Name: dissection and observation of honey bee dorsal vessel for studies of cardiac function
Uploaded: 2016-12-12T13:00:00
Description: Watch this Scientific Journal Video about Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function at JoVE.com

The authors thank Drs. Jeffrey Bloomquist and Daniel Swale for their technical comments and suggestions. This project was partially funded by the Department of Entomology and the College of Agriculture and Life Sciences at Virginia Tech.

1. Collection and Preparation of Test Subjects
<ol>
	<li>Collect the appropriate number of bees from the colony. 
	NOTE: The number needed depends upon not only the size and scope of the experiment, but also the skill of the investigator. For example, if there are 2 treatment groups with a desired sample size of 10 bees per group, a reasonably skilled investigator might collect at least 30 bees to account for unsuccessful dissections and end up with 20 useful videos to score.</li>
	<li>Minimize the amount of time ...

<ol>
	<li>Klowden, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulatory+Systems.">Circulatory Systems.</a> Physiological Systems in Insects, 3rd Edition. , 365-413 (2013).</li><li>League, G. P., Onuh, O. C., Hillyer, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+structural+and+functional+analysis+of+the+larval+and+adult+dorsal+vessel+and+its+role+in+hemolymph+circulation+in+the+mosquito+Anopheles+gambiae.">Comparative structural and functional analysis of the larval and adult dorsal vessel and its role in hemolymph circulation in the mosquito Anopheles gambiae.</a> J Exp Biol. 218 (Pt 3), 370-380 (2015).</li><li>Wasserthal, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillating+Hemolymph+Circulation+in+the+Butterfly+Papilio-Machaon+L+Revealed+by+Contact+Thermography+and+Photocell+Measurements.">Oscillating Hemolymph Circulation in the Butterfly Papilio-Machaon L Revealed by Contact Thermography and Photocell Measurements.</a> J Comp Physiol. 139 (2), 145-163 (1980).</li><li>Wasserthal, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+circulation+and+tracheal+ventilation+in+holometabolous+insects.">Interaction of circulation and tracheal ventilation in holometabolous insects.</a> Adv Insect Physiol. 26, 297-351 (1996).</li><li>Schwab, E. R., Chilson, R. A., Eddleman, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heartbeat+Rate+Modulation+Mediated+by+the+Ventral+Nerve+Cord+in+the+Honey-Bee,+Apis-Mellifera.">Heartbeat Rate Modulation Mediated by the Ventral Nerve Cord in the Honey-Bee, Apis-Mellifera.</a> J Comp Physiol B-Biochem Syst Environ Physiol. 161 (6), 602-610 (1991).</li><li>Kaiser, W., Weber, T., Otto, D., Miroschnikow, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+supply+of+the+heart+and+electrocardiogram+potentials+with+reversed+polarity+in+sleeping+and+resting+honey+bees.">Oxygen supply of the heart and electrocardiogram potentials with reversed polarity in sleeping and resting honey bees.</a> Apidologie. 45 (1), 73-87 (2014).</li><li>Papaefthimiou, C., Theophilidis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Octopamine--a+single+modulator+with+double+action+on+the+heart+of+two+insect+species+(Apis+mellifera+macedonica+and+Bactrocera+oleae):+Acceleration+vs.+inhibition.">Octopamine--a single modulator with double action on the heart of two insect species (Apis mellifera macedonica and Bactrocera oleae): Acceleration vs. inhibition.</a> J Insect Physiol. 57 (2), 316-325 (2011).</li><li>Castro, C. A., Hogan, J. B., Benson, K. A., Shehata, C. W., Landauer, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral-Effects+of+Vehicles+-+Dmso,+Ethanol,+Tween-20,+Tween-80,+and+Emulphor-620.">Behavioral-Effects of Vehicles - Dmso, Ethanol, Tween-20, Tween-80, and Emulphor-620.</a> Pharmacol Biochem Behav. 50 (4), 521-526 (1995).</li><li>Papaefthimiou, C., Papachristoforou, A., Theophilidis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biphasic+responses+of+the+honeybee+heart+to+nanomolar+concentrations+of+amitraz.">Biphasic responses of the honeybee heart to nanomolar concentrations of amitraz.</a> Pestic Biochem Phys. 107 (1), 132-137 (2013).</li><li>Roeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Octopamine+in+invertebrates.">Octopamine in invertebrates.</a> Prog Neurobiol. 59 (5), 533-561 (1999).</li><li>Johnson, E., Ringo, J., Dowse, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Drosophila+heartbeat+by+neurotransmitters.">Modulation of Drosophila heartbeat by neurotransmitters.</a> J Comp Physiol B. 167 (2), 89-97 (1997).</li></ol>

