The authors would like to thank Mariko Yamatake and Masako Takashima for technical assistance. This work was supported in part by KAKENHI grant numbers 16H00759 and 17H04017 (SO).

1. Prepare Liposomes
<ol>
	<li>Disperse phospholipids (e.g., 10 mg in powder) in chloroform at a desired concentration (e.g., 10 mg/mL).</li>
	<li>Evaporate chloroform.
	<ol>
		<li>Place the phospholipid solution in a round-bottom flask and set it on a rotary evaporator (see Table of Materials) connected to a N2 gas cylinder. Rotate the flask under N2 flow at room temperature until a thin phospholipid film appears (after ~30 min).</li>
		<li>Place the open flask into a desiccator that is connected to a vacuum pump. Using the vacuum pump, aspirate the inside of the desiccator for several hours to remove the chloroform thoroughly.</li>
	</ol>
	</li>
	<li>Add an appropriate volume of an electrolyte solution to the flask and suspend the phospholipid to obtain a 2 mg/mL phospholipid suspension.</li>
	<li>Sonicate the suspension for several tens of seconds using a bath sonicator (see Table of Materials) to obtain a multilayered vesicle (MLV) suspension.</li>
	<li>For preparation of proteoliposomes that contain ion channel proteins, add a protein solution (with the proteins solubilized using appropriate detergents; 2% volume is the maximum) to the MLV suspension and sonicate for several seconds using the bath sonicator.</li>
</ol>
2. Prepare Large-Bore Glass Pipettes
<ol>
	<li>Set a glass capillary to a pipette puller and fabricate micropipettes with a fine tapered tip through two-step pulling.</li>
	<li>Set the micropipette on a microforge and contact the tip of the micropipette to a platinum filament at the tapered portion with a diameter of 30 to 50 &#181;m.</li>
	<li>Heat the filament briefly (5 s) and immediately turn it off. 
	NOTE: This manipulation forms a crack at the heating point, whereby the tip of the micropipette is cut off, leaving a wide bore with a diameter of 30 to 50 &#181;m.</li>
</ol>
3. Treat the Surface of Glass Slide with a Shallow Concave Well (Siliconization for a Water-Repellent Finish)
<ol>
	<li>Clean the surface of the glass slide with a shallow well with distilled water and ethanol.</li>
	<li>Apply an appropriate volume (e.g., 100 &#181;L) of a siliconizing reagent (water-repellent) onto the hole slide glass.</li>
	<li>Dry the reagent completely in the air.</li>
	<li>Place the glass slide on the stage of an inverted microscope.</li>
</ol>
4. Form the CBB and Perform Electrophysiological Measurement
<ol>
	<li>Add 100 &#181;L of hexadecane into the shallow well of the siliconized hole slide glass. 
	&#8203;NOTE: For the lipid-out method, phospholipids are dispersed in hexadecane (20 mg/mL) beforehand.</li>
	<li>Fill the electrolyte solution up to half the length of the micropipette, using a tuberculin syringe.</li>
	<li>Set the micropipette onto the micropipette holder with a pressure port, allowing the Ag/AgCl wire electrode to soak into the pipette electrolyte solution.</li>
	<li>Connect one of the micropipette holders to the head stage of a patch-clamp amplifier and the other one to the electrical ground.</li>
	<li>Connect a microinjector to the pressure port of the micropipette holder.</li>
	<li>Set the micropipette to an appropriate position above the stage of an inverted microscope by manipulating the micromanipulator.</li>
	<li>Adjust the electrode offset potential.
	<ol>
		<li>Place 1 &#181;L of the same electrolyte solution used to fill the micropipette on the flat surface around the shallow well of the hole slide glass, creating an electrolyte dome.</li>
		<li>Soak the tip of both micropipettes into the electrolyte dome by manipulating the micromanipulator.</li>
		<li>Adjust the electrode offset potential of the patch-clamp amplifier.</li>
		<li>Confirm the correct offset at the end of experiments by breaking the CBB via application of a high membrane potential (electrical breakdown; using Zap on the amplifier), causing the two bubbles to be fused into one (bubble fusion).</li>
		<li>Correct the liquid junction potential54 in the cases where asymmetric electrolyte solutions are used, such that the calculated value is added to the applied membrane potential for the true membrane potential. 
		NOTE: The liquid junction potential is calculated using the program JPCalc55.</li>
	</ol>
	</li>
	<li>Draw the liposome solution from the tip. 
	&#8203;NOTE: When water-soluble channels are examined using the lipid-out method, suctioning of the liposome solution is not necessary.
	<ol>
		<li>Place 1 &#181;L of liposome solution on the flat surface around the shallow well of the hole slide glass (liposome-containing dome).</li>
		<li>Manipulate the micromanipulator and insert the tip of the micropipette into the liposome-containing dome.</li>
		<li>Aspirate the liposome-containing solution by lowering the pressure inside the micropipette holder using the microinjector.</li>
		<li>Repeat the procedure for the other pipette.</li>
	</ol>
	</li>
	<li>Manipulate the micromanipulator and dip the tip of the micropipette into the hexadecane in the shallow well.</li>
	<li>Blow a water bubble slowly by increasing the pressure until the bubble reaches the desired size (e.g., 50 &#181;m in diameter) and maintain the same pressure thereafter.</li>
	<li>Discard the bubbles by passing the tip through the oil-air interface if it is hard to keep the size of the bubbles stable.</li>
	<li>Repeat steps 4.8 to 4.9 until stable bubbles are formed.</li>
	<li>Manipulate the bubbles to allow contact between them (Figure 5). 
	NOTE: Sometimes, the bubbles approach each other spontaneously to form the CBB. In other cases, the bubbles are close but do not contact each other. In this case, push the bubbles each other mechanically.</li>
	<li>Fine-tune the pressure to maintain the bubble size, because the size may gradually change even at the constant intra-bubble pressure.</li>
	<li>Set the membrane potential to the appropriate value using the patch-clamp amplifier and wait for the channel current to emerge (Figure 6).</li>
</ol>
5. Measure Bilayer Capacitance
<ol>
	<li>Measure the bilayer electrical capacitance (Cel) by applying a ramp potential. 
	NOTE: When the rate of the voltage change in the ramp command is 10 mV/10 ms (or 1 V/s) followed by -10 mV/10 ms, the amplitude of the current jump upon changes in the slope corresponds to the read of the membrane&#39;scapacitance value (e.g., 100 pA &#8594; 100 pF).</li>
	<li>Evaluate the bilayer area of two bubbles stacked one on the other and focus the microscope at the bilayer level to view the edge of the bilayer (Figure 6). 
	NOTE: The bilayer shape is mostly circular, and the area is calculated from the radius.</li>
	<li>Calculate the specific membrane capacitance (Csp) by dividing the electrical capacitance by the bilayer area (Csp = Cel/A).</li>
	<li>Calculate the bilayer thickness (thickness of the hydrophobic core) using Dc = (&#949;r&#949;0)/Csp (where &#949;r and &#949;0 represent the permittivity of the hydrophobic region of the bilayer and the permittivity of a vacuum, respectively).</li>
</ol>

