to an electrophysiological setup.
<ol>
	<li>Stereotactically inject recombinant adeno-associated virus with mosaic serotype 1 and 2 (rAAV1/2) into the brain. Determine the stereotactic coordinates for the brain region of interest using stereotactic atlases for mouse&#160;or rat brain.
	<ol>
		<li>Use a micropipette puller to pull injection micropipettes with long shanks of &#216; 7&#8211;9 &#181;m; clip the injection micropipettes on the shanks with scissors. Use a thin marker and graph paper to place calibrating marks on the injection micropipettes every 2 mm.</li>
		<li>Anesthetize the animal with isoflurane and fix it in the stereotactic apparatus.</li>
		<li>Keep the animal warm throughout the operation with a heating pad set at 37 &#176;C.</li>
		<li>Protect the eyes with ocular lubricant.</li>
		<li>Shave the fur on the head with an electric razor.</li>
		<li>Spread povidone iodine on the shaved head using a cotton bud.</li>
		<li>Under a dissecting microscope, make a midline incision.</li>
		<li>Clean the skull surface with a cotton bud, so to make the bregma and lambda visible.</li>
		<li>Place the injection micropipette into its holder. Attach the holder to the stereotactic arm.</li>
		<li>Determine the x and y coordinates of the site of injection relative to the bregma and/or the lambda.</li>
		<li>Use a drill to thin the skull over the target area. 
		NOTE: Use gentle circular movements and avoid drilling through the skull as this will damage the surface of the brain.</li>
		<li>If there is bleeding, absorb excessive blood with towel paper. 
		NOTE: Excessive blood will make it difficult to determine correctly the z coordinate.</li>
		<li>Load &#8804;2 &#181;L of virus into the injection micropipette by capillary action.</li>
		<li>Bring the injection micropipette to the x and y coordinates of the site of injection.</li>
		<li>Calculate the z coordinate from the dura and lower the pipette slowly into the brain.</li>
		<li>When the z coordinate is reached, wait 3 min to allow for tissue adjustment.</li>
		<li>Apply low positive pressure using a 1 mL syringe connected via a flexible tube to the back of the injection micropipette. Visually monitor the speed of ejection of the virus from the injection micropipette through the dissection microscope and using the calibrating marks as reference points. 
		NOTE: Virus needs to be injected at slow rate (&#60;100 nL/min) to avoid tissue damage.</li>
		<li>When injection is finished, wait 5 min, withdraw the injection micropipette by 0.2 mm, wait for another 5 min, to avoid backflow of the virus.</li>
		<li>Withdraw the injection micropipette slowly and completely from the brain and dispose of it into a container filled with bleach.</li>
		<li>Wet the skull with physiological solution and suture the skin with 3-4 stitches.</li>
		<li>Apply gentamicin ointment to the wound.</li>
		<li>Single-house the animal in a clean cage with food pellets and under a heat lamp till it fully recovers.</li>
	</ol>
	</li>
	<li>&#8805;15 days post-injection, decapitate animals under deep isoflurane anesthesia.</li>
	<li>Prepare acute brain slices of the brain region of interest with a vibratome using gassed (95% O2, 5% CO2) ice-cold artificial cerebrospinal Fluid (aCSF) solution, containing (in mM): 123 NaCl, 1.25 KCl, 1.25 KH2PO4, 1.5 MgCl2, 1 CaCl2, 25 NaHCO3, 2 sodium pyruvate and 18 glucose (osmolarity adjusted to 300 mOsm). For example, if the aim is to analyze synaptic transmission from CA3 to CA1 pyramidal neurons, prepare sagittal slices of the hippocampal formation (350 &#181;m thick). 
	NOTE: From this step onwards work under low-light conditions to avoid activation of the optogenetic probe by environmental light.</li>
	<li>Let the slices recover for 30 min at 37 &#176;C in the same aCSF in a chamber designed for holding brain slices. Keep the slices in the same brain slice chamber at room temperature till recording. 
	NOTE: Using these conditions, slices can be maintained healthy for up to 6&#8211;8 h.</li>
	<li>Transfer one slice to a submerged recording chamber and superfuse it with 2 mL/min of the same aCSF used for recovery supplemented with 1.5 mM CaCl2&#160;(total Ca2+: 2.5 mM) and without sodium pyruvate. 
	NOTE: Slices are prepared and maintained in low concentration of Ca2+&#160;(1 mM) to minimize toxicity. Recordings are performed in 2.5 mM Ca2+&#160;to favor vesicle release.</li>
	<li>Check briefly the signal of the expressed fluorescent reporter (e.g. TdTomato;&#160;Figure 1B) to ascertain the localization and intensity of infection.</li>
	<li>Fill a patch electrode with an intracellular solution containing (in mM): 110 K-gluconate, 22 KCl, 5 NaCl, 0.5 ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#8242;,N&#8242;-tetraacetic acid (EGTA), 3 MgCl2, 4 adenosine 5&#8242;-triphosphate magnesium salt (Mg-ATP), 0.5 Guanosine 5&#39;-triphosphate trisodium salt (Na3-GTP), 20 K2-creatine phosphate, 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer adjusted with KOH (HEPES-KOH, pH 7.28, 290 m&#937;). 