The protocol presented here provides a simple and effective approach to testing pharmacological compounds for their effects on honey bee heart rate. As observed in prior experiments that either transect the ventral nerve cord of a living insect5 or dissect out the ventral nerve cord when exposing the dorsal vessel7, the loss of central nervous system regulation results in a stable, low frequency heartbeat. The low frequency of beats allows the investigator to visually assess heart rate without havin...

The overall goal of this methodology is to allow the investigator to quickly and easily observe and quantify the effect that a pharmacological agent has on the heart rate of honey bees. Bees, like other insects, have an open circulatory system that disseminates hemolymph, the insect equivalent of blood, throughout the body cavity, known as the hemocoel. The circulation of hemolymph is essential for the transport of nutrients, immune factors, waste products, as well neurohormones and other signaling molecules1. Circulation is facilitated by the dorsal vessel, which extends along the dorsal midline of the insect, as well as accessory pulsatile organs. The dorsal vessel is divided into two functionally distinct sections, designated the heart in the abdomen and the aorta in the thorax and head. Propagated contractions in the heart pump hemolymph towards the thorax and head, while accessory pulsatile organs ensure hemolymph flow to the extremities.
Insect cardiac function can be observed using a variety of methods, depending upon the size, physiology, or life stage of the insect. A common approach for observing heart rate in larvae or smaller insects is the use of intravital imaging2. This method is less useful in adult bees, however, as it can be difficult to clearly view the dorsal vessel through the abdominal wall. An established approach for recording heart rate in a variety of insects, including bees, is the use of contact thermography, which utilizes thermistors applied to the exterior of the insect to detect cardiac pulsations3,4. Heart rate in adult bees has also been recorded using an electrophysiological technique to measure an electrical impedance signal4,5. This technique requires the insertion of electrodes into the animal next to the heart and the use of an impedance converter to record heartbeats4. Similarly, electrocardiograms have been used to detect electrical signals produced by the heart and combined with video recording of the bee to observe changes in cardiac activity6. A distinct advantage to these approaches is that heart rate is assessed in an intact, living bee, rather than in a dissected specimen, which helps to ensure the availability of the full range of physiological responses in the subject. The challenges of these approaches include accounting for immobilization or anesthetization of the subject, the need to limit outside variables and stimuli that might alter heart rate, as well as determining an appropriate delivery method when testing pharmacological agents.
Another approach that has been used for studying bee cardiac activity is to partially dissect the insect in order to expose the heart, then measure dorsal vessel contractions using a force displacement transducer7. In this protocol, the heart is continually bathed with running physiological saline and test compounds can be dissolved in this solution for application to the subject7. A significant difference between this method and those previously described is that the ventral nerve cord is removed, eliminating the role that the central nervous system has been shown to play in modulating heart rate5. The result is that the baseline heartbeat, which is usually quite erratic, stabilizes at a much lower frequency and amplitude than is typically observed in a living insect5,7. What all of these methods have in common is that they require highly specialized and often expensive equipment, in addition to a certain level of expertise, in order to be conducted. Perhaps the greatest disadvantage is that none of these approaches are particularly well suited to experiments that involve testing a large number of subjects, such as screening a library of potentially cardiomodulatory compounds.
The greatest strength of the approach described here is its simplicity. The protocol is relatively easy to master, the setup requires little space, and only a minimal financial input is necessary. The method requires little more than some bees, a few surgical instruments, an isotonic solution, and either a digital microscope or a traditional microscope with a digital camera. Bees are dissected to visualize the dorsal vessel and digital videos are used to record heart rate before and after treatment with pharmacological agents. Although video recording is not actually necessary to observe changes in heart rate, it will greatly increase throughput (i.e., the number of subjects that can be processed in a given amount of time). The investigator can maximize efficiency by recording a large number of videos at once and then later scoring these videos at a more convenient time. Another advantage of this approach is that videos allow the investigator to start over, should the scoring process be interrupted, and make it easier for the viewer to be blinded to the treatment in order to reduce bias.