The authors have no conflict of interest to disclose.

The CBB method of lipid bilayer formation is based on the principle of a water-in-oil droplet lined by a monolayer20. Technically, the procedures for forming CBBs are easy, especially for patch-clamp researchers, who are proficient in manipulating glass micropipettes. The electrophysiological setup for the patch clamp is readily used in the CBB when two pipette manipulators with microinjectors are available. On the other hand, because the CBB is a successor of the conventional PLB, for which a large amount of physicochemical knowledge has been accumulated8, this background as well as knowledge of surface chemistry38 is helpful for operating and manipulating CBBs. The CBB serves a versatile platform for studying channel-membrane interplays with its ability of modifying chemical composition and physical status61. In the CBB, lipids lining the bubble can diffuse freely across the border of the monolayer phase and leaflet of the bilayer, and we have developed techniques such as the bilayer attach-detach and the membrane perfusion methods45. We have extended CBBs for in vitro channel protein synthesis, where channel synthesis was performed in the bubble, and newly synthesized channel proteins were subjected to spontaneous transfer to the bilayer under application of a membrane potential where nascent channel functions of the KcsA channel was traced from the time of initiating the transcription/translation of KcsA DNA62.
Among the steps of the protocol, maintaining the bubble size is critical. The bubbles may slowly swell or shrink spontaneously because lipid transfer to the interface is slow and the monolayer tension is prone to change. In particular, the first bubble inflated just after suction of the liposome solution (steps 4.8.3 and 4.10) is hard to maintain because the monolayer tension is lowered by the liposomes accumulated at the tip of the pipette. Discarding the initial bubbles and subsequent formation of the bubbles provide the stable CBB. Visual tracing of the bubble size and fine tuning of the pressure are necessary.
There are experimental limitations to the CBB method. Although the CBB is designed for electrophysiological measurements, the electrical resistance of the bilayer for certain lipid compositions may not be high enough (e.g., 100 G&#937;) for single-channel recordings. For example, dioleoyl phosphatidylcholine is frequently used for liposomes, but it forms electrically leaky CBBs at 25 &#176;C63. CBBs cannot even be formed with lipid species whose phase transition temperature is above the recording temperature. Indeed, CBB formation was difficult with dipalmitoyl phosphatidylcholine at room temperature, but raising the temperature above Tm circumvented the problem64. In these experiments, the temperature was controlled by using a transparent heat plate (see Table of Materials) beneath the slide glass62. The phase transition temperature of the frequently used lipid diphytanoyl phosphatidylcholine is below 0 &#176;C 65, and CBBs are readily formed32,45,51 at a broad temperature range.
Phospholipids of the water-oil interface are provided from either the lipid-in or lipid-out method, where the transfer rate is relatively slower in the lipid-out than in the lipid-in method63. In addition, for reasons unknown, the electrical resistance of the bilayer membrane is relatively smaller with the lipid-out method than with the lipid-in method.
In the protocol (step 4.8), a solution of liposomes and channel-reconstituted liposomes (1 &#181;L) is loaded from the tip of the pipette (steps 4.9 and 4.10), and the rest of the pipette contains the previously filled electrolyte solution. This is merely for conserving materials, such as lipids and channel molecules. Consequently, the liposomes diffuse gradually toward the upper pipette solution, and after a while, the lipid concentration at the tip of the pipette becomes insufficient to form lipid monolayers. In this case, fresh liposome solution should be aspirated from the tip (step 4.8). This temporal concentration change can be circumvented when the whole pipette is filled with the solution containing lipids and channel molecules, whereas liposomes gradually settle down into the bubbles. In this case, bubbles are renewed.
Electrophysiologically, the CBB size is larger than that of the patch clamp, thus yielding a larger membrane capacitance. However, the series resistance (the electrical resistance in a series of membrane resistance) is much lower (~100 k&#937;) than that for the patch clamp (pipette resistance &#62; 1 M&#937;), which accelerates the speed of the voltage clamp and attenuates the background noise and series resistance errors in the voltage-clamped membrane potentials66,67,68. The background noise is a function of the membrane capacitance and the series resistance, where a low series resistance complements a high membrane capacitance, resulting in low background noise9.
As a rule of lipid bilayer experiments, detergents should not be used to wash glassware. Even a trace amount of detergent perturbs the integrity of the bilayer. Organic solvents such as chloroform/methanol and ethanol should be used for such cleaning purposes instead.
Overall, the CBB integrates the benefits of both the patch clamp (e.g., the mechanical manipulation of the membrane) and the PLB (e.g., the ability to modify the lipid composition of the membrane). Various types of channel-forming substances and channel proteins have been studied52,53,69,70. The development of this method is opportune since increasingly more researchers are focusing on channel-membrane interplay, and the CBB provides a versatile platform for various experiments. Further experimental developments in the CBB method are expected.

