The authors thank Arnim Jenett, Aljoscha Nern, and other members of the Rubin laboratory for advice and sharing of unpublished reagents and the Janelia Fly Light Project Team for generating confocal images. The authors also thank the members of the Gaul laboratory for comments on the manuscript.

1. Preparing Flies for MultiColor FlpOut (MCFO) Experiments
NOTE: The MCFO technique refers to a modified version of the so called Flp-mediated stop cassette excision (FlpOut). Transgenic MCFO flies carry a heat shock promoter (hsp)-Flp recombinase and different reporters under UAS control. Each reporter consists of a common backbone of a myristoylated (myr) super folder green fluorescent protein (sfGFP), in which 10 copies of an epitope tag (e.g., HA, FLAG or V5) have been inserted. The resulting non-fluorescent proteins are named &#34;spaghetti monster GFPs&#34; (smGFPs)14 and can be detected using specific antibodies against the different epitope tags.
<ol>
	<li>Cross flies with the MCFO cassette and the hsp-Flp (hsp-Flp; 10XUAS-FRT-stop-FRT-myr-smGFP-HA,10XUAS-FRT-stop-FRT-myr-smGFP-FLAG, 10XUAS-FRT-stop-FRT-myr-smGFP-V5) to the appropriate glial GAL4 driver lines (see Table of Materials).
	<ol>
		<li>As the hsp is already active at 25 &#176;C and few cells may undergo recombination leading to an unspecific labeling, set up crosses at 18 &#176;C in order to avoid leakiness of the system. Wait around 20 days at 18 &#176;C to obtain the F1 generation. After the heat shock (see step 2), maintain flies at 25 &#176;C (Figure 3).</li>
	</ol>
	</li>
</ol>
2. Heat Shock
NOTE: Depending on the insertion site, the efficiency of the hsp-Flp may differ; therefore, the optimal heat shock time has to be optimized. For this protocol, a MCFO fly stock in which the hsp-Flp was inserted on the X chromosome has been used (see Table of Materials).
<ol>
	<li>Transfer the progeny of the cross in step 1.1 (F1 generation flies, 3-4 days old) into new vials containing food and heat shock them at 37 &#176;C in a water bath. 
	NOTE: Young flies (1-2 days old) are very sensitive to heat shock. Wait 1 or 2 more days before performing the heat shock in order to reduce the mortality rate caused by the treatment. Use some of the F1 generation flies as a control, without heat shock.</li>
	<li>During the heat shock, maintain flies in normal vials with food in order to decrease the rate of stress-induced mortality. Put females and males in separate vials.</li>
	<li>Make sure to immerse the entire vial in the water to ensure a homogeneous heat shock. Use a 5-8 min shock as a suitable starting point for sparse labeling of glia cells with many GAL4 driver lines; the shock duration must be optimized for each driver and experiment (shorter or longer heat shock times may be necessary).</li>
	<li>After the heat shock, lay the vials horizontally on the bench for a few minutes in order to allow the flies to recover from the heat shock. 
	NOTE: It is not necessary to change the vials.</li>
	<li>Maintain the flies at 25 &#176;C and dissect (see step 3 and 4) them 2 or more days after the heat shock for optimal reporter expression.</li>
</ol>
3. Dissection Preparation and Solutions
<ol>
	<li>The day of the dissection, prepare fresh fixative solution in 200 &#181;L PCR tubes by mixing 180 &#181;L of S2 cell culture medium with 20 &#181;L of 20% paraformaldehyde (PFA) to obtain a 2% PFA solution. Maintain the fixative solution on ice before and during the dissection. 
	Caution: PFA is toxic and must be handled with care. 
	NOTE: Mix 160 &#181;L of S2 cell culture medium with 40 &#181;L of 20% paraformaldehyde (PFA) in a 200 &#181;L PCR tube to prepare a 4% PFA solution instead of a 2% solution.</li>
	<li>Use one PCR tube per genotype with a maximum of 8-10 brains per tube for optimal staining results.</li>
	<li>Prepare the adult brain washing solution: 0.5% bovine serum albumin, 0.5% Triton X-100 in PBS. 