<table border="0" cellpadding="0" cellspacing="0" width="834">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:200px;">Dino-Lite Edge digital USB microscope</td>
			<td style="width:111px;">Dino-Lite</td>
			<td style="width:119px;">AM4815ZT</td>
			<td style="width:405px;">Any digital microscope or similar setup will suffice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Microscope stand</td>
			<td style="width:111px;">Dino-Lite</td>
			<td style="width:119px;">RK-10</td>
			<td>Any stand appropriate for the digital microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Laptop or PC</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>Necessary for digital microscope</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:200px;">Microdissection scissors (Vannas, 8cm, Straight, 5mm Blades)</td>
			<td style="width:111px;">World Precision Instruments</td>
			<td style="width:119px;">14003</td>
			<td>Any similar scissors suitable for microdissection will suffice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:200px;">Microdissecting Forceps, 10.2cm, Angled (2 pair)</td>
			<td style="width:111px;">World Precision Instruments</td>
			<td style="width:119px;">504482</td>
			<td>Any similar forceps suitable for microdissection will suffice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:200px;">Ringers solution 1/4 strength tablets&#160;</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">96724-100TAB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Dissecting tray</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>Any surface suitable for microdissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Single channel 10 &#181;l pipette</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>Any device capable of accurately delivering 10 &#181;l volume</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Pipette tips</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:200px;">Small beaker or container of water</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>Used to rinse instruments between subjects</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Hand tally counter</td>
			<td style="width:111px;">Office Depot</td>
			<td style="width:119px;">295033</td>
			<td>Any similar product will suffice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Timer</td>
			<td style="width:111px;">Office Depot</td>
			<td style="width:119px;">644219</td>
			<td>Any similar product will suffice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:200px;">Deionized water</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>Preparation of Ringers solution and rinsing instruments</td>
		</tr>
	</tbody>
</table>

dissection and observation of honey bee dorsal vessel for studies of cardiac function

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination services, as well as an important model social insect used to study memory and learning, aging, and more. Here we describe a detailed protocol for the dissection of the dorsal abdominal wall of a bee in order to visualize its dorsal vessel, which serves the role of the heart in the insect. A successful dissection will expose a functional heart that, under the proper conditions, can maintain a steady heartbeat for an extended period of time. This allows the investigator to manipulate heart rate through the application of cardiomodulatory compounds to the dorsal vessel. By using either a digital microscope or a microscope equipped with a digital camera, the investigator can make video recordings of the dorsal vessel before and after treatment with test compounds. The videos can then be scored at a time convenient to the user in order to determine changes in heart rate, as well as changes in the pattern of heartbeats, following treatment. The advantages of this protocol are that it is relatively inexpensive to set up, easy to learn, requires little space or equipment, and takes very little time to conduct.

Since many of the pharmacologically active compounds that might be tested using this protocol are not soluble in water, it is necessary to have a reliable solvent that will allow test compounds to be delivered via the isotonic solution used to bathe the dorsal vessel. Dimethyl sulfoxide (DMSO) is a solvent that is commonly used as a vehicle for delivering experimental drugs and other compounds in animals8, and it has been used successfully for this purpose in studies examining ...

Watch this Scientific Journal Video about Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function at JoVE.com

Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function

The abdominal dorsal vessel of the honey bee and other insects serves as the functional equivalent of the mammalian heart and plays an important role in nutrient transport, waste removal, immune function, and more. Here we describe a protocol for the visualization and pharmacological manipulation of bee heart rate.

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination ...