For ion channels, the cell membrane is not simply a supporting material but a partner for generating the ion flux. Functionally, the membrane is an electrical insulator in which ion channels are embedded, and all cell membranes are imparted with a resting membrane potential. Conventionally, an arbitrary membrane potential was imposed from an external circuit by which electrical current through the channels was measured. This quantitative evaluation of the ion flux at different membrane potentials revealed the molecular properties of these channels, such as their ion-selective permeation and gating functions1,2. The membrane platform for functional studies of ion channels is either the cell membrane or the lipid bilayer membrane. Historically, single-channel electrical current recordings were first performed in lipid bilayers3,4, and the relevant techniques were developed for cell membranes, such as the patch-clamp method (Figure 1A)5,6. Since then, these two techniques have evolved separately for different purposes (Figure 1)7,8.
Membrane lipids and bilayer membranes are currently the focus of research for their roles in supporting the structure and function of channel proteins. Therefore, the ready availability of methods to vary the lipid composition in bilayers is in high demand. Lipid bilayer formation methods such as the planar lipid bilayer (PLB)8,9,10,11, water-in-oil droplet bilayer12, and droplet interface bilayer (DIB)13,14,15,16,17,18,19 techniques (Figure 1) are common choices, providing an opportunity for examining the channel function under varying lipid compositions20. Although the DIB is technically much easier to produce than the conventional PLB, the large size of the DIB has created a disincentive for patch-clampers to apply it for studying single-channel current recordings with usual-sized conductance (&#60;100 pS).
To circumvent the background noise, the bilayer area must be minimized. This issue recalls the repetitions of history in developing electrophysiological techniques for lipid bilayers (Figure 1). In the early days, a small-sized bilayer (1-30 &#181;m in diameter) was formed at the tip of a pipette (tip-dip method; Figure 1C)21,22,23, rather than using a free-standing bilayer (~100 &#181;m in diameter) on a hydrophobic septum in a chamber (Figure 1B). The tip-dip method allowed for electrical measurements with much lower background noise24. Our experiences with the PLB25,26, tip-dip22,23,27, and patch-clamp28,29,30,31 methods led us to a novel idea of forming lipid bilayers by using the principles of the water-in-oil bilayer. We have referred to this as the contact bubble bilayer (CBB) method20,32. In this method, rather than hanging the water droplets in an oil phase (Figure 1D), a water bubble is blown from a glass pipette (with tip diameter of approximately 30 &#181;m) into the oil phase (Figure 1E and 2), where the bubble is maintained by applying a steady pressure. A monolayer forms spontaneously at the water-oil interface at the surface of the bubble. Then, two bubbles are docked through the manipulation of two glass pipettes, and the bilayer is formed as the two monolayers approach each other, yielding an equilibrium bilayer area. The size of the bubble is controlled by the intra-bubble pressure (holding pressure), and likewise the bilayer size. An average diameter of 50 &#181;m is frequently used. Although the volume of the bubble is small (&#60;100 pL), it is connected to the larger volume of the pipette solution that is in the microliter range, constituting the bulk electrolyte phase.
There are many benefits to use the CBB method (Table 1). As a lipid bilayer formation technique, membranes of various lipid compositions can be produced, and asymmetric membranes are more readily formed32 than are those by the conventional folding method33. The bilayer can be mechanically manipulated, unlike the conventional PLB that can only be bent with a hydrostatic pressure difference34,35. By changing the holding pressure, the bubbles either expand or shrink, leading to increased or decreased membrane tension32. The bilayer is mechanically detachable into monolayers, similar to the freeze-fracture technique36,37 of membranes in morphological studies, but with the CBB, a maneuver allows for repeated detach and attach cycles32. The small volume of the electrolyte solution within the bubble allows efficient fusion of channel-reconstituted liposomes into the bilayer, and the probability of getting channel recordings is much higher than with the conventional PLB technique. The small bubble volume also allows rapid perfusion (within ~20 ms) once another injection pipette is inserted into either of the bubbles. Unlike the patch-clamp method, once broken, a CBB membrane is re-formed immediately and repeatedly, and pipettes can be used several times a day. By integrating benefits of the patch-clamp and PLB methods, the CBB provides a versatile platform to vary the physicochemical conditions of the membrane, allowing for unprecedented studies of channel-membrane interactions.
Before presenting a detailed protocol of the CBB formation process, the physicochemical background of the bilayer formation is presented first,which will be helpful for patch-clampers to resolve experimental difficulties relating to the membrane formation that are encountered.
CBB experiments impart lessons of surface chemistry science38. The CBB is similar to a soap bubble blown from a straw into the air, where likewise, a water bubble is blown into an organic solvent. One will notice that a water bubble is hardly inflated when membrane lipids are not included in either the water bubble or the organic solvent. In the absence of amphipathic lipids, the surface tension at a water-oil interface is high, and the intra-bubble pressure for blowing a bubble will be high. This is a realization of the Laplace equation (&#916;P = 2 &#947;/R, where &#916;P is the intra-bubble pressure, &#947; is the surface tension, and R is the bubble radius). When the concentration of lipids in either the organic phase or the electrolyte solution is high, the density of lipids in the monolayer increases, as dictated by the Gibbs adsorption isotherm (-d&#947; = &#915;i d&#181;i, where &#915;i is the surface excess of compound i, and &#181;i is the chemical potential of component i)39, leading to a lower surface tension and ease of bubble formation. In the CBB, the bilayer can be observed from a tangential angle (Figure 2), and the contact angle between the monolayer and bilayer is measurable. This angle represents an equilibrium between the surface tensions of the monolayer and bilayer (Young equation: &#947;bi = &#947;mo cos(&#952;), where &#947;bi is the bilayer tension, &#947;mo is the monolayer tension, and &#952; is the contact angle). The changes in the contact angle indicate changes in the bilayer tension, given that the monolayer tension is evaluated from changes in the contact angle as a function of the membrane potential (Young-Lippmann equation: &#947;mo = Cm V2/4 (cos(&#952;0) - cos(&#952;v)), where Cm is the membrane capacitance, V is the membrane potential, and &#952;0 and &#952;v are the contact angles at 0 and V mV, respectively)40,41,42. When two bubbles are close enough, they approach each other spontaneously. This is due to the van der Waals force, and we can visually observe this dynamic process in CBB formation.
A CBB system consists of distinct phases: namely, a bulk oil phase, water bubbles coated with a monolayer, and a contacting bilayer (Figure 3). These are reminiscent of the multiple phases observed in a PLB, such as a solvent-containing torus around the bilayer phase and a thin organic phase sandwiched by two monolayers43,44. In the CBB, the monolayer phase is continuous with the bilayer leaflet, and lipid molecules readily diffuse between the monolayer and the leaflet. The monolayer phase covers most of the bubble surface, constituting the major phase that serves as a lipid reservoir. Because the hydrophobic tail of lipids in the monolayer extends outward to the bulk oil phase, the bilayer interior or the hydrophobic core opens to the bulk oil phase. Thus, a hydrophobic substance injected into the oil phase close to the bilayer is able to readily access the bilayer interior. This is the membrane perfusion technique we had developed recently45, by which the lipid composition in the bilayer is changed rapidly (within a second) during single-channel current recordings. We found that the cholesterol content in the bilayer could be reversibly controlled by switching the cholesterol perfusion on and off45. In the event that the concentration of the relevant substance in the monolayer and bilayer differs, the concentration gradient of the relevant substance is immediately dissolved through diffusion, which is known as the Marangoni effect46,47. On the other hand, flip-flops across the monolayers are slow48,49,50.
Using the CBB method, the bilayer is formed under versatile physicochemical conditions, such as an electrolyte pH as low as 1 51, a salt (K+, Na+, etc.) concentration up to 3 M, a membrane potential as high as &#177;400 mV, and a system temperature of up to 60 &#176;C.
There are several options for formation of the CBB and incorporation of channel molecules therein. For formation of the monolayer at the water-oil interface, lipids are added either in an organic solvent (lipid-out method; Figure 4A, 4C) or in a bubble as liposomes (lipid-in method; Figure 4B, 4D). Notably, the lipid-in method allows for the formation of asymmetric membranes15,32. Channel molecules soluble in aqueous solution (e.g., channel-forming peptides) are directly added into the bubble (Figure 4A, B)52,53, whereas channel proteins are reconstituted into liposomes, which are then added into the bubble (Figure 4C, D). Herein, the formation of CBBs by the lipid-in method for either a channel peptide (polytheonamide B (pTB); Figure 4A) or a protein (KcsA potassium channel, Figure 4C) is shown.