	NOTE: Depending on the staining, a solution containing 1% Triton X-100 may be needed. A higher concentration of Triton X-100 increases the penetration of the antibody into the tissue and it is necessary to stain regions that lie deep inside the tissue (e.g., synaptic regions in the adult Drosophila brain).</li>
	<li>Prepare the blocking solution: 3% normal goat serum, 3% normal donkey serum, and 0.5% Triton X-100 in PBS.</li>
	<li>The adult brain washing solution and the blocking solution can be stored at 4 &#176;C for a few weeks.</li>
	<li>Put deep depression wells glass plate on ice and fill each well with the following solutions: one with 70% ethanol, one with PBS, and one with S2 cell culture medium.</li>
</ol>
4. Adult Brain Dissection
<ol>
	<li>Position a 3 cm dissecting dish on the stage of a stereomicroscope and fill it with cold S2 cell culture medium. Conduct all the dissections in this medium to ensure that the tissue remains healthy during the dissection procedure. 
	NOTE: Use a dissecting dish lined with several millimeters of black silicone which provides a good background contrast (in the images), and preserve the forceps during the dissection.</li>
	<li>Anesthetize the flies of the desired genotype with CO2.</li>
	<li>With forceps, grab the fly by the wings and wash it in cold 70 % ethanol for 30 s, then with cold PBS for additional 30 s.</li>
	<li>Transfer the fly into the deep depression well that is filled with S2 cell culture medium.</li>
	<li>Repeat the same procedure for all flies of the same genotype and maintain them in cold S2 cell culture medium until dissection, which will preserve the tissue. 
	NOTE: Anesthetize only the number of flies that can be dissected in a time period of about 30 min. Long incubation times in S2 cell culture medium may damage the tissue.</li>
	<li>Grab the fly with forceps and, under a stereomicroscope, submerge the fly in S2 cell culture medium.</li>
	<li>Detach the head from the rest of the body. Discard the body and keep the head submerged in S2 cell culture medium. It is extremely important to hold the head with forceps for the entire duration of the dissection. If the head starts to float, it will be difficult to retrieve it without damaging the tissue.</li>
	<li>Begin the dissection by pulling out one eye, while holding the head with at least one forceps at all times. Make sure that the tip of the forceps is just below the retina to avoid damaging the tissue underneath. Pull out the other eye and begin removing the cuticle until the brain appears.</li>
	<li>Remove all tracheal tissue and cuticle around the brain until the tissue appears clean. Make sure to remove all tracheal tissue around the brain for optimal staining results; the tissues are now ready to be fixed and stained.</li>
	<li>Maintain all the dissected brains in cold S2 cell culture medium before transferring them into the PFA solution in order to time the fixation step.</li>
</ol>
5. Adult Brain Staining
<ol>
	<li>Transfer the isolated brain with a P10 pipette into the PFA solution at room temperature (RT). To avoid tissue damage, never use forceps for transferring the brains after the dissection. Perform all steps in 200 &#181;L PCR tubes on a nutator.</li>
	<li>For all steps, before discarding the old solution with a P200 pipette, allow the brains to settle on the bottom of the tube. Try to remove the supernatant without aspirating the tissue. Protect the tissue from light exposure.</li>
	<li>Fix the brains in 200 &#181;L of 2% PFA in S2 cell culture medium for 1 h or in 4% PFA for 30 minutes. Keep fixation times identical between samples in order to increase reproducibility.</li>
	<li>After 3 (or more) washes of 15 minutes each in 200 &#181;L of adult brain washing solution, block the tissues with 200 &#181;L of blocking solution for 30 minutes. Longer incubation times in blocking solution are possible.</li>
	<li>Incubate the brains overnight at 4 &#176;C with primary antibodies diluted in adult brain washing solution in a total volume of 200 &#181;L. 
	NOTE: The incubation times may vary depending on the type of antibody used. Longer incubations may be necessary.
	<ol>
		<li>For labeling of the three MCFO markers, incubate the brains with anti-HA (1:500), anti-FLAG (DYKDDDDK epitope) (1:100), and anti-V5 primary antibodies.</li>
		<li>Primary directly-conjugated antibodies could be used instead of the normal primary + secondary combination (e.g., anti-V5:DyLight 549 (1:200)). In this case, treat the directly-conjugated antibodies as secondary antibodies. 
		NOTE: For each genotype, keep some brains as controls to verify the specificity of the staining. Skip the primary antibody incubation and go directly to the next step.</li>
	</ol>
	</li>
	<li>Wash the tissues 3 times for 1 h in 200 &#181;L of adult brain washing solution (step 3.3) and incubate them with secondary fluorophore-conjugates/primary directly-conjugated antibodies diluted in adult brain washing solution overnight at 4 &#176;C or for 4 h at RT, in a total volume of 200 &#181;L 
	NOTE: Examples of appropriate antibodies: AlexaFluor 488 (1:250), anti-V5:DyLight 549 (1:200) and DyLight 647 (1:100)-conjugated antibodies.</li>
	<li>Wash the brains 3 times for 1 h in 200 &#181;L of adult brain washing solution, followed by a final wash in 200 &#181;L of PBS overnight at 4 &#176;C or for 1-2 h at RT; the final washing step in PBS removes any traces of Triton X-100 and is necessary for optimal staining results. Mount the brains in mounting medium with an anti-fade agent on glass coverslips.</li>
</ol>
6. Mounting of Brains for Imaging
<ol>
	<li>Trim an imaging spacer to the appropriate size using scissors. For high-resolution microscopy, sandwich the specimen and imaging spacer between two glass coverslips. 
	NOTE: Imaging spacers are thin (0.12 mm thick) adhesive spacers that peel and stick to glass coverslips or microscope slides, allowing the mounting of specimens without compression.</li>
	<li>Using forceps, remove the adhesive liner from one surface and apply the spacer, adhesive side down, on the surface of a glass coverslip (22 mm x 60 mm). Apply 10 &#181;L of mounting medium to a new glass coverslip.</li>
	<li>With a P10 pipette, transfer the brains from the 200 &#181;L tube to the coverslip, next to the drop of mounting medium. Together with the tissues, transfer some PBS in order to maintain the brains in solution.</li>
	<li>With a P10 pipette, move the brains from the PBS to the drop of mounting medium trying to limit the amount of PBS. Transfer the specimens in mounting medium on the coverslip with the imaging spacer. Use enough mounting media to cover the specimen.</li>
	<li>Remove the other adhesive liner from the upper side of the spacer and add a glass coverslip on top of the specimens. With the tip of a forceps, apply a gentle pressure over the adhesive area to seal. Image the mounted tissues immediately or store the slides at -20 &#176;C for later analysis.</li>
</ol>
7. Image Acquisition
<ol>
	<li>Acquire stacks of confocal fluorescence images using a confocal microscope with a 40X (NA = 1.2, water immersion) objective or 63X (NA =1.4, oil immersion) objective (pixel sizes = 1,024 x 1,024 in the xy plane; Distance between two confocal sections = 0.5 &#181;m). Adjust detector gain and offset in each channel in such a way that no saturated pixels and zero pixel values are present in the images; this avoids information loss and ensures use of the full dynamic range of the detector. 
	NOTE: Since 3D imaging is time consuming, the measurements can be automated by scanning multiple samples in parallel at different locations. To avoid evaporation with the water immersion objective, use special oil immersion medium with refractive index n = 1.33.</li>
	<li>Process the confocal stacks for maximum density projections, changes in channel hue, and adjustment of brightness and contrast using any standard image analysis software (see Table of Materials).</li>
</ol>

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None of the authors have competing or conflicting interests.

This protocol describes an easy and efficient method to study the morphology of different cell types within a tissue of interest at high resolution. With the MCFO technique, multiple reporters with different epitope tags are used in combination for multicolor stochastic labeling (Figure 2). Similar to other methods such as Brainbow/Flybow15,16,17, MCFO increases the label diversity through marker co...

Glia, the non-neuronal cell population of the nervous system (NS), were long believed to provide a static framework for neurons and therefore were not studied in detail. However, in humans, glia constitute the vast majority of cells in the NS (~90%) and fall into several different categories, including astrocytes, oligodendrocytes, microglia and Schwann cells. In Drosophila, glia constitute about 10% of the cells in the NS. Intriguingly, their morphologies and functions are remarkably similar to those found in vertebrates1,2. Their morphologies include blood-brain barrier (BBB) forming epithelia, ensheathing, and astrocyte-like cells.
The Drosophila central nervous system (CNS) consists of the following principal structures: cortex regions that contain the neuronal cell bodies; neuropils that harbor synaptic connections; small and large axon tracts that connect the different neuropiles; peripheral nerves that connect sensory organs and muscles with the CNS (Figure 1). Glia are found associated with all these anatomical structures: Cortex glia (CG) in the cortical regions, astrocyte-like glia (ALG) and ensheathing glia (EG) in the neuropile regions, ensheathing glia are also associated with central axon tracts and peripheral nerves (EGN), and finally, two sheet-like glia, perineurial glia (PG) and subperineurial (SPG), which together form a contiguous layer that covers the entire NS (Figure 2).
Previous studies have shown that glia play important roles in the development of the NS; they monitor neuronal cell numbers by reacting to systemically circulating insulin-like peptides, provide trophic support to neurons, such as the astrocyte-neuron lactate shuttle, and eliminate dying neurons by phagocytosis3,4,5,6. In the mature NS, glia maintain the BBB, take up neurotransmitters and maintain ionic homeostasis, act as the major immune cells in the NS, since macrophages cannot breach the BBB, and modulate synaptic activity as well as animal behavior6,7,8,9,10,11.
Whether the different glial subtypes perform specialized functions remains an important open question. However, a systematic genome-wide analysis of glia, especially in the adult, has been hampered by a lack of appropriate genetic tools for their manipulation. Here, a method that allows the efficient and easy characterization of cell shapes to study complex cell-cell interactions is presented. This technique has been applied to characterize the morphology of the different glial subtypes in the adult Drosophila brain, but, depending on the specific GAL4 driver used, it could be adapted to study neurons12,13, any kind of intermingling cells, and in principle any tissue in any developmental stages.