The overall goal of this protocol is to observe changes in bee heart rate, resulting from the direct application of cardiomodulatory agents to the dorsal vessel. This method can answer key questions about insect physiology, especially those relating to pollinator health. The main advantage of this technique is its simplicity which makes it cost effective and easy to perform.Collect the appropriate number of bees from the colony. A prudent measure would be to include a 50 percent surplus to account for failed dissections, which even skilled investigators must plan for. Provide the bees with a source of water and food while housed in the lab.For housing durations that are less than six hours, at a minimum provide access to a 50 percent sucrose solution. For longer periods, also provide access to honey. House the bees overnight at approximately 32 degrees celsius with 60 to 80 percent relative humidity.Freshly collected bees are easier to dissect, so proceed with dissecting as soon as possible. Prior to the dissections, anesthetize the bees just enough to reduce their movement by briefly exposing them to carbon dioxide. The depth of the anesthesia can make the dissection more difficult, so use as little gas as possible.The bees must be alive at the time of dissection. To begin, use forceps or micro-dissection scissors to remove the legs and wings. After each manipulation, rinse the tools in distilled water.Next, restrain the bee with forceps and use the scissors to cut laterally along the dorsal abdominal wall, between the first and second tergites. Then, lightly grip the posterior edge of the second tergite with the forceps and carefully cut longitudinally along each side of the bee from the initial incision to the stinger. Do not puncture the digestive tract.Next, use fine forceps to carefully separate the dorsal abdominal wall from the rest of the abdomen. Then, gently remove the stinger and carefully remove any portion of the digestive tract that remains attached to the dorsal abdominal wall. Don't release the gut's contents, as they can obscure visualization of the dorsal vessel.Now remove the final turgite in order to improve visualization of the dorsal vessel. The next step is to adjust the stage to view the dorsal vessel and trim away any excess abdominal wall that impedes its view. The shape of the dorsal abdominal wall should resemble a shallow cup or bowl when properly situated.Cover the dorsal vessel with 10 microliters of the recommended isotonic solution to maintain its phsyiological conditions, and remove any remaining membrane that impedes visualization of the dorsal vessel. Addition of the isotonic solution can cause loose membrane to float and obscure the view of the dorsal vessel. These membrane pieces can be removed with fine forceps, but use caution to avoid damaging the dorsal vessel.Initially, there may appear to be no heartbeat but in a few minutes in isotonic solution, the heart will usually resume beating. If kept bathed in solution, the heart can continue beating for hours. Allow the dorsal vessel to sit undisturbed until a stable, continuous heartbeat is achieved.This usually occurs within five minutes. Measure the baseline heart rate using a hand tally counter, or for more accuracy, make a video recording of the experiment and take the measurements later. Record at least one minute of stable heartbeats prior to the first treatment.Now, prepare to apply the potential cardiomodulators, which are ideally dissolved in the same isotonic solution as the bath. For this reason, the recommended solution contains some DMSO, which allows most compounds to be solubilized. After adding a test compound, wait at least one minute before measuring the change in heart rate.To get an accurate measure of the heart rate, at least a minute of data is needed, and several minutes of data are usually required. A variety of isotonic solutions containing a range of DMSO concentrations were tested for any effect on heart rate. Five percent and 10 percent DMSO significantly decreased the heart rate, whereas a one percent DMSO isotonic solution did not.So the one percent solution was used for experiments. Octopamine, a well characterized demodulator in invertebrates, was tested against the vehicle. Higher concentrations of octopamine significantly increased the heart rate, thus demonstrating the utility of the assay.After watching this video, you should have a good understanding of how to perform a dissection to visualize the dorsal vessel as well as how to test the effects of potential cardiomodulators on bee heart rate. Once this technique has been mastered, a dissection can be completed in a matter of minutes if performed properly.

dissection-observation-honey-bee-dorsal-vessel-for-studies-cardiac

Research

JoVE Journal

Biology

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to measure odor evoked activity in the antennal lobe1. From then on, it has been continuously refined and applied to different neuropiles in the bee brain. Here, we describe the preparation currently used in the lab to measure activity in mushroom body neurons using a dextran coupled calcium-sensitive dye (Fura-2). We retrogradely stain mushroom body neurons by injecting dye into their axons or soma region. We focus on reducing the invasiveness, to achieve a preparation in which it is still possible to train the bee using PER conditioning. We are able to monitor and quantify the behavioral response by recording electro-myograms from the muscle which controls the PER (M17)2.
After the physiological experiment the imaged structures are investigated in greater detail using confocal scanning microscopy to address the identity of the neurons.

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

Bees can be conditioned in an appetitive olfactory learning paradigm (PER-conditioning). Using odors as stimuli, we established a method in which behavior is recorded while simultaneously Calcium Imaging is used to measure odor evoked activity in mushroom body neurons in vivo.

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best established model to study how plasticity in lifespan and aging is explained by social factors.
The worker caste of honey bees includes nurse bees, which tend the brood, and forager bees, which collect nectar and pollen. Previous work has shown that brain functions and flight performance senesce more rapidly in foragers than in nurses. However, brain functions can recover, when foragers revert back to nursing tasks. Such patterns of accelerated and reversed functional senescence are linked to changed metabolic resource levels, to alterations in protein abundance and to immune function. Vitellogenin, a yolk protein with adapted functions in hormonal control and cellular defense, may serve as a major regulatory element in a network that controls the different aging dynamics in workers.
Here we describe how the emergence of nurses and foragers can be monitored, and manipulated, including the reversal from typically short-lived foragers into longer-lived nurses. Our representative results show how individuals with similar chronological age differentiate into foragers and nurse bees under experimental conditions. We exemplify how behavioral reversal from foragers back to nurses can be validated. Last, we show how different cellular senescence can be assessed by measuring the accumulation of lipofuscin, a universal biomarker of senescence.
For studying mechanisms that may link social influences and aging plasticity, this protocol provides a standardized tool set to acquire relevant sample material, and to improve data comparability among future studies.

In honey bee workers, aging depends on social behaviors rather than on chronological age. Here we show how worker-types with very different aging patterns can be obtained and analyzed for cellular senescence.

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees collect and convert to beebread by placing colonies in a specially constructed enclosed flight area. Methods were developed to analyze the protein and amino acid composition of the pollen and beebread. We also describe how consumption of the beebread was measured and methods used to determine adult worker bee hemolymph protein titers after feeding on beebread for 4, 7 and 11 days after emergence. Methods were applied to determine if genotype affects the conversion of pollen to beebread and the rate that bees consume and acquire protein from it. Two subspecies (European and Africanized honey bees; EHB and AHB respectively) were provided with the same pollen source. Based on the developed methods, beebread made by both subspecies had lower protein concentrations and pH values than the pollen. In general, amino acid concentrations in beebread made by either EHB or AHB were similar and occurred at higher levels in beebread than in pollen. Both AHB and EHB consumed significantly more of the beebread made by AHB than by EHB. Though EHB and AHB consumed similar amounts of each type of beebread, hemolymph protein concentrations in AHB were higher than in EHB. Differences in protein acquisition between AHB and EHB might reflect environmental adaptations related to the geographic region where each subspecies evolved. These differences could contribute to the successful establishment of AHB populations in the New World because of the effects on brood rearing and colony growth.

Here are methods to quantify nutrients in pollen before and after its conversion to beebread by two subspecies of honeybees. We describe techniques to measure beebread consumption and resulting protein titers in both subspecies.

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

Monitoring Colony-level Effects of Sublethal Pesticide Exposure on Honey Bees

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment methods. Here we describe methods to measure the quantities of adult bees, brood, and food resources by weighing hives and hive parts, by photographing frames, and by installing hives on scales and with internal sensors. Data from these periodic evaluations are then combined with running average and daily detrended data on hive weight and internal hive temperature. The resulting datasets have been used to detect colony-level effects of imidacloprid applied in a sugar syrup as low as 5 parts per billion. The methods are objective, require little training, and provide permanent records in the form of sensor output and photographs.

Sublethal doses of pesticides may affect colonies in ways that are difficult to detect using only periodic or visual methods. Methods to measure total adult bee mass, brood, and food resources by weighing hives and hive parts, photographing frames, and installing sensors, are provided. Data analysis is also addressed.

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...