<table border="0" cellpadding="0" cellspacing="0" style="width:607px;" width="605">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:219px;">Azolectin (L-&#945;-Phosphatidylcholine, Type IV-S)</td>
			<td style="width:85px;">Sigma-Aldrich</td>
			<td style="width:124px;">P3644</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:219px;">A/D Converter</td>
			<td style="width:85px;">Molecular Divices</td>
			<td style="width:124px;">Digidata1550A</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:219px;">Ag/AgCl electrode</td>
			<td style="width:85px;">Warner Instruments</td>
			<td style="width:124px;">64-1317</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Bath Sonicator</td>
			<td style="width:85px;">Branson</td>
			<td style="width:124px;">M1800H-J</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:219px;">Camera</td>
			<td style="width:85px;">Hamamatsu Photonics</td>
			<td style="width:124px;">C11440-10C</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:219px;">Glass Capillary</td>
			<td style="width:85px;">Harvard Apparatus</td>
			<td style="width:124px;">30-0062</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Hepes</td>
			<td style="width:85px;">Dojindo</td>
			<td style="width:124px;">342-01375</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:219px;">Hole Slideglass</td>
			<td style="width:85px;">Matsunami Glass</td>
			<td style="width:124px;">S339929</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Inverted Microscope</td>
			<td style="width:85px;">Olympus</td>
			<td style="width:124px;">IX73</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Isolation Table</td>
			<td style="width:85px;">Herz</td>
			<td style="width:124px;">TDI-86LA(Y)2</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Micro Injenctor</td>
			<td style="width:85px;">Narishige</td>
			<td style="width:124px;">IM-11-2</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Micro Manipulator</td>
			<td style="width:85px;">Narishige</td>
			<td style="width:124px;">EMM</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Microforge</td>
			<td style="width:85px;">Narishige</td>
			<td style="width:124px;">MF-830</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Micropipette holder</td>
			<td style="width:85px;"></td>
			<td style="width:124px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">n-Hexadecane</td>
			<td style="width:85px;">Nacalai</td>
			<td style="width:124px;">07819-32</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Patch-Clamp Amplifier</td>
			<td style="width:85px;">HEKA</td>
			<td style="width:124px;">EPC800</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:219px;">Pipette Puller</td>
			<td style="width:85px;">Sutter Instrument Co.</td>
			<td style="width:124px;">P-87</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:219px;">POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine)</td>
			<td style="width:85px;">Avanti Polar Lipids</td>
			<td style="width:124px;">850457</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:219px;">POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine 
			)</td>
			<td style="width:85px;">Avanti Polar Lipids</td>
			<td style="width:124px;">850757</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:219px;">POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) )</td>
			<td style="width:85px;">Avanti Polar Lipids</td>
			<td style="width:124px;">840457</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Potassium Chloride</td>
			<td style="width:85px;">Nacalai</td>
			<td style="width:124px;">28514-75</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Rotary Evapolator</td>
			<td style="width:85px;">Iwaki</td>
			<td style="width:124px;">REN-1000</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Succinic Acid</td>
			<td style="width:85px;">Nacalai</td>
			<td style="width:124px;">32402-05</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Vacuum Pump</td>
			<td style="width:85px;">Buchi</td>
			<td style="width:124px;">V-100</td>
			<td style="width:179px;"></td>
		</tr>
	</tbody>
</table>

lipid bilayer experiments with contact bubble bilayers for patch-clampers

Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions under various membrane lipid compositions. Among them, the droplet interface bilayer has gained popularity; however, the large membrane size hinders the recording of low electrical background noise. We have established a contact bubble bilayer (CBB) method that combines the benefits of planar lipid bilayer and patch-clamp methods, such as the ability to vary the lipid composition and to manipulate the bilayer mechanics, respectively. Using the setup for conventional patch-clamp experiments, CBB-based experiments can be readily performed. In brief, an electrolyte solution in a glass pipette is blown into an organic solvent phase (hexadecane), and the pipette pressure is maintained to obtain a stable bubble size. The bubble is spontaneously lined with a lipid monolayer (pure lipids or mixed lipids), which is provided from liposomes in the bubbles. Next, the two monolayer-lined bubbles (~50 &#181;m in diameter) at the tip of the glass pipettes are docked for bilayer formation. Introduction of channel-reconstituted liposomes into the bubble leads to the incorporation of channels in the bilayer, allowing for single-channel current recording with a signal-to-noise ratio comparable to that of patch-clamp recordings. CBBs with an asymmetric lipid composition are readily formed. The CBB is renewed repeatedly by blowing out the previous bubbles and forming new ones. Various chemical and physical perturbations (e.g., membrane perfusion and bilayer tension) can be imposed on the CBBs. Herein, we present the basic procedure for CBB formation.

A typical CBB had a diameter of 50 &#181;m (Figure 5, 6) and the specific membrane capacitance in hexadecane was 0.65 &#181;F/cm2. The bubble size was arbitrarily controlled by the intra-bubble pressure. When small bubbles are necessary for low-noise recordings, the tip diameter should be correspondingly small. For example, for a bubble size of 50 &#181;m in diameter, the tip diameter should be 30 &#181;m.
Once the CBB had formed, the channel molecules either in aqueous solution or in the liposome were spontaneously inserted into the bilayer within a span of a few to dozens of minutes. Insertion of the channels was confirmed by the stepwise increase of current amplitude (Figure 7) under the applied membrane potential. The smaller membrane area (&#60;1,000 &#181;m2) relative to that in the conventional PLB and DIB systems substantially improved the electrical signal-to-noise ratio.
Current recordings could be continued until the membrane was disrupted and the two bubbles merged. New bubbles were blown and the CBB formed immediately and repeatedly. The pipette could be used repeatedly within a day.
Duty cycle of attach and detach. A bilayer formed by the CBB method can be disintegrated into two monolayers. Detach-attach of the CBB can be repeated by manipulating the two bubbles (duty cycle of detach-attach). This process was monitored by the appearance of the channel current of pTB, a peptide channel from a marine sponge, as it was readily inserted into the lipid bilayer to form a monovalent cation-selective pore52,56. The pTB channel current emerged immediately after attaching the two monolayers, and the current became larger as the area of contact increased (Figure 8). The amplitude of the current was synchronized with the detach-attach manipulation of the CBB.
Single-channel measurements of the KcsA channel. The KcsA potassium channel is pH sensitive, being activated by acidic intracellular pH57. Thus, the electrolyte solution was set to be asymmetric58,59,60. In step 4.2 of CBB formation, the pipette solution of the left side was set at pH 4, whereas that of the right side was set at pH 7.525. In step 4.7.1, a proteoliposome suspension, rather than liposome suspension, was placed onto the slide glass for aspiration into the left pipette. Accordingly, the KcsA channel was oriented in the membrane with its cytoplasmic domain facing the left bubble. Liposomes with the protein:lipid weight ratio of 1:2000 are suitable for single-channel current recording, and those with a 1:10 ratio are for macroscopic current recording.
Asymmetric membrane. An asymmetric lipid bilayer can be formed using different liposome suspensions for each bubble (lipid-in)15,32. The orientation of the KcsA channel in the membrane was regulated by setting an asymmetric solution pH (pH 4in/pH 7out), leading to its cytoplasmic side being toward the left side. Accordingly, the left side was assigned as &#34;in,&#34; whereas the right side was assigned as &#34;out.&#34; To examine lipid dependence on the gating of the KcsA channel, four types of CBB (including an asymmetric CBB) were used; namely, PGin/PGout, PGin/PCout, PCin/PGout, and PCin/PCout (PG: phosphatidylglycerol) (Figure 9). The KcsA channel exhibited high open probability (&#62;90%) only in the PGin/PGout and PGin/PCout membranes. The KcsA channel requires the existence of anionic phospholipids in the inner leaflet of the membrane for a high open probability26.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58840/58840fig1.jpg" /> 
Figure 1:&#160;Lipid bilayer and patch-clamp methods. Various methods have been developed for forming the lipid bilayer. (A) Patch-clamp method. (B) Conventional planar lipid bilayer method, which provides a free-standing, mostly vertical, bilayer on a small hole. (C) Tip-dip method. A monolayer at the air-water interface is positioned on the tip of a glass electrode. Various modifications have been developed. (D) Droplet interface bilayer method. (E) Contact bubble bilayer method. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58840/58840fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58840/58840fig2.jpg" /> 
Figure 2:&#160;Contact bubble bilayer formation. As a bubble is blown into an oil phase, lipid molecules transfer spontaneously to the water-oil interface. Different types of lipids as liposomes are included in each bubble (lipid-in), and monolayers are formed exclusively by the relevant lipids. Docking two monolayers generates an asymmetric bilayer membrane. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58840/58840fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58840/58840fig3.jpg" /> 
Figure 3:&#160;Distinct phases in the contact bubble bilayer, and the dynamics of lipids therein. Lipids lining the bubble constitute a phase where they belong to either a monolayer or a bilayer. Flip-flop of lipids across the bilayer is infrequent, and the asymmetric membrane is retained for a long time. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58840/58840fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58840/58840fig4.jpg" /> 
Figure 4:&#160;Delivering lipids with the lipid-out or lipid-in method. Lipids are added either in an organic solvent (lipid-out; A and C) or in aqueous solution as liposomes (lipid-in; B and D). In the lipid-in method, an asymmetric membrane is formed. Channel-forming substances that are soluble in aqueous solution (blue), such as peptides, are added into one of the bubbles and spontaneously inserted into the bilayer. In the lipid-in method, a part of the channels is inserted into liposomes, which are then fused to the bilayer. Channel proteins (red) are reconstituted into liposomes in either the lipid-out or lipid-in method, which are then added into one of the bubbles and spontaneously fused to the bilayer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58840/58840fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58840/58840fig5.jpg" /> 
Figure 5:&#160;Formation and microscopic image of the contact bubble bilayer. The bilayer is observed from a tangential direction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58840/58840fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58840/58840fig6v2.jpg" /> 
Figure 6:&#160;Observation of the bilayer for measuring the bilayer area. Two bubbles are stacked one on the other through pipette manipulation, and the microscope is focused at the level of the contact bubble bilayer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58840/58840fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58840/58840fig7v2.jpg" /> 
Figure 7:&#160;Stepwise insertion of the KcsA channel into the contact bubble bilayer membrane. The E71A mutant of the KcsA channel was used to show the immediate detection of the inserted channels. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58840/58840fig7v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58840/58840fig8.jpg" /> 
Figure 8:&#160;Detach-attach of monolayers and responses of channels. A peptide channel, polytheonamide B, was added into one of the bubbles, which was then spontaneously incorporated into the membrane. When detached, channels in the membrane withdrew but were retained at the membrane leaflet of the channel-added side. Upon attachment, the channels were inserted into the membrane. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58840/58840fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/58840/58840fig9.jpg" /> 
Figure 9:&#160;Formation of an asymmetric membrane and channel activity dependent on the composition of the leaflet. The KcsA channel is regulated by acidic lipids, such as PG, in the inner leaflet of the membrane. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58840/58840fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>Conventional PLB</td>
			<td>Liposome patch</td>
			<td>DIB</td>
			<td>CBB</td>
		</tr>
		<tr>
			<td>Success rate1</td>
			<td>high</td>
			<td>low</td>
			<td>very high</td>
			<td>very high</td>
		</tr>
		<tr>
			<td>Immediate reformability2</td>
			<td>yes</td>
			<td>no</td>
			<td>intermediate</td>
			<td>yes</td>
		</tr>
		<tr>
			<td>Asymmetric membrane</td>
			<td>yes</td>
			<td>no</td>
			<td>yes</td>
			<td>yes</td>
		</tr>
		<tr>
			<td>Solution exchange</td>
			<td>slow</td>
			<td>very fast</td>
			<td>slow</td>
			<td>fast</td>
		</tr>
		<tr>
			<td>S/N ratio3</td>
			<td>low</td>
			<td>high</td>
			<td>low</td>
			<td>high</td>
		</tr>
		<tr>
			<td>Membrane deformation</td>
			<td>no</td>
			<td>no</td>
			<td>no</td>
			<td>yes4</td>
		</tr>
		<tr>
			<td>Mechanical manipulation</td>
			<td>no</td>
			<td>yes5</td>
			<td>yes</td>
			<td>yes</td>
		</tr>
		<tr>
			<td>Membrane perfusion</td>
			<td>no</td>
			<td>no</td>
			<td>yes</td>
			<td>yes</td>
		</tr>
		<tr>
			<td>Lipid compositions</td>
			<td>diverse</td>
			<td>limited6</td>
			<td>diverse</td>
			<td>diverse</td>
		</tr>
		<tr>
			<td colspan="5">1The success rate of forming stable lipid bilayer per trial.</td>
		</tr>
		<tr>
			<td colspan="5">2Formation of a new membrane quickly after break down of a membrane. Also, membrane is formed repeatedly.</td>
		</tr>
		<tr>
			<td colspan="5">3The signal-to-noise (S/N) ratio represents the electrical background noise primary generated from the membrane capacitance.</td>
		</tr>
		<tr>
			<td colspan="5">4Concave or convex membrane can be formed by applying different inner pressure between two bubbles.</td>
		</tr>
		<tr>
			<td colspan="5">5Only increasing the membrane tension.</td>
		</tr>
		<tr>
			<td colspan="5">6Phospholipids that can form giant unilamellar vesicles are applicable, but the giga-seal may not be attained.</td>
		</tr>
	</tbody>
</table>
Table 1: Characteristics of various lipid bilayer membranes.