	NOTE: Use patch electrode with a pipette resistance of 5&#8211;6 m&#8486;.</li>
	<li>Under infrared illumination, reach tight-seal whole-cell configuration from a neuron receiving synaptic inputs from the infected neurons. For example, if CA3 pyramidal neurons were infected, patch pyramidal neurons in the proximal to medial tract of the CA1 region (Figure 1C). 
	NOTE: Series resistance can be left uncompensated, but it should be constant and low (&#8804;20 M&#8486;).</li>
	<li>Use pharmacology to isolate the synaptic currents under investigation. For example, if the aim is to investigate excitatory synaptic transmission, block inhibitory synaptic transmission with 10 &#181;M bicuculline.</li>
	<li>Evoke synaptic currents, for example, excitatory postsynaptic currents (EPSCs), using a 473 nm blue laser coupled to an optical fiber (&#216; &#8804;250 &#181;m) positioned on the somata of the infected neurons (e.g. CA3 pyramidal neurons).
	<ol>
		<li>Adjust stimulation length to a minimum, to reduce the possibility of evoking more than one action potential per light pulse. If using ChETA, set it to 2 ms.</li>
		<li>Adjust stimulation strength of the laser to yield small but clearly detectable synaptic currents (&#8804;50 pA peak amplitude for EPSCs recorded at a holding potential of -70 mV between CA3 and CA1 pyramidal neurons;&#160;Figure 1D). If using ChETA and an optical fiber of 250 &#181;m in diameter, stimulation strengths of 1&#8211;3 mW at fiber exit should be suitable. 
		NOTE: if laser strength cannot be regulated, use neutral density filters.</li>
		<li>Shine the 473 nm laser light on the somata of the infected neurons, but not on their axons. For example, shine the light on CA3 somata, and away from the Schaffer collaterals, to avoid direct depolarization of the axons.</li>
		<li>Apply tetrodotoxin (TTX; 0.5 &#181;M), a blocker of sodium channels, to the sample to confirm the channels are action potential driven.</li>
		<li>In different recordings, apply a selective blocker to the synaptic current under investigation to confirm the stimulation is selective. 
		NOTE: For example, apply 2,3-dioxo-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX, 10 &#181;M) to &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor-mediated EPSCs.</li>
		<li>Compare optically and electrically evoked synaptic currents in the same acute brain slice in response to repetitive stimulation (&#8805;2 stimuli at &#8804;20 Hz). A similar degree of synaptic facilitation or depression should be observed between electrical and optical stimulation when using a control microRNA (miR), thus suggesting that similar cellular mechanisms are activated by the two types of stimulations. 
		NOTE: The above steps are necessary to ensure that optical stimulation does not induce a direct depolarization of presynaptic boutons, thus bypassing some mechanisms of synaptic transmission.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Brain slices Prechamber</td>
			<td>Harvard Apparatus</td>
			<td>BSC-PC</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera-based imager</td>
			<td>Bio-Rad</td>
			<td>ChemiDoc&#8482; MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture reagents</td>
			<td>Life Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>Foredom</td>
			<td>K.1030</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin ointment</td>
			<td>Local pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>54459</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection micropipettes</td>
			<td>Narishige</td>
			<td>GD1</td>
			<td></td>
		</tr>
		<tr>
			<td>Inorganic salts &#38; detergents</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>K2-creatine phosphate</td>
			<td>Calbiochem</td>
			<td>237911</td>
			<td></td>
		</tr>
		<tr>
			<td>KGluconate</td>
			<td>Fluka</td>
			<td>60245</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser</td>
			<td>Laserglow Technologies</td>
			<td>LRS-0473-GFM-00100-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane</td>
			<td>Amersham</td>
			<td>Protran&#8482; 0.2 &#181;m NC</td>
			<td></td>
		</tr>
		<tr>
			<td>MgATP</td>
			<td>Sigma</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette holder</td>
			<td>Narishige</td>
			<td>IM-H1</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Narishige</td>
			<td>PC-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Na3GTP</td>
			<td>Sigma</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr>
			<td>NaPyruvate</td>
			<td>Sigma</td>
			<td>P5280</td>
			<td></td>
		</tr>
		<tr>
			<td>Ocular lubricant</td>
			<td>Local pharmacy</td>
			<td>Lacrigel</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone iodine</td>
			<td>Local pharmacy</td>
			<td>Betadine</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotactic apparatus</td>
			<td>WPI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrodotoxin</td>
			<td>Tocris</td>
			<td>1069</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1200S</td>
			<td></td>
		</tr>
	</tbody>
</table>

microrna-mediated inhibition of excitatory postsynaptic currents in mouse hippocampal slices

Source: Thalhammer, A., et al. <a href="https://app.jove.com/v/57223">Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits.</a> J. Vis. Exp. (2018)
This video demonstrates microRNA-mediated inhibition of excitatory synaptic currents in mouse hippocampal brain slices. The CA3 neurons were infected with a recombinant viral vector harboring a construct for expressing a fluorescent reporter, a light-activated membrane channel, and a microRNA that downregulates the expression of voltage-gated calcium channels. Upon placing a slice in a recording chamber and patching a CA1 neuron with a recording pipette, light pulses were used to stimulate the light-activated channel, resulting in an ion influx and generating an action potential. The absence of voltage-gated calcium channels in CA3 neurons inhibits neurotransmitter release, causing inhibition of excitatory postsynaptic currents in the synaptically-connected CA1 neurons.

<img alt="Figure 1" src="/files/ftp_upload/22765/22765_Figure1.jpg" /> 
Figure 1. Assessing the role of Cav2.1[EFa] and Cav2.1[EFb] in the native hippocampus by targeted stimulation of knocked-down neurons with optogenetics. (A) Scheme of the expression cassette of the rAAV constructs used for in vivo infection. (B) Hippocampal section showing that TdTomato fluorescence is limited to the CA3 region and ...

MicroRNA-Mediated Inhibition of Excitatory Postsynaptic Currents in Mouse Hippocampal Slices

1. Assessing the Role of Presynaptic Proteins in Intact Neuronal Circuits by Targeted Stimulation of Knocked-down Neurons with Optogenetics
NOTE: The ...

Take mouse hippocampal brain slices containing CA3 neurons infected with a recombinant viral vector.
The viral genome expresses a light-activated channel tagged with a fluorescent reporter and a microRNA that downregulates the expression of voltage-gated calcium channels.
Place a slice in a recording chamber under dark conditions. Using a microscope, identify the fluorescent CA3 neurons.
Patch a recording electrode onto a CA1 neuron that receives input from infected CA3 neurons. Rupture the membrane to record intracellular ionic currents.
Apply an inhibitor to block inhibitory neurotransmitter receptors, allowing only excitatory signals.
Using light pulses, stimulate the light-activated channels in the infected CA3 neurons, generating action potentials.
In uninfected mice, the action potential activates voltage-gated calcium channels, causing a calcium ion influx that triggers neurotransmitter release. The neurotransmitters trigger excitatory postsynaptic currents, or EPSCs, in the CA1 neurons.
In infected mice, the microRNA-mediated reduction of voltage-gated calcium channels results in EPSC inhibition.

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40 Views. Source: Thalhammer, A., et al. Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits. J. Vis. Exp. (2018) This video demonstrates microRNA-mediated inhibition of excitatory synaptic currents in mouse hippocampal brain slices. The CA3 neurons were infected with a recombinant viral vector harboring a construct for expressing a fluorescent reporter, a light-activated membrane channel, and a microRNA th...

Video: MicroRNA-Mediated Inhibition of Excitatory Postsynaptic Currents in Mouse Hippocampal Slices - Concept

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate ...

JoVE Journal

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic multiplicity). Synaptic multiplicity is plastic and changes throughout development and in different physiological conditions, being an important determinant for the efficacy of synaptic transmission. Here, we outline experiments for estimating the degree of multiplicity of synapses terminating onto a given postsynaptic neuron using whole-cell patch clamp electrophysiology in acute brain slices. Specifically, voltage-clamp recording is used to compare the difference between the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). The theory behind this method is that afferent inputs that exhibit multiplicity will show large, action potential-dependent sEPSCs due to the synchronous release that occurs at each synaptic contact. In contrast, action potential-independent release (which is asynchronous) will generate smaller amplitude mEPSCs. This article outlines a set of experiments and analyses to characterize the existence of synaptic multiplicity and discusses the requirements and limitations of the technique. This technique can be applied to investigate how different behavioral, pharmacological or environmental interventions in vivo affect the organization of synaptic contacts in different brain areas.

Here, we present a protocol for evaluating the functional synaptic multiplicity using whole-cell patch clamp electrophysiology in acute brain slices.

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic ...

Functional Site-Directed Fluorometry in Native Skeletal Muscle Cells

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability

Functional site-directed fluorometry has been the technique of choice to investigate the structure-function relationship of numerous membrane proteins, including voltage-gated ion channels. This approach has been used primarily in heterologous expression systems to simultaneously measure membrane currents, the electrical manifestation of the channels&#39; activity, and fluorescence measurements, reporting local domain rearrangements. Functional site-directed fluorometry combines electrophysiology, molecular biology, chemistry, and fluorescence into a single wide-ranging technique that permits the study of real-time structural rearrangements and function through fluorescence and electrophysiology, respectively. Typically, this approach requires an engineered voltage-gated membrane channel that contains a cysteine that can be tested by a thiol-reactive fluorescent dye. Until recently, the thiol-reactive chemistry used for the site-directed fluorescent labeling of proteins was carried out exclusively in Xenopus oocytes and cell lines, restricting the scope of the approach to primary non-excitable cells. This report describes the applicability of functional site-directed fluorometry in adult skeletal muscle cells to study the early steps of excitation-contraction coupling, the process by which muscle fiber electrical depolarization is linked to the activation of muscle contraction. The present protocol describes the methodologies to design and transfect cysteine-engineered voltage-gated Ca2+ channels (CaV1.1) into muscle fibers of the flexor digitorum brevis of adult mice using in vivo electroporation and the subsequent steps required for functional site-directed fluorometry measurements. This approach can be adapted to study other ion channels and proteins. The use of functional site-directed fluorometry of mammalian muscle is particularly relevant to studying basic mechanisms of excitability.

Author Spotlight: Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability  

Functional site-directed fluorometry is a method to study protein domain motions in real time. Modification of this technique for its application in native cells now allows the detection and tracking of single voltage-sensor motions from voltage-gated Ca2+ channels in murine isolated skeletal muscle fibers.

Functional site-directed fluorometry has been the technique of choice to investigate the structure-function relationship of numerous membrane proteins, ...

MicroRNA-Mediated Inhibition of Excitatory Postsynaptic Currents in Mouse Hippocampal Slices

Transcript

MicroRNA-Mediated Inhibition of Excitatory Postsynaptic Currents in Mouse Hippocampal Slices

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability