This work is supported by the following grants: NIH/NIGMS T32GM008464 (K.K.G.), Columbia University Irving Medical Center Target of Opportunity Provost award to the Department of Anesthesiology (K.K.G.), Society of Pediatric Anesthesia Young Investigator Research Award (K.K.G.), and NIH/NINDS R01NS112706 (R.J.L.)

Institutional Animal Care and Use Committee of Columbia University Medical Center approval was obtained for all methods described. FXS (Fmr1 KO) (FVB.129P2-Pde6b+ Tyrc-ch Fmr1tm1Cgr/J) and control (FVB) (FVB.129P2-Pde6b+ Tyrc-ch/AntJ) mice used as the model systems for this study were commercially acquired (see the Table of Materials). Five to eleven animals were used in each experimental group. Postnatal day 10 (P10) mice were used to model for a time point in human infancy.
1. Mitochondrial isolation from mouse heart
<ol>
	<li>Prepare buffers for mitochondrial isolation and respiration experiments as described in Table 1. Store at 4 &#176;C if made in advance.
	<ol>
		<li>Prepare 15% density gradient medium: dilute commercial density gradient medium to 80% volume/volume (v/v) with density gradient diluent. Further dilute 80% density gradient medium 15% v/v by adding mitochondrial isolation buffer (MI)/bovine serum albumin (BSA).</li>
	</ol>
	</li>
	<li>Isolate mitochondria from fresh, not frozen tissue for these experiments and use on the day of preparation, typically within five hours26. Carry out all isolation steps on ice.
	<ol>
		<li>Decapitate the mouse and excise the heart. Immediately wash 1-2 times in a Petri dish with ice-cold MI/BSA. Cut off the atrium and mince the tissue.</li>
		<li>Transfer the minced heart tissue to a glass-glass homogenizer containing 1 mL of MI/BSA. Homogenize with a looser (A) pestle (10 strokes), then a tighter (B) pestle (10 strokes).</li>
		<li>Centrifuge the homogenate at 1,100 &#215; g for 2 min at 4 &#176;C to remove nuclear and cellular debris.</li>
		<li>Take the supernatant gently without touching any fluffy pellet, and carefully layer it on top of 700 &#956;L of 15% density gradient medium in a centrifuge tube. Centrifuge at 18,500 &#215; g for 15 min at 4 &#176;C.</li>
		<li>Resuspend the pellet in 1 mL of sucrose buffer (SB)/BSA and centrifuged at 10,000 &#215; g for 10 min at 4 &#176;C.</li>
		<li>Completely remove and discard the supernatant. Resuspend the pellet in SB/BSA to a final volume of 55 &#956;L.</li>
		<li>Quantify mitochondrial protein content using a standard assay such as the bicinchoninic acid (BCA) protein assay.</li>
	</ol>
	</li>
</ol>
2. Mitochondrial oxygen (O2) consumption and &#916;&#936;
<ol>
	<li>Oxygen electrode assembly and calibration
	<ol>
		<li>Set up and calibrate the oxygen electrode (see the Table of Materials) according to the manufacturer&#39;s instructions.
		<ol>
			<li>Place a drop of 50% potassium chloride (KCl) electrolyte solution on top of the dome of the electrode disc.</li>
			<li>Place a small piece ~2 cm2 cigarette paper spacer covered with a slightly larger piece of the provided polytetrafluoroethylene (PTFE) membrane over the electrolyte drop.</li>
			<li>Using the applicator tool provided, push the small electrode disc O-ring over the dome of the electrode. 
			NOTE: Ensure that there are no air bubbles and that the membrane is smooth.</li>
		</ol>
		</li>
		<li>Top up the reservoir well with the electrolyte solution.</li>
		<li>Place the larger O-ring in the recess around the electrode disc well.</li>
		<li>Install the disc into the electrode chamber and connect it to the control unit.</li>
		<li>Add 2 mL of air saturated deionized water to the reaction chamber and the PTFE-coated magnet to the chamber.</li>
		<li>Connect the chamber to the rear of the control unit.</li>
		<li>Set the temperature to 37 &#176;C and the stirring speed to 100.</li>
		<li>Allow 10 min for the system temperature to equilibrate before commencing calibration.</li>
