This work was supported by the National Science Foundation under grant #IOS-1025627 and by the National Institutes of Health under grant #1R21NS078582-01 to J.A.W.

This protocol uses two setups (Figure 1) to acquire four different readouts collected from flies subjected to a bacterial infection. These outputs include 1) sleep/wake behavior; 2) survival outcome; 3) bacterial load in the fly; and 4) real-time measurement of NF&kappa;B reporter activity in vivo. In combination with the genetic tools that are available in Drosophila, these measurements provide mechanistic insight into the molecular link between immune function and behavior.
<p cla...

<ol>
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No conflicts of interest declared.

This protocol outlines an approach to investigate how behavior, particularly sleep, is linked to immune response parameters. These parameters include bacterial load, survival outcome, and NF&kappa;B activity as measured by a luciferase reporter in vivo. Together these parameters provide information about how well a fly can fight an infection. Bacterial load and survival outcome are immune response parameters that involve a straightforward measurement in Drosophila. RelE20 muta...

<table border="1">
 <tbody>
 <tr>
 <td>Material Name </td>
 <td>Company </td>
 <td>Catalogue number </td>
 <td>Comments </td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>Incubators</td>
 <td>Percival Scientific, Inc.</td>
 <td>I30BLLC8 
 I36VLC8</td>
 <td>Any incubator capable of running programmed light/temperature schedules is appropriate.</td>
 </tr>
 <tr>
 <td>Drosophila Activitiy Monitors</td>
 <td>Trikinetics Inc., Waltham, MA</td>
 <td>DAM2</td>
 <td>As described elsewhere6, this system requires a computer interface, software, and other accessories.</td>
 </tr>
 <tr>
 <td>Pyrex Glass Tubes</td>
 <td>Trikinetics Inc., Waltham, MA</td>
 <td>PGT-5x65</td>
 <td></td>
 </tr>
 <tr>
 <td>Microplate scintillation and luminescence counter</td>
 <td>Perkin Elmer</td>
 <td>TopCount NXT 
 12 detector </td>
 <td>Any microplate reader capable of detecting luminescence can be used for this type of reporter assay. TopCount contains multiple detectors and an automated stacker; it is capable of being programmed to read continuously from multiple plates. </td>
 </tr>
 <tr>
 <td>FluorChem 8900</td>
 <td>Alpha Innotech</td>
 <td></td>
 <td>Imaging of bacterial cultures is optional; any digital imaging system with visual light capability is sufficient.</td>
 </tr>
 <tr>
 <td>Micropipette Puller</td>
 <td>Tritech Research, Inc.</td>
 <td>Narishige PC-10</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Supplies </td>
 </tr>
 <tr>
 <td>Borosilicate Glass Capillaries</td>
 <td>World Precision Instrument Inc.</td>
 <td>1B100F-4</td>
 <td></td>
 </tr>
 <tr>
 <td>3 ml Syringe</td>
 <td>Fisher Scientific</td>
 <td>BD 305482</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe Needles</td>
 <td>Fisher Scientific</td>
 <td>BD 305196</td>
 <td>18 G - cut off the tip of the needle to prevent damage to the tubing. </td>
 </tr>
 <tr>
 <td>Silicone Tubing, i.d. (0.030") o.d. (0.065") Wall Thickness (0.018")</td>
 <td>VWR</td>
 <td>60985-706</td>
 <td>Used for attaching glass capillary needles to a syringe</td>
 </tr>
 <tr>
 <td>3 Way Stopcock</td>
 <td>American Pharmaseal Company</td>
 <td>K75</td>
 <td></td>
 </tr>
 <tr>
 <td>Kontes Pellet Pestle Cordless Motor</td>
 <td>Fisher Scientific</td>
 <td>K749540-0000</td>
 <td></td>
 </tr>
 <tr>
 <td>Kontes Pellet Pestle </td>
 <td>Fisher Scientific </td>
 <td>K749521-1590</td>
 <td></td>
 </tr>
 <tr>
 <td>Glass balls 3mm</td>
 <td>VWR</td>
 <td>26396-630</td>
 <td></td>
 </tr>
 <tr>
 <td>Microplate Microlite 1+</td>
 <td>Thermo Scientific</td>
 <td>7571</td>
 <td>Select 96-well plates that are appropriate for luminescence - they must be opaque.</td>
 </tr>
 <tr>
 <td>TopSeal-A:96-well Microplates</td>
 <td>PerkinElmer</td>
 <td>6005185</td>
 <td>Microplate Press-On Adhesive Sealing Film</td>
 </tr>
 <tr>
 <td>D-Luciferin, Potassium Salt</td>
 <td>Gold BioTechnology, Inc.</td>
 <td>LUCNA</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Software </td>
 </tr>
 <tr>
 <td>Insomniac2</td>
 <td>Available upon request to the authors</td>
 <td>custom; written by Lesley Ashmore, Ph.D. (Westminster College) </td>
 <td>Matlab based software that has been used routinely for analysis of sleep2,6,11 </td>
 </tr>
 <tr>
 <td>Drosonex</td>
 <td>Available upon request to the authors</td>
 <td>custom; written by Thomas Coradetti (Sidewalk Software) </td>
 <td>A PC MSVC6 program used for survival analysis from raw data files collected with the Trikinetics system</td>
 </tr>
 <tr>
 <td>Photoshop CS3</td>
 <td>Adobe</td>
 <td></td>
 <td>Useful for obtaining numbers of cfu/plate from digital images (optional)</td>
 </tr>
 </tbody>
</table>

quantitative measurement of the immune response and sleep in drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

<ol>
	<li>Infection promotes sleep. In this example, Canton-S (CS) wild type flies and mutant flies lacking an NF&kappa;B gene, Relish (RelE20) 14, were loaded into two DAM2 activity monitors (n=32 for each genotype) and infected as described above. Flies were maintained in constant light to eliminate the influence of the circadian clock on behavior and infection2,7,8. The RelE20 mutants were isogenized to CS as described previously11. Both sets of flies were infe...

Watch this Scientific Journal Video about Quantitative Measurement of the Immune Response and Sleep in Drosophila at JoVE.com

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

quantitative-measurement-immune-response-sleep

Research

JoVE Journal

Immunology and Infection

15.3K visualizações.  University of Pennsylvania Perelman School of Medicine. Para entender uma ligação entre a resposta imune e comportamento, nós descrevemos um método para medir o comportamento locomotor em Drosophila Durante a infecção bacteriana, bem como a capacidade das moscas de montar uma resposta imune pela sobrevivência de monitorização, a carga bacteriana e, em tempo real, a actividade de um regulador chave da imunidade inata, NFkB.