<table>
	<tbody>
		<tr>
			<td>Water bath</td>
			<td>Grant</td>
			<td>GD100</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>Sarstedt</td>
			<td>72.737.002</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dumont</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting dish 30 mm x 12 mmm</td>
			<td>Electron Microscopy Sciences</td>
			<td>70543-30</td>
			<td>Glass dissection dish</td>
		</tr>
		<tr>
			<td>Pyrex 3 Depression Glass Spot Plate</td>
			<td>Corning</td>
			<td>7223-34</td>
			<td>Glass dissection plates</td>
		</tr>
		<tr>
			<td>Sylgard Black</td>
			<td>SYLGARD, Sigma-Aldrich</td>
			<td>805998</td>
			<td>home made with charcoal</td>
		</tr>
		<tr>
			<td>ExpressFive S2 cell culture medium</td>
			<td>Invitrogen</td>
			<td>10486-025</td>
			<td></td>
		</tr>
		<tr>
			<td>20% PFA</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Roth</td>
			<td>3051.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Jackson Laboratories</td>
			<td>005-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>Jackson Laboratories</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit HA-tag</td>
			<td>Cell Signaling</td>
			<td>C29F4</td>
			<td>Primary antibody, dilution 1:500</td>
		</tr>
		<tr>
			<td>Rat FLAG-tag</td>
			<td>Novus Biologicals</td>
			<td>NBP1-06712</td>
			<td>Primary antibody, dilution 1:100</td>
		</tr>
		<tr>
			<td>Mouse V5-tag:DyLight 549</td>
			<td>AdSerotec</td>
			<td>0411</td>
			<td>Conjugated antibody, dilution 1:200</td>
		</tr>
		<tr>
			<td>anti-rabbit AlexaFluor 488</td>
			<td>Invitrogen</td>
			<td>A11034</td>
			<td>Secondary antibody, dilution 1:250</td>
		</tr>
		<tr>
			<td>anti-rat DyLight 647</td>
			<td>Jackson Laboratories</td>
			<td>712-605-153</td>
			<td>Secondary antibody, dilution 1:100</td>
		</tr>
		<tr>
			<td>Vecta Shield</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>SlowFate Gold</td>
			<td>Invitrogen</td>
			<td>S36937</td>
			<td></td>
		</tr>
		<tr>
			<td>Secure Seal Spacer</td>
			<td>Grace Biolabs</td>
			<td></td>
			<td>Contact company for ordering</td>
		</tr>
		<tr>
			<td>Microscope cover glass 22 X 60 mm</td>
			<td>Marienfeld</td>
			<td>101152</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass 22 x 22 mm</td>
			<td>Roth</td>
			<td>H874</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope, Leica MZ6</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope LSM710</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Immersol</td>
			<td>Zeiss</td>
			<td>518 F</td>
			<td>Immersion oil for fluorescence-microscopy, halogen free</td>
		</tr>
		<tr>
			<td>Immersol</td>
			<td>Zeiss</td>
			<td>W 2010</td>
			<td>Immersion fluid for water-immersion objectives, halogen free</td>
		</tr>
		<tr>
			<td>R56F03-GAL4 (EG)</td>
			<td>Bloomington Stock Center</td>
			<td>39157</td>
			<td>GAL4 driver</td>
		</tr>
		<tr>
			<td>R86E01-GAL4 (ALG)</td>
			<td>Bloomington Stock Center</td>
			<td>45914</td>
			<td>GAL4 driver</td>
		</tr>
		<tr>
			<td>hspFlpPestOpt; UAS-FRT-stop-FRT-myr-smGFP-HA, UAS-FRT-stop-FRT-myr-smGFP-FLAG, UAS-FRT-stop-FRT-myr-smGFP-V5</td>
			<td>Bloomington Stock Center</td>
			<td>64085</td>
			<td>UAS reporter (https://bdscweb.webtest.iu.edu/stock/misc/mcfo.php)</td>
		</tr>
		<tr>
			<td>Fiji (Image J)</td>
			<td></td>
			<td></td>
			<td>Image analysis software</td>
		</tr>
		<tr>
			<td>Multi Time Macro</td>
			<td>Zeiss</td>
			<td></td>
			<td>Software for automated scanning</td>
		</tr>
	</tbody>
</table>

application of multicolor flpout technique to study high resolution single cell morphologies and cell interactions of glia in drosophila

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between cell types or even within a given type? What kinds of spatial rules do they follow? The answers to such fundamental questions in vivo have been hampered so far by a lack of tools for high resolution single cell labeling. Here, a detailed protocol to target single cells with a MultiColor FlpOut (MCFO) technique is provided. This method relies on three differently tagged reporters (HA, FLAG and V5) under UAS control that are kept silent by a transcriptional terminator flanked by two FRT sites (FRT-stop-FRT). A heat shock pulse induces the expression of a heat shock-induced Flp recombinase, which randomly removes the FRT-stop-FRT cassettes in individual cells: expression occurs only in cells that also express a GAL4 driver. This leads to an array of differently colored cells of a given cell type that allows the visualization of individual cell morphologies at high resolution. As an example, the MCFO technique can be combined with specific glial GAL4 drivers to visualize the morphologies of the different glial subtypes in the adult Drosophila brain.

This section illustrates examples of results that can be obtained by using the MCFO technique in the adult Drosophila brain. Figure 3 shows a schematic of the method. Three differently membrane-tagged reporters (myr-smGFP-HA, myr-smGFP-FLAG and myr-smGFP-V5) under UAS control are kept silent by a transcriptional terminator flanked by two FRT sites (FRT-stop-FRT). A heat shock pulse induces the expression of Flp recombinase which rand...

Watch this Scientific Journal Video about Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila at JoVE.com

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Cells display different morphologies and establish a variety of interactions with their neighbors. This protocol describes how to reveal the morphology of single cells and to investigate cell-cell interaction by using the well-established Gal4/UAS expression system.

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between ...

application-multicolor-flpout-technique-to-study-high-resolution

Research

JoVE Journal

Behavior

8.1K Views.  Ludwig-Maximilians-University Munich. Cells display different morphologies and establish a variety of interactions with their neighbors. This protocol describes how to reveal the morphology of single cells and to investigate cell-cell interaction by using the well-established Gal4/UAS expression system.

Video: Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Assessment and Evaluation of the High Risk Neonate: The NICU Network Neurobehavioral Scale

There has been a long-standing interest in the assessment of the neurobehavioral integrity of the newborn infant. The NICU Network Neurobehavioral Scale (NNNS) was developed as an assessment for the at-risk infant. These are infants who are at increased risk for poor developmental outcome because of insults during prenatal development, such as substance exposure or prematurity or factors such as poverty, poor nutrition or lack of prenatal care that can have adverse effects on the intrauterine environment and affect the developing fetus. The NNNS assesses the full range of infant neurobehavioral performance including neurological integrity, behavioral functioning, and signs of stress/abstinence. The NNNS is a noninvasive neonatal assessment tool with demonstrated validity as a predictor, not only of medical outcomes such as cerebral palsy diagnosis, neurological abnormalities, and diseases with risks to the brain, but also of developmental outcomes such as mental and motor functioning, behavior problems, school readiness, and IQ. The NNNS can identify infants at high risk for abnormal developmental outcome and is an important clinical tool that enables medical researchers and health practitioners to identify these infants and develop intervention programs to optimize the development of these infants as early as possible. The video shows the NNNS procedures, shows examples of normal and abnormal performance and the various clinical populations in which the exam can be used.

The NICU Network Neurobehavioral Scale (NNNS) was developed as an assessment for the at-risk infant. The purpose of this article is to describe the NNNS, provide video examples of the NNNS procedures and discuss the ways in which the exam has been used.

There has been a long-standing interest in the assessment of the neurobehavioral integrity of the newborn infant.  The NICU Network Neurobehavioral Scale ...

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

Study Motor Skill Learning by Single-pellet Reaching Tasks in Mice

Reaching for and retrieving objects require precise and coordinated motor movements in the forelimb. When mice are repeatedly trained to grasp and retrieve food rewards positioned at a specific location, their motor performance (defined as accuracy and speed) improves progressively over time, and plateaus after persistent training. Once such reaching skill is mastered, its further maintenance does not require constant practice. Here we introduce a single-pellet reaching task to study the acquisition and maintenance of skilled forelimb movements in mice. In this video, we first describe the behaviors of mice that are commonly encountered in this learning and memory paradigm, and then discuss how to categorize these behaviors and quantify the observed results. Combined with mouse genetics, this paradigm can be utilized as a behavioral platform to explore the anatomical underpinnings, physiological properties, and molecular mechanisms of learning and memory.

Persistent practice improves the precision of coordinated movements.&#160;Here we introduce a single-pellet reaching task, which is designed to assess the learning and memory of forelimb skill in mice.&#160;

Reaching for and retrieving objects require precise and coordinated motor movements in the forelimb. When mice are repeatedly trained to grasp and ...

Uncovering Beat Deafness: Detecting Rhythm Disorders with Synchronized Finger Tapping and Perceptual Timing Tasks