		<li>Under the Calibration tab, select Liquid Phase Calibration to perform liquid phase calibration. Confirm that the temperature is set to 37 &#176;C , the stirring speed is 100, and the pressure is set to atmospheric pressure (101.32 kPa).</li>
		<li>Press OK and wait for the signal to plateau.</li>
		<li>When a plateau is reached, press OK.</li>
		<li>Establish zero O2 in the chamber by adding a small amount (~20 mg) of sodium dithionite.</li>
		<li>Press OK and wait for the signal to plateau.</li>
		<li>When a plateau is reached, press Save to accept the calibration.</li>
	</ol>
	</li>
	<li>TPP+-selective electrode assembly
	<ol>
		<li>Fill the TPP+- selective electrode tip with 10 mM TPP solution using the provided syringe and flexible needle, taking care to avoid air bubbles.</li>
		<li>Assemble the TPP+-selective electrode apparatus, including the reference electrode and electrode holder, (see Table of Materials) according to the manufacturer&#39;s instructions.</li>
		<li>Briefly, loosen the electrode holder cap and insert the internal reference electrode into the TPP+&#160;tip. Tighten the cap to secure the tip. Connect the provided cable to the electrode holder and the auxiliary port of the control box.</li>
		<li>Insert the TPP+-selective electrode and reference electrodes into the adapted plunger assembly for ion-selective electrodes. Connect the reference electrode to the reference port of the control box.</li>
		<li>Insert the TPP+-selective and reference electrodes into the adapted plunger assembly for ion-selective electrodes.</li>
	</ol>
	</li>
	<li>Reaction chamber preparation
	<ol>
		<li>Add the reaction mixture to the reaction chamber: 10 mM succinate (complex II substrate), 5 &#956;M rotenone (complex I inhibitor), 80 ng mL&#8722;1 nigericin (to collapse pH gradient across IMM), 2.5 &#956;g mL-1 oligomycin (to induce state 4 respiration), and respiration buffer (RB)/BSA reaction buffer to a final volume of 1 mL. Take care to avoid introducing air bubbles into the chamber.</li>
		<li>Close the chamber with the adapted plunger assembly with the TPP+-selective and the reference electrode in place. Select GO to start recording. Once the chamber is closed, introduce additional reagents directly into the reaction solution using separate microsyringes modified with plastic tubing to adjust the needle length.</li>
	</ol>
	</li>
	<li>TPP+ calibration
	<ol>
		<li>Once a stable voltage signal is obtained, calibrate the TPP+-selective electrode by adding 1 &#956;M increments of a 0.1 mM TPP solution to a final concentration of 3 &#956;M. Observe that there is a logarithmic decline in the TPP+ voltage signal with each addition. 
		NOTE: Calibrate the TPP+-selective electrode at the start of each experiment.</li>
	</ol>
	</li>
	<li>Data acquisition
	<ol>
		<li>Allow the O2 and TPP+ traces to stabilize, and add 100 &#956;g of freshly prepared cardiomyocyte mitochondria to the reaction chamber to a final concentration of 0.1 mg mL-1 via the reagent addition port in the plunger assembly. Observe the decrease in the O2 levels in the chamber as mitochondria become energized and consume O2 and the abrupt increase in the TPP+ voltage signal as the mitochondria generate a membrane potential and take up TPP+ from the solution.</li>
		<li>Add 2.5 &#956;g mL-1 oligomycin to induce state 4 respiration. 
		NOTE: O2 consumption rates will now represent the rate of proton leak respiration. Oligomycin can be added to the reaction chamber prior to TPP+ as an alternative.</li>
	</ol>
	</li>
	<li>Assessing the open probability of the mPTP
	<ol>
		<li>Observe that &#916;&#936; declines over time during leak respiration. Once the desired &#916;&#936; is reached, add 1 &#956;M CsA (mPTP inhibitor) to the reaction chamber to assess open probability of the mPTP at that specific &#916;&#936;.</li>
		<li>Measure the effect of CsA on O2 consumption and &#916;&#936; before and after CsA addition</li>
		<li>Determine the voltage dependence of mPTP opening by varying the &#916;&#936; at which CsA is added in successive experiments.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to disclose.

This paper describes a method to assess the open probability of the mPTP. Specifically, the voltage threshold for low-conductance mPTP opening was determined by assessing the effect of CsA inhibition on proton leak over a range of &#916;&#936;s. Using this technique, we could identify differences in voltage gating of the mPTP between FXS mice and FVB controls consistent with their differences in tissue-specific CoQ content. Critical to the success of this methodology is that mitochondria are freshly isolated prior to use...

The mPTP mediates the permeability transition (PT), whereby the IMM becomes abruptly permeable to small molecules and solutes1,2. This striking phenomenon is a distinct departure from the characteristic impermeability of the IMM, which is fundamental for establishing the electrochemical gradient necessary for oxidative phosphorylation3. PT, unlike other mitochondrial transport mechanisms, is a high-conductance, nonspecific, and nonselective process, allowing the passage of a range of molecules up to 1.5 kDa4,5. The mPTP is a voltage-gated channel within the IMM whose opening alters &#916;&#936;, ATP production, calcium homeostasis, reactive oxygen species (ROS) production, and cell viability4.
At the pathologic extreme, uncontrolled and prolonged high-conductance opening of mPTP leads to the collapse of the electrochemical gradient, matrix swelling, depletion of matrix pyridine nucleotides, outer membrane rupture, release of intermembrane proteins (including cytochrome c), and ultimately, cell death4,6. Such pathological mPTP opening has been implicated in cardiac ischemia-reperfusion injury, heart failure, traumatic brain injury, various neurodegenerative diseases, and diabetes1,7. However, low-conductance mPTP opening is physiological in nature and, in contrast to high-conductance opening, does not lead to profound depolarization or mitochondrial swelling4.
Low-conductance opening of the pore restricts permeability to ~300 Da, allows the passage of protons independent of ATP synthesis, and is a potential source of physiological proton leak5. Physiologic mPTP opening causes a controlled decline in &#916;&#936;, increases electron flux through the respiratory transport chain, and results in a short burst or flash of superoxide, contributing to ROS signaling8. Regulation of such transient mPTP opening is important for calcium homeostasis and normal cellular development and maturation4,9,10,11. Transient pore opening in developing neurons, for example, triggers differentiation, while the closure of the mPTP induces maturation in immature cardiomyocytes4,5.
Although the functional significance of the mPTP in health and disease is well established, its precise molecular identity remains debated. Progress on the molecular structure and function of the mPTP has been comprehensively reviewed elsewhere12. Briefly, currently, high- and low- conductance states of the mPTP have been hypothesized to be mediated by distinct entities12. The leading candidates are the F1/F0 ATP synthase (ATP synthase) and adenine nucleotide transporter (ANT) for high- and low-conductance modes, respectively12.
Despite the lack of consensus regarding the exact identity of the pore-forming component of the mPTP, certain key characteristics have been detailed. A well-established feature of the mPTP is that it is regulated by the electrochemical gradient such that depolarization of the IMM leads to pore opening13. Prior work has shown that the redox state of vicinal thiol groups alters the voltage gating of the mPTP, such that oxidation opens the pore at relatively higher &#916;&#936;s, and thiol group reduction results in closed mPTP probability14. However, the identity of the proteinaceous voltage sensor is unknown.
Various small molecules that modulate the open probability of the pore have been identified. For example, the mPTP can be stimulated to open with calcium, inorganic phosphate, fatty acids, and ROS and can be inhibited by adenine nucleotides (particularly ADP), magnesium, protons, and CsA5,12. The mechanisms of action of some of these regulators have been elucidated. Mitochondrial calcium triggers mPTP opening at least in part by binding to the &#946;-subunit of the ATP synthase15. ROS can activate the mPTP by decreasing its affinity for ADP and enhancing its affinity for cyclophilin D (CypD), the best-studied proteinaceous mPTP activator16. The mechanism of activation of the mPTP by inorganic phosphate and fatty acids is less clear. As for endogenous inhibitors, ADP is thought to inhibit the mPTP by binding at the ANT or ATP synthase, while magnesium exerts its inhibitory effect by displacing calcium from its binding site15,17,18,19.
Low pH inhibits mPTP opening by protonating histidine 112 of the regulatory oligomycin sensitivity-conferring protein (OSCP) subunit of the ATP synthase12,20,21. The prototypical pharmacologic inhibitor of the mPTP, CsA, acts by binding CypD and preventing its association with OSCP22,23. Previous work has also shown that a variety of CoQ analogs interact with the mPTP, inhibiting it or activating it24. In recent work, we found evidence of a pathologically open mPTP, excessive proton leak, and inefficient oxidative phosphorylation due to a CoQ deficiency in forebrain mitochondria of newborn FXS mouse pups25.
Closure of the pore with exogenous CoQ blocked the pathologic proton leak and induced morphologic maturity of dendritic spines25. Interestingly, in the same animals, FXS cardiomyocytes had excessive CoQ levels and closed mPTP probability compared to wildtype controls26. Although the cause of these tissue-specific differences in CoQ levels is unknown, the findings underscore the concept that endogenous CoQ is likely a key regulator of the mPTP. However, there is a major gap in our knowledge because the mechanism of CoQ-mediated inhibition of the mPTP remains unknown.
Regulation of the mPTP is a critical determinant of cell signaling and survival4. Thus, detecting mPTP opening within mitochondria is key when considering specific pathophysiological mechanisms. Typically, the threshold for high-conductance pore opening is determined using calcium to trigger the permeability transition. Such calcium loading leads to the collapse of the membrane potential, rapid uncoupling of oxidative phosphorylation, and mitochondrial swelling27,28. We sought to develop a method to detect low-conductance mPTP opening in situ, without inducing it per se.
The approach exploits the role of the mPTP as a proton leak channel. To do so, Clark-Type and TPP+ ion-selective electrodes were employed to simultaneously measure oxygen consumption and membrane potential, respectively, in isolated mitochondria during leak respiration29. The threshold for mPTP opening was determined by the onset of CsA-mediated inhibition of proton leak at specific membrane potentials. Using this approach, differences in voltage gating of the mPTP in the context of CoQ excess were precisely defined.

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			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
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			<td>Adapted plunger assembly for pH or ion-selective electrodes for use with OXYT1</td>
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			<td>BD Intramedic PE Tubing, PE 50, 0.023 in. 10 ft.</td>
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			<td>to modify the length of the hamilton synringe as needed</td>
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			<td>Bovine Serum Albumin (BSA). Fatty acid free</td>
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			<td>A7030-10G</td>
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			<td>Dri-Ref Reference Electrode, 2 mm</td>
			<td>World Precision Inst. LLC</td>
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			<td>Electrode Holder for KWIK-Tips</td>
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			<td>Ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#8242;,N&#8242;-tetraacetic acid&#160; (EGTA)</td>
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			<td>FVB.129P2-Pde6b+ Tyrc-ch Fmr1tm1Cgr/J</td>
			<td>Jackson Laboratory, Bar Harbor, ME</td>
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			<td>FXS mice, Fmr1 KO&#160;</td>
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			<td>FVB.129P2-Pde6b+ Tyrc-ch/AntJ</td>
			<td>Jackson Laboratory, Bar Harbor, ME</td>
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			<td>Hamilton 80366 Standard Syringes, 10 uL, Cemented-Needle, 6/pk</td>
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			<td>EW-07938-30</td>
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			<td>1374248</td>
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			<td>P1,P5-diadenosine-5&#8242; pentaphosphate pentasodium (AP5A)</td>
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			<td>D4022-10MG</td>
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			<td>P1644</td>
			<td>medium for density gradient separation</td>
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			<td>P3911</td>
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			<td>Potassium dihydrogen phosphate (KH2PO4)</td>
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			<td>Sigma</td>
			<td>S0389</td>
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			<td>TPP+ Electrode Tips (3)</td>
			<td>World Precision Inst. LLC</td>
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assessment of open probability of the mitochondrial permeability transition pore in the setting of coenzyme q excess