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Lipid Bilayer Experiments with Contact Bubble Bilayers for Patch-Clampers

Here, we present a protocol for the formation of lipid bilayers using a contact bubble bilayer method. A water bubble is blown into an organic solvent, whereby a monolayer is formed at the water-oil interface. Two pipettes are manipulated to dock the bubbles to form a bilayer.

Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions ...

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Channels and the Electrical Properties of Membranes

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9.5K Views.  University of Fukui Faculty of Medical Sciences. Here, we present a protocol for the formation of lipid bilayers using a contact bubble bilayer method. A water bubble is blown into an organic solvent, whereby a monolayer is formed at the water-oil interface. Two pipettes are manipulated to dock the bubbles to form a bilayer.

Video: Lipid Bilayer Experiments with Contact Bubble Bilayers for Patch-Clampers

Combining Computer Game-Based Behavioural Experiments With High-Density EEG and Infrared Gaze Tracking

Experimental paradigms are valuable insofar as the timing and other parameters of their stimuli are well specified and controlled, and insofar as they yield data relevant to the cognitive processing that occurs under ecologically valid conditions. These two goals often are at odds, since well controlled stimuli often are too repetitive to sustain subjects' motivation. Studies employing electroencephalography (EEG) are often especially sensitive to this dilemma between ecological validity and experimental control: attaining sufficient signal-to-noise in physiological averages demands large numbers of repeated trials within lengthy recording sessions, limiting the subject pool to individuals with the ability and patience to perform a set task over and over again. This constraint severely limits researchers' ability to investigate younger populations as well as clinical populations associated with heightened anxiety or attentional abnormalities. Even adult, non-clinical subjects may not be able to achieve their typical levels of performance or cognitive engagement: an unmotivated subject for whom an experimental task is little more than a chore is not the same, behaviourally, cognitively, or neurally, as a subject who is intrinsically motivated and engaged with the task. A growing body of literature demonstrates that embedding experiments within video games may provide a way between the horns of this dilemma between experimental control and ecological validity. The narrative of a game provides a more realistic context in which tasks occur, enhancing their ecological validity (Chaytor &amp; Schmitter-Edgecombe, 2003). Moreover, this context provides motivation to complete tasks. In our game, subjects perform various missions to collect resources, fend off pirates, intercept communications or facilitate diplomatic relations. In so doing, they also perform an array of cognitive tasks, including a Posner attention-shifting paradigm (Posner, 1980), a go/no-go test of motor inhibition, a psychophysical motion coherence threshold task, the Embedded Figures Test (Witkin, 1950, 1954) and a theory-of-mind (Wimmer &amp; Perner, 1983) task. The game software automatically registers game stimuli and subjects' actions and responses in a log file, and sends event codes to synchronise with physiological data recorders. Thus the game can be combined with physiological measures such as EEG or fMRI, and with moment-to-moment tracking of gaze. Gaze tracking can verify subjects' compliance with behavioural tasks (e.g. fixation) and overt attention to experimental stimuli, and also physiological arousal as reflected in pupil dilation (Bradley et al., 2008). At great enough sampling frequencies, gaze tracking may also help assess covert attention as reflected in microsaccades - eye movements that are too small to foveate a new object, but are as rapid in onset and have the same relationship between angular distance and peak velocity as do saccades that traverse greater distances. The distribution of directions of microsaccades correlates with the (otherwise) covert direction of attention (Hafed &amp; Clark, 2002).

Procedures for recording high-density EEG and gaze data during computer game-based cognitive tasks are described. Using a video game to present cognitive tasks enhances ecological validity without sacrificing experimental control.

Experimental paradigms are valuable insofar as the timing and other parameters of their stimuli are well specified and controlled, and insofar as they ...

Examining Local Network Processing using Multi-contact Laminar Electrode Recording

Cortical layers are ubiquitous structures throughout neocortex1-4 that consist of highly recurrent local networks. In recent years, significant progress has been made in our understanding of differences in response properties of neurons in different cortical layers5-8, yet there is still a great deal left to learn about whether and how neuronal populations encode information in a laminar-specific manner.
Existing multi-electrode array techniques, although informative for measuring responses across many millimeters of cortical space along the cortical surface, are unsuitable to approach the issue of laminar cortical circuits. Here, we present our method for setting up and recording individual neurons and local field potentials (LFPs) across cortical layers of primary visual cortex (V1) utilizing multi-contact laminar electrodes (Figure 1; Plextrode U-Probe, Plexon Inc).
The methods included are recording device construction, identification of cortical layers, and identification of receptive fields of individual neurons. To identify cortical layers, we measure the evoked response potentials (ERPs) of the LFP time-series using full-field flashed stimuli. We then perform current-source density (CSD) analysis to identify the polarity inversion accompanied by the sink-source configuration at the base of layer 4 (the sink is inside layer 4, subsequently referred to as granular layer9-12). Current-source density is useful because it provides an index of the location, direction, and density of transmembrane current flow, allowing us to accurately position electrodes to record from all layers in a single penetration6, 11, 12.

A fundamental issue in our understanding of cortical circuitry is how networks in different cortical layers encode sensory information. Here we describe electrophysiological techniques utilizing multi-contact laminar electrodes to record single-units and local field potentials and present analyses to identify cortical layers.

Cortical layers are ubiquitous structures throughout neocortex1-4 that consist of highly recurrent local networks. In recent years, significant progress ...

Closed-loop Neuro-robotic Experiments to Test Computational Properties of Neuronal Networks

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation of a given stimulus might be dependent on context and history. If this is actually the case, bi-directional interactions between the brain (or if need be a reduced model of it) and sensory-motor system can shed a light on how encoding and decoding of information is performed. Here an experimental system is introduced and described in which the activity of a neuronal element (i.e., a network of neurons extracted from embryonic mammalian hippocampi) is given context and used to control the movement of an artificial agent, while environmental information is fed back to the culture as a sequence of electrical stimuli. This architecture allows a quick selection of diverse encoding, decoding, and learning algorithms to test different hypotheses on the computational properties of neuronal networks.

In this paper, an experimental framework to perform closed-loop experiments is presented, in which information processing (i.e., coding and decoding) and learning of neuronal assemblies are studied during the continuous interaction with a robotic body.

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation ...

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, &#34;kiss-and-run&#34; fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore &#34;openness&#34;, the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic applications. This protocol describes a fabrication technique of a modified cylindrical nerve cuff electrode to meet these criteria. Minimum computer-aided design and manufacturing (CAD and CAM) skills are necessary to consistently produce cuffs with high precision (contact placement 0.51 &#177; 0.04 mm) and various cuff sizes. The precision in spatially distributing the contacts and the ability to retain a predefined geometry accomplished with this design are two criteria essential to optimize the cuff&#39;s interface for selective recording and stimulation. The presented design also maximizes the flexibility in the longitudinal direction while maintaining sufficient rigidity in the transverse direction to reshape the nerve by using materials with different elasticities. The expansion of the cuff&#39;s cross sectional area as a result of increasing the pressure inside the cuff was observed to be 25% at 67 mm Hg. This test demonstrates the flexibility of the cuff and its response to nerve swelling post-implant. The stability of the contacts&#39; interface and recording quality were also examined with contacts&#39; impedance and signal-to-noise ratio metrics from a chronically implanted cuff (7.5 months), and observed to be 2.55 &#177; 0.25 k&#8486; and 5.10 &#177; 0.81 dB respectively.

This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic ...

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding diseases, and future therapies hinge on fundamental understandings about how different retinal cells function normally. Gaining such information with biochemical methods has proven difficult because contributions of particular cell types are diminished in the retinal cell milieu. Live retinal imaging can provide a view of numerous biological processes on a subcellular level, thanks to a growing number of genetically encoded fluorescent biosensors. However, this technique has thus far been limited to tadpoles and zebrafish larvae, the outermost retinal layers of isolated retinas, or lower resolution imaging of retinas in live animals. Here we present a method for generating live ex vivo retinal slices from adult zebrafish for live imaging via confocal microscopy. This preparation yields transverse slices with all retinal layers and most cell types visible for performing confocal imaging experiments using perfusion. Transgenic zebrafish expressing fluorescent proteins or biosensors in specific retinal cell types or organelles are used to extract single-cell information from an intact retina. Additionally, retinal slices can be loaded with fluorescent indicator dyes, adding to the method&#39;s versatility. This protocol was developed for imaging Ca2+ within zebrafish cone photoreceptors, but with proper markers it could be adapted to measure Ca2+ or metabolites in M&#252;ller cells, bipolar and horizontal cells, microglia, amacrine cells, or retinal ganglion cells. The retinal pigment epithelium is removed from slices so this method is not suitable for studying that cell type. With practice, it is possible to generate serial slices from one animal for multiple experiments. This adaptable technique provides a powerful tool for answering many questions about retinal cell biology, Ca2+, and energy homeostasis.

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

Imaging retinal tissue can provide single-cell information that cannot be gathered from traditional biochemical methods. This protocol describes preparation of retinal slices from zebrafish for confocal imaging. Fluorescent genetically encoded sensors or indicator dyes allow visualization of numerous biological processes in distinct retinal cell types.

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding ...

Conducting Hyperscanning Experiments with Functional Near-Infrared Spectroscopy

Concurrent brain recordings of two or more interacting persons, an approach termed hyperscanning, are gaining increasing importance for our understanding of the neurobiological underpinnings of social interactions, and possibly interpersonal relationships. Functional near-infrared spectroscopy (fNIRS) is well suited for conducting hyperscanning experiments because it measures local hemodynamic effects with a high sampling rate and, importantly, it can be applied in natural settings, not requiring strict motion restrictions. In this article, we present a protocol for conducting fNIRS hyperscanning experiments with parent-child dyads and for analyzing brain-to-brain synchrony. Furthermore, we discuss critical issues and future directions, regarding the experimental design, spatial registration of the fNIRS channels, physiological influences and data analysis methods. The described protocol is not specific to parent-child dyads, but can be applied to a variety of different dyadic constellations, such as adult strangers, romantic partners or siblings. To conclude, fNIRS hyperscanning has the potential to yield new insights into the dynamics of the ongoing social interaction, which possibly go beyond what can be studied by examining the activities of individual brains.

The present protocol describes how to conduct fNIRS hyperscanning experiments and analyze brain-to-brain synchrony. Further, we discuss challenges and possible solutions.

Concurrent brain recordings of two or more interacting persons, an approach termed hyperscanning, are gaining increasing importance for our understanding ...

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 days a year. Participants consent to donate their brain and spinal cord. Most patients were followed by neurologists at the Cleveland Clinic Mellen Center for MS Treatment and Research. Their clinical courses and neurological disabilities are well-characterized. Soon after death, the body is transported to the MS Imaging Center, where the brain is scanned in situ by 3 T magnetic resonance imaging (MRI). The body is then transferred to the autopsy room, where the brain and spinal cord are removed. The brain is divided into two hemispheres. One hemisphere is immediately placed in a slicing box and alternate 1 cm-thick slices are either fixed in 4% paraformaldehyde for two days or rapidly frozen in dry ice and 2-methylbutane. The short-fixed brain slices are stored in a cryopreservation solution and used for histological analyses and immunocytochemical detection of sensitive antigens. Frozen slices are stored at -80 &#176;C and used for molecular, immunocytochemical, and in situ hybridization/RNA scope studies. The other hemisphere is placed in 4% paraformaldehyde for several months, placed in the slicing box, re-scanned in the 3 T magnetic resonance (MR) scanner and sliced into centimeter-thick slices. Postmortem in situ MR images (MRIs) are co-registered with 1 cm-thick brain slices to facilitate MRI-pathology correlations. All brain slices are photographed and brain white-matter lesions are identified. The spinal cord is cut into 2 cm segments. Alternate segments are fixed in 4% paraformaldehyde or rapidly frozen. The rapid procurement of postmortem MS tissues allows pathological and molecular analyses of MS brains and spinal cords and pathological correlations of brain MRI abnormalities. The quality of these rapidly-processed postmortem tissues (usually within 6 h of death) is of great value to MS research and has resulted in many high-impact discoveries.

Multiple sclerosis is an inflammatory demyelinating disease with no cure. Analysis of brain tissue provides important clues to understanding the pathogenesis of the disease. Here we discuss the methodology and downstream analysis of MS brain tissue collected through a unique rapid autopsy program in operation at the Cleveland Clinic.

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 ...

Using Neuron Spiking Activity to Trigger Closed-Loop Stimuli in Neurophysiological Experiments

Closed-loop neurophysiological systems use patterns of neuronal activity to trigger stimuli, which in turn affect brain activity. Such closed-loop systems are already found in clinical applications, and are important tools for basic brain research. A particularly interesting recent development is the integration of closed-loop approaches with optogenetics, such that specific patterns of neuronal activity can trigger optical stimulation of selected neuronal groups. However, setting up an electrophysiological system for closed-loop experiments can be difficult. Here, a ready-to-apply Matlab code is provided for triggering stimuli based on the activity of single or multiple neurons. This sample code can be easily modified based on individual needs. For instance, it shows how to trigger sound stimuli and how to change it to trigger an external device connected to a PC serial port. The presented protocol is designed to work with a popular neuronal recording system for animal studies (Neuralynx). The implementation of closed-loop stimulation is demonstrated in an awake rat.

This protocol demonstrates how to use an electrophysiological system for closed-loop stimulation triggered by neuronal activity patterns. Sample Matlab code that can be easily modified for different stimulation devices is also provided.

Closed-loop neurophysiological systems use patterns of neuronal activity to trigger stimuli, which in turn affect brain activity. Such closed-loop systems ...

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 &#176;C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

Herein, we display an efficient method for the purification of oligodendrocytes and production of oligodendrocyte-conditioned medium that can be used for co-culture experiments.

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...

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