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here with the goal of uncovering rhythm disorders, such as beat deafness. Beat deafness is characterized by poor performance in perceiving durations in auditory rhythmic patterns or poor synchronization of movement with auditory rhythms (e.g., with musical beats). These tasks include the synchronization of finger tapping to the beat of simple and complex auditory stimuli and the detection of rhythmic irregularities (anisochrony detection task) embedded in the same stimuli. These tests, which are easy to administer, include an assessment of both perceptual and sensorimotor timing abilities under different conditions (e.g., beat rates and types of auditory material) and are based on the same auditory stimuli, ranging from a simple metronome to a complex musical excerpt. The analysis of synchronized tapping data is performed with circular statistics, which provide reliable measures of synchronization accuracy (e.g., the difference between the timing of the taps and the timing of the pacing stimuli) and consistency. Circular statistics on tapping data are particularly well-suited for detecting individual differences in the general population. Synchronized tapping and anisochrony detection are sensitive measures for identifying profiles of rhythm disorders and have been used with success to uncover cases of poor synchronization with spared perceptual timing. This systematic assessment of perceptual and sensorimotor timing can be extended to populations of patients with brain damage, neurodegenerative diseases (e.g., Parkinson&#8217;s disease), and developmental disorders (e.g., Attention Deficit Hyperactivity Disorder).

Behavioral tasks that allow for the assessment of perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) are presented. Synchronization of finger tapping to the beat of an auditory stimuli and detecting rhythmic irregularities provides a means of uncovering rhythm disorders.

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here ...

A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to screen different neurostimulation patterns in hemiparkinsonian (unilateral lesioned) rats. It consists of creating a unilateral Parkinson&#39;s lesion by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle, implanting chronic stimulation electrodes into the subthalamic nucleus and evaluating motor outcomes at the end of 24 hr periods of cable-bound external neurostimulation. The stimulation was conducted with constant current stimulation. The amplitude was set 20% below the individual threshold for side effects. The motor outcome evaluation was done by the assessment of spontaneous paw use in the cylinder test according to Shallert and by the assessment of skilled reaching in the staircase test according to Montoya. This protocol describes in detail the training in the staircase box, the cylinder test, as well as the use of both in hemiparkinsonian rats. The use of both tests is necessary, because the staircase test seems to be more sensitive for fine motor skill impairment and exhibits greater sensitivity to change during neurostimulation. The combination of the unilateral Parkinson model and the two behavioral tests allows the assessment of different stimulation parameters in a standardized way.

Deep brain stimulation (DBS) is an effective treatment option for Parkinson's disease. We established a study design to screen novel stimulation paradigms in rats. The protocol describes the use of the staircase test and cylinder test for motor outcome assessment in DBS treated hemiparkinsonian rats.

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to ...

Reversible Cooling-induced Deactivations to Study Cortical Contributions to Obstacle Memory in the Walking Cat

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For example, in the cat, visual information about an impending obstacle can modulate stepping for avoidance. Locomotor adaptation can also occur independent of vision, as sudden tactile inputs to the leg by an expected obstacle can modify the stepping of all four legs for avoidance. Such complex locomotor coordination involves supraspinal structures, such as the parietal cortex. This protocol describes the use of reversible, cooling-induced cortical deactivation to assess parietal cortex contributions to memory-guided obstacle locomotion in the cat. Small cooling loops, known as cryoloops, are specially shaped to deactivate discrete regions of interest to assess their contributions to an overt behavior. Such methods have been used to elucidate the role of parietal area 5 in memory-guided obstacle avoidance in the cat.

Complex locomotion in naturalistic environments requiring careful coordination of the limbs involves regions of the parietal cortex. The following protocol describes the use of reversible cooling-induced deactivation to demonstrate the role of parietal area 5 in memory-guided obstacle avoidance in the walking cat.

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For ...

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, and issues commands to start or stop feeding. Deciphering the processes underlying the decision-making of timely and moderate feeding carries major implications in our understanding of physiological and psychological disorders related to feeding control. Simple, quantitative, and robust methods are required to measure the food ingestion of animals after experimental manipulation, such as forcibly increasing the activities of certain target neurons. Here, we introduced dye-labeling-based feeding assays to facilitate the neurogenetic study of feeding control in adult fruit flies. We review available feeding assays, and then describe our methods step-by-step from setup to analysis, which combine thermogenetic and optogenetic manipulation of neurons controlling feeding motivation with dye-labeled food intake assay. We also discuss the advantages and limitations of our methods, compared with other feeding assays, to help readers choose an appropriate assay.