The mitochondrial permeability transition pore (mPTP) is a voltage-gated, nonselective, inner mitochondrial membrane (IMM) mega-channel important in health and disease. The mPTP mediates leakage of protons across the IMM during low-conductance opening and is specifically inhibited by cyclosporine A (CsA). Coenzyme Q (CoQ) is a regulator of the mPTP, and tissue-specific differences have been found in CoQ content and open probability of the mPTP in forebrain and heart mitochondria in a newborn mouse model of fragile X syndrome (FXS, Fmr1 knockout). We developed a technique to determine the voltage threshold for mPTP opening in this mutant strain, exploiting the role of the mPTP as a proton leak channel.
To do so, oxygen consumption and membrane potential (&#916;&#936;) were simultaneously measured in isolated mitochondria using polarography and a tetraphenylphosphonium (TPP+) ion-selective electrode during leak respiration. The threshold for mPTP opening was determined by the onset of CsA-mediated inhibition of proton leak at specific membrane potentials. Using this approach, differences in voltage gating of the mPTP were precisely defined in the context of CoQ excess. This novel technique will permit future investigation for enhancing the understanding of physiological and pathological regulation of low-conductance opening of the mPTP.

Typical O2 consumption and &#916;&#936; curves generated in these experiments are shown (Figure 1A,B). The logarithmic decline in the voltage signal with TPP+ calibration is shown at the start of each experiment. The absence of this logarithmic pattern may suggest a problem with the TPP+ selective electrode. Mitochondria typically generate &#916;&#936; immediately upon addition to respiratory buffer. &#916;&#936; can be interpreted from chang...

Watch this Scientific Journal Video about Assessment of Open Probability of the Mitochondrial Permeability Transition Pore in the Setting of Coenzyme Q Excess at JoVE.com

Assessment of Open Probability of the Mitochondrial Permeability Transition Pore in the Setting of Coenzyme Q Excess

This method exploits the contribution of the mitochondrial permeability transition pore to low-conductance proton leak to determine the voltage threshold for pore opening in neonatal fragile X syndrome mice with increased cardiomyocyte mitochondrial coenzyme Q content compared to wildtype control.

The mitochondrial permeability transition pore (mPTP) is a voltage-gated, nonselective, inner mitochondrial membrane (IMM) mega-channel important in ...

assessment-open-probability-mitochondrial-permeability-transition

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

2.2K Views.  Columbia University Medical Center. This method exploits the contribution of the mitochondrial permeability transition pore to low-conductance proton leak to determine the voltage threshold for pore opening in neonatal fragile X syndrome mice with increased cardiomyocyte mitochondrial coenzyme Q content compared to wildtype control. 

Video: Assessment of Open Probability of the Mitochondrial Permeability Transition Pore in the Setting of Coenzyme Q Excess

The Structure of Skilled Forelimb Reaching in the Rat: A Movement Rating Scale

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and primates. The video describes a test situation that allows filming of repeated acts of reaching for food by the rat that has been mildly food deprived. A rat is trained to reach through a slot in a holding box for food pellet that it grasps and then places in its mouth for eating. Reaching is accomplished in the main by proximally driven movements of the limb but distal limb movements are used for pronating the paw, grasping the food, and releasing the food into the mouth. Each reach is divided into at least 10 movements of the forelimb and the reaching act is facilitated by postural adjustments. Each of the movements is described and examples of the movements are given from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation. Because the reaching act for the rat is very similar to that displayed by humans and nonhuman primates, the scale can be used for comparative purposes. from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation.

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the University of Lethbridge Animal Care Committee in accordance with the regulations of the Canadian Council on Animal Care.

The skilled reaching scale divides the movement by a forelimb in a reach for food act into composite elements each of which are evaluated with a three-point scale. The rating scale is described for a normal rat and can be applied toward evaluating neurological motor disorders.

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and ...

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Assessment of Open Probability of the Mitochondrial Permeability Transition Pore in the Setting of Coenzyme Q Excess

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The Structure of Skilled Forelimb Reaching in the Rat: A Movement Rating Scale

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

Measuring the Osmotic Water Permeability Coefficient (P_f) of Spherical Cells: Isolated Plant Protoplasts as an Example

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy