The work was supported by the Lundbeck Foundation (Grant R133-A12270) and the Novo Nordisk Foundation (Grant NNF0C0028664). The authors thank Dr. Ales&#780; Marek for the supply of [3H]radioligand.

All animal handling was performed in compliance with the guidelines from The Danish Animal Experimentation Inspectorate.
NOTE: The protocol described here covers tissue preparation (i.e., mouse brain tissue), the in vitro autoradiographic assay in sufficient detail for setting up the method in a new lab, the exposure to phosphor imaging plates as well as subsequent densitometric analysis of autoradiograms (Figure 2) with the aim of localizing and quantifying radioligand binding in distinct anatomical structures. For histological comparison, a protocol for cresyl violet staining is included. Moreover, the determination of non-specific binding with a competing ligand is included within the protocol. For a detailed description on how to determine Kd, Bmax or Ki, see previous publication4.
1. Tissue Preparation by Cryosectioning
<ol>
	<li>Euthanize the mouse by cervical dislocation and immediately dissect out the brain using scissors and forceps. Directly proceed to the next step to avoid tissue damage.</li>
	<li>Snap-freeze the tissue by submersion in powdered dry ice, gaseous CO2 or isopentane. Directly transfer the frozen tissue to a cryostat with the temperature set to -20 &#176;C. Alternatively, store the tissue at -80 &#176;C until processing. 
	NOTE: Avoid repeated thawing/freezing to reduce tissue damage.</li>
	<li>Let the tissue acclimate to -20 &#176;C in the cryostat for 20 min before further processing to avoid tissue shattering.</li>
	<li>Cover the tissue holder with embedding medium outside the cryostat and quickly place the frozen tissue specimen in the desired orientation while the embedding medium is still liquid. For instance, place the mouse brain vertically onto cerebellum in order to achieve rostral coronal sections. Transfer the tissue holder back to the cryostat and expose the embedding medium to temperatures below -10 &#176;C for hardening. 
	NOTE: Fragile tissue specimen should be coated in embedding medium within the tissue molds prior to mounting.</li>
	<li>Position the tissue holder in the microtome of the cryostat. Adjust the orientation of the tissue to avoid sloped sections.</li>
	<li>Cut the tissue with the guidance of a stereotaxic atlas18 in sections of desired thickness (12 &#181;m recommended for [3H] labelled ligands). Carefully straighten and unfold the section with a brush of small size if necessary and thaw-mount the section onto a microscope slide. Sequentially collect the sections from the region of interest for desired technical replication (e.g., 4 sections per slide).</li>
	<li>Allow the sections on the slides to air-dry for 1 h before further handling. 
	NOTE: Addition of desiccant material to slide boxes minimizes moisture build up on the tissue sections. Procotol can be paused here by storing sections long-term in slide boxes at -80 &#176;C.</li>
</ol>
2. In vitro Autoradiography
CAUTION: Radioactivity. Work in a certified laboratory according to local regulations. Wear protective clothing. Dispose in accordance with radioactive decay or outsource to a certified company.
<ol>
	<li>Thaw the sections for at least 30 min at room temperature (RT). Label the slides with experimental conditions. Use a pencil because the slides will be bathed in ethanol during subsequent staining.</li>
	<li>Place the slides horizontally in plastic trays. 
	NOTE: Positioning slides on an elevated platform within plastic trays facilitates their handling.</li>
	<li>Pre-incubate the sections mounted on the slides in assay buffer adjusted to target in question (for GHB protocol, 50 mM KHPO4 buffer pH 6.0 is used) by carefully applying an appropriate volume onto the slide (700 &#181;L for 3-4 rodent coronal sections). 
	NOTE: Make sure that every section is covered completely with liquid.
	<ol>
		<li>Cover the plastic trays with a lid in order to avoid evaporation and pre-incubate at relevant temperature (for GHB protocol pre-incubate for 30 min at RT) under constant gentle (20 rpm) shaking on a plate shaker.</li>
		<li>For the determination of non-specific binding, supplement assay buffer with relevant concentration of unlabelled compound (for GHB protocol, 1 mM GHB). 
		NOTE: Pre-incubation may not be necessary.</li>
	</ol>
	</li>
	<li>Pour off pre-incubation liquid from each slide and transfer the slides back to the plastic tray.</li>
	<li>To avoid dehydration, immediately incubate the sections with relevant concentration of radioligand in assay buffer under desired conditions (for GHB protocol, 1 nM [3H]HOCPCA for 1 h at RT) by covering the sections completely with the radioligand solution (700 &#181;L for 3-4 rodent coronal sections).
	<ol>
		<li>Incubate under under constant gentle (20 rpm) shaking of plastic trays with closed lid. 
		NOTE: The radioligand concentration can be validated by counting an aliquot in a liquid scintillation counter.</li>
	</ol>
	</li>
	<li>Remove the incubation solution by pouring off the liquid and transfer the slides into a microscope slide rack. Immediately proceed to the next step to avoid section dehydration.</li>
	<li>Wash the slides. For the GHB protocol, wash with ice-cold assay buffer twice for 20 s and then rinse twice by dipping the slide rack into the trays filled with ice-cold distilled water to remove salts. Position the slides vertically in racks for air-drying for at least 1 h at RT or dry the slides for 5 min with a blower set to cold temperature. 
	NOTE: Washing must be optimized, e.g., extensive washing may be useful for decreasing non-specific binding.</li>
	<li>Transfer the slides to a fixator containing paraformaldehyde (PFA) powder for overnight fixation with PFA vapours at RT in order to protect the integrity of the ligand-target complex. 
	CAUTION: PFA is toxic. Positionthe fixator in fume hood and avoid skin/eye contact with PFA.</li>
	<li>The following day, transfer the slides to a desiccator box containing silica gel for 3 h at RT to eliminate moisture.</li>
</ol>
3. Exposure to Phosphor Imaging Plates and Scanning
<ol>
	<li>Place the sections in a radiation-shielded imaging plate cassette with the tissue facing up. For subsequent quantification of radioligand binding, include a [3H]microscale in every cassette. Arrange the sections randomly and expose the sections for direct comparison in the same cassette.</li>
	<li>Erase the tritium-sensitive phosphor imaging plate immediately before usage in order to remove accumulated signals from storage and to eliminate background signals. Therefore, load the plate into phosphor imaging machine and expose it to visible/infrared light according to the instructions of the manufacturer.</li>
	<li>Remove the plate from phosphor imaging machine and immediately place it onto the sections in the cassette. Make sure that the cassette is closed completely. Expose the sections to the phosphor imaging plate for 3 days at RT shielded from light.</li>
	<li>Because light erases signal from the imaging plate, carefully open the cassette in the dark and immediately transfer the imaging plate into the dark box of a phosphor imager or place the phosphor imager in a dark room. 
	NOTE: Make sure to notate the spatial arrangement of the slides during exposure in order to identify individual specimen on the digital image after analysis. Therefore, phosphor imaging plates also display one corner cut in a distinct angle in order to identify the correct orientation of the plate on the digital picture.</li>
	<li>Scan the plate at the highest resolution possible to obtain a digital image.</li>
</ol>
4. Optional: Cresyl Violet Staining of Tissue Sections
<ol>
	<li>Prepare 1% cresyl violet solution by mixing 5 g of cresyl violet acetate in 500 mL of deionized water (dH2O) until dissolved (approximately 2 h). Filter through a filter paper using a funnel into a new 500 mL bottle. Adjust pH to 3.5-3.8.</li>
	<li>Position the slide staining set under fume hood. Prepare trays with the following solutions in white polypropylene trays (except for xylene): 
	a. 50% ethanol/50% dH2O 
	b. 70% ethanol/30% dH2O 
	c. 100% ethanol 
	d. 100% ethanol 
	e. 100% dH2O 
	f. 1% cresyl violet 
	g. 0.07% acetic acid (add 175 &#181;L of acetic acid to 250 mL of dH2O). 
	h. 100% xylene in green solvent-resistant trays 