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Quantitative food-intake assays with dyed food provide a robust and high-throughput means to evaluate feeding motivation. Combining the food consumption assay with thermogenetic and optogenetic screens is a powerful approach to investigate the neural circuits underlying appetite in adult Drosophila melanogaster.

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, ...

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific responses to changing temperatures according to their physiological tolerance and adaptability. Drosophila flies also possess a temperature sensing system that has become fundamental to understanding the neural basis of temperature processing in ectotherms. We present here a temperature-controlled arena that permits fast and precise temperature changes with temporal and spatial control to explore the response of individual flies to changing temperatures. Individual flies are placed in the arena and exposed to pre-programmed temperature challenges, such as uniform gradual increases in temperature to determine reaction norms or spatially distributed temperatures at the same time to determine preferences. Individuals are automatically tracked, allowing the quantification of speed or location preference. This method can be used to rapidly quantify the response over a large range of temperatures to determine temperature performance curves in Drosophila or other insects of similar size. In addition, it can be used for genetic studies to quantify temperature preferences and reactions of mutants or wild-type flies. This method can help uncover the basis of thermal speciation and adaptation, as well as the neural mechanisms behind temperature processing.

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Here we present a protocol to automatically determine the locomotor performance of Drosophila at changing temperatures using a programmable temperature-controlled arena that produces fast and accurate temperature changes in time and space.

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific ...

Assessing the Coherence of Parents' Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with parents. This paper focuses on such a method, namely Five-Minute Speech Sample-Coherence (FMSS-Coherence). In this method, a parent is asked to speak for five uninterrupted minutes about her/his child and their relationship. The resulted narrative is coded for coherence, namely the extent to which the parent provides&#160;in the narrative&#160;a clear, consistent, multidimensional and well-supported portrayal of the child. FMSS-Coherence is based on attachment research that shows that the coherence of parents&#8217; narratives is indicative of the quality of the parent-child relationship and child adjustment. It overcomes the limitations of attachment narrative measures which are typically labor intensive. FMSS-Coherence is less nuanced than extant attachment narrative measures. Yet, studies of families from different cultural backgrounds, across different child ages and in the context of typically developing children as well as children with special needs suggest that coherence can be reliably evaluated using the FMSS procedure. Furthermore, parents&#8217; FMSS-Coherence is associated with parenting quality and children&#8217;s socio-emotional adjustment. Thus, it holds promise for researchers and practitioners who seek a relatively time- and cost-effective method for assessing the coherence of parents&#8217; narratives regarding their child.

This paper introduces a method for assessing the coherence of parents' short narratives regarding their child and their relationship using the five-minute speech sample procedure.

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with ...

Radiotracer Administration for High Temporal Resolution Positron Emission Tomography of the Human Brain: Application to FDG-fPET

Functional positron emission tomography (fPET) provides a method to track molecular targets in the human brain. With a radioactively-labelled glucose analogue, 18F-fluordeoxyglucose (FDG-fPET), it is now possible to measure the dynamics of glucose metabolism with temporal resolutions approaching those of functional magnetic resonance imaging (fMRI). This direct measure of glucose uptake has enormous potential for understanding normal and abnormal brain function and probing the effects of metabolic and neurodegenerative diseases. Further, new advances in hybrid MR-PET hardware make it possible to capture fluctuations in glucose and blood oxygenation simultaneously using fMRI and FDG-fPET.
The temporal resolution and signal-to-noise of the FDG-fPET images is critically dependent upon the administration of the radiotracer. This work presents two alternative continuous infusion protocols and compares them to a traditional bolus approach. It presents a method for acquiring blood samples, time-locking PET, MRI, experimental stimulus, and administering the non-traditional tracer delivery. Using a visual stimulus, the protocol results show cortical maps of the glucose-response to external stimuli on an individual level with a temporal resolution of 16 s.

This manuscript describes two radiotracer administration protocols for FDG-PET (constant infusion and bolus plus infusion) and compares them to bolus administration. Temporal resolutions of 16 s are achievable using these protocols.

Functional positron emission tomography (fPET) provides a method to track molecular targets in the human brain. With a radioactively-labelled glucose ...