	i. 100% xylene in green solvent-resistant trays</li>
	<li>Transfer the slides to the fume hood and place them in a slide rack.</li>
	<li>Dissolve the lipids through increasing graded series of ethanol in dH2O into 100% ethanol (tray a-d) by dipping the slides for 1 min.</li>
	<li>Rehydrate the specimens to dH2O through descending concentrations of ethanol (tray a-d in reverse order, followed by tray e) by dipping the slides for 1 min.</li>
	<li>Immerse the specimens in cresyl violet solution for 10 min.</li>
	<li>Rinse the specimens in 0.07% acetic acid by lifting the slides up and down gently for 4-8 s. Wash the slides by dipping in dH2O for 1 min.</li>
	<li>Dehydrate the specimens by immersion of the slides for 30 s in ascending concentrations of ethanol (tray a-d).</li>
	<li>Transfer the specimens through two trays of 100% xylene (tray h and i) to quench the ethanol.</li>
	<li>Rehydrate the specimens to dH2O through descending concentrations of ethanol (tray a-d in reverse order, followed by tray e) by dipping the slides for 1 min.</li>
	<li>Remove the slides from saline with forceps. Add a few drops of organic solvent mounting media per slide and add a 24 x 60 mm coverslip on top to protect specimens. Remove air bubbles between the specimen and coverslip by gently pressing onto the coverslip. 
	NOTE: Keep the remaining slides in xylene during mounting to prevent drying.</li>
	<li>Dry the slides overnight in a fume hood at RT.</li>
	<li>Obtain a picture of specimen with a microscope and 1.25X objective.</li>
</ol>
5. Densitometric Analysis of Digital Image
<ol>
	<li>Measure relative optical densities (RODs) of each calibration standard from the [3H]microscale with an image analysis software.
	<ol>
		<li>Select an area of equal size for each point of the [3H]microscale using a tool for Region creation from the menu item Region determination. Assign a number to each selected area by clicking on Number under the menu item Label.</li>
		<li>Export OD values for each point of the calibration standard by clicking File | Export | 2D region report. Transfer ROD values to a spreadsheet and normalize by the size of the selected area. Perform linear regression to obtain a standard curve for further densitometric analysis. 
		NOTE: Make sure that the selected areas are labelled in order to identify matching ROD values and samples.</li>
	</ol>
	</li>
	<li>Perform quantification of autoradiograms using the proprietary imaging software by selecting the region of interest (ROI) using a Region creation tool in every section and measuring its ODs. Select the same region in every section by creating a template for the region of interest, which is copied and manually adjusted to minor variations in brain anatomy for each autoradiogram. Identify the anatomy of the ROI by comparison of autoradiograms with a brain atlas18. When multiple treatments are compared, perform the analysis blinded and randomized in order to avoid biased selection of ROIs.</li>
	<li>Export ROD values and sizes of selected areas into a spreadsheet by clicking File | Export | 2D region report.</li>
	<li>Divide the measured ROD of the selected ROI by its area to obtain the density per specific area.</li>
	<li>Measure the ROD of the background of the plate and export corresponding ROD values and area size into a spreadsheet. Subtract the average background signal from every ROD value of each ROI.</li>
	<li>Average the RODs of technical replicates, i.e., section replicates using tissue from the same animal.</li>
	<li>Use the standard curve to convert RODs into units of radioligand binding, i.e., nCi/mg tissue equivalents (TE). 
	NOTE: The term TE is used because standards are generated with materials simulating tissue.</li>
	<li>Express binding by conversion of nCi/mg to nmol/mg TE according to the specific activity of the radioligand (Equation 1). 
	<img alt="Equation" src="/files/ftp_upload/58879/58879eq1v2.jpg" /> (1)</li>
	<li>To obtain specific binding values, subtract non-specific binding from total binding.</li>
	<li>Average the binding of every biological replicate by using the average of the technical replicates of each animal (obtained in Step 5.6).</li>
</ol>

The authors declare no conflicts of interest.

The quality of an autoradiographic assay is most often determined by the sensitivity of the radioligand. A major contributing factor is the selected radioisotope, which is given by the availability of known ligands or by the feasibility of specific labelling techniques to yield ligands with appropriate specific activity (i.e., the amount of radioactivity per unit mole of a radioligand)23and with limited amounts of chemical degradation. A large number of radioligands of known ligands are l...

Autoradiography is a method which provides images of radioactive decay. The technique is routinely used to study the tissue distribution of a protein of interest in vitro based on a specific pharmacological interaction between a radiolabelled compound and its target. This provides direct information about the selectivity of the ligand for the target. In vitro autoradiography may also be used for quantitative determination of pharmacological binding parameters of radioligands, such as the dissociation constant (Kd) and density of binding sites (Bmax), as well as for determining the inhibition constant (Ki) of competing ligands1,2. Compared to traditional homogenate radioligand binding, autoradiography has the advantage of being able to visualize spatial anatomy and giving succinct details of regional expression patterns3. The method of autoradiography is therefore a relevant alternative to immunocytochemistry, especially in the absence of a validated antibody. Autoradiography is easily implemented in a standard radioisotope laboratory given the availability of a suitable radioligand with the required pharmacological specificity, access to a microtome cryostat for preparing tissue sections, and a suitable imaging device that is able to analyze the distribution of radioactivity in the respective tissue sections. Notably, an important selection criterion for the radioligand is a limited amount of binding to non-target sites. This can be to other proteins, membranes or materials such as plastic or filters, and is collectively referred to as non-specific binding. Usually, non-specific binding is non-saturable but can be saturable if it involves a specific off-target protein. The best way of validating true specific binding is to compare to tissues lacking the target, e.g., genetically engineered (knock-out) tissue4.
Here, the methodology is illustrated with the autoradiographic characterization of the high-affinity binding site for &#947;-hydroxybutyric acid (GHB) in the mammalian brain. Understanding the pharmacological interaction between GHB and its binding site is of relevance as GHB is both a clinically useful drug in the treatment of narcolepsy and alcoholism5, but also a natural constituent of the mammalian brain and a recreational drug6. High-affinity GHB binding sites were first described using [3H]GHB binding to rat brain homogenate7. Over the years, further autoradiography studies with [3H]GHB and the analogue [3H]NCS-382 has showed a high density of binding sites in forebrain regions of rat8,9,10, mouse9, pig11and monkey/human brain12. However, the molecular identity and exact functional relevance of these binding sites have remained elusive.
With the intention to further characterize the binding sites, and to facilitate studies on the physiological role of GHB, multiple radioligands incorporating different isotopes endowed with different affinities have been developed ([3H]GHB, [3H]NCS-382, [3H]HOCPCA and [125I]BnOPh-GHB)13,14,15,16(reviewed in17) (Figure 1). The combination of selective high-affinity radioligands and a very high tissue density of the binding sites have allowed for the production of high-quality images using the phosphor imaging technique9,11. Along with an outline of the practical points in setting up an autoradiographic experiment and an illustration to exemplify details, the discussion section will emphasize i) the choice of radionuclide, ii) the choice of assay conditions, and iii) the use of phosphor imaging plates versus X-ray film. The overall goal of this paper is to provide technical, methodological and scientific details on the autoradiography technique for informing about tissue distribution and pharmacological analysis of protein targets.

<table>
	<tbody>
		<tr>
			<td>Absolute ethanol</td>
			<td>Merck Millipore</td>
			<td>107017</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>BAS-TR2040 Imaging Plate</td>
			<td>GE Healthcare Life Science</td>
			<td>28956481</td>
			<td>20x40 cm - Sensitive to tritium</td>
		</tr>
		<tr>
			<td>Cresyl violet acetate</td>
			<td>Sigma-Aldrich</td>
			<td>C5042-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>DPX (non-aqueous mounting medium for microscopy)</td>
			<td>Merck Millipore</td>
			<td>100579</td>
			<td></td>
		</tr>
		<tr>
			<td>O.C.T. Compound, 12 x 125 mL</td>
			<td>Sakura</td>
			<td>4583</td>
			<td>Tissue-Tek</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>16005-1KG-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus slides</td>
			<td>VWR</td>
			<td>631-9483</td>
			<td>microscope slides</td>
		</tr>
		<tr>
			<td>Tissue-Tek Manual Slide Staining Set</td>
			<td>Sakura Finetek Denmark ApS</td>
			<td>4451</td>
			<td></td>
		</tr>
		<tr>
			<td>Tritium Standard on Glas</td>
			<td>American Radiolabeld Chemicals, Inc.</td>
			<td>ART 0123</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene substitute</td>
			<td>Sigma-Aldrich</td>
			<td>A5597</td>
			<td></td>
		</tr>
	</tbody>
</table>

autoradiography as a simple and powerful method for visualization and characterization of pharmacological targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

Using the described protocol, the anatomical distribution of the high-affinity GHB binding sites was visualized with the radiolabelled GHB analogue [3H]HOCPCA in mouse brain, which was cut into coronal, sagittal and horizontal sections (Figure 3). High levels of binding were observed in hippocampus and cortex, lower binding in striatum and no binding was detected in cerebellum, corresponding to previous reported expression patterns of the high-affi...

Watch this Scientific Journal Video about Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets at JoVE.com

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

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Neuroscience

43.7K Views.  University of Copenhagen. The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

Video: Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously published phenotype assessments in several disease models, including spinocerebellar ataxias, Huntington s disease and spinobulbar muscular atrophy. Measures include hind limb clasping, ledge test, gait and kyphosis. Each measure is recorded on a scale of 0-3, with a combined total of 0-12 for all four measures. The results effectively discriminate between affected and non-affected individuals, while also quantifying the temporal progression of neurodegenerative disease phenotypes. Measures may be analyzed individually or combined into a composite phenotype score for greater statistical power. The ideal combination of the four described measures will depend upon the disorder in question. We present an example of the protocol used to assess disease severity in a transgenic mouse model of spinocerebellar ataxia type 7 (SCA7).
Albert R. La Spada and Gwenn A. Garden contributed to this manuscript equally.

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebellar ataxia. Measures include hind limb clasping, ledge test, gait and kyphosis. This protocol effectively discriminates between affected and non-affected individuals, and detects the progression of affected individuals over time.

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously ...

Novel Apparatus and Method for Drug Reinforcement

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction. Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in animal research to model various components of drug reinforcement, consumption, and addiction in humans. For this study, we used a novel approach to studying drug reinforcement in rats by combining traditional CPP and self-administration methodologies. We assembled an apparatus using two Med Associate operant chambers, sensory stimuli, and a Plexiglas-constructed neutral zone. These modifications allowed our experiments to encompass motivational aspects of drug intake through self-administration and drug-free assessment of drug/cue conditioning strength with the CPP test. In our experiments, rats self-administered cocaine (0.75 mg/kg/inj, i.v.) during either four (e.g., the "short-term") or eight (e.g., the "long-term") alternating-day sessions in an operant environment containing distinctive sensory cues (e.g., olfactory and visual). On the alternate days, in the other (differently-cued) operant environment, saline was available for self-infusion (0.1 ml, i.v.). Twenty-four hours after the last self-administration/cue-pairing session, a CPP test was conducted. Consistent with typical CPP findings, there was a significant preference for the chamber associated with cocaine self-administration. In addition, in animals undergoing the long-term experiment, a significant positive correlation between CPP magnitude and the number of cocaine-reinforced lever responses. In conclusion, this apparatus and approach is time and cost effective, can be used to examine a wide array of topics pertaining to drug abuse, and provides more flexibility in experimental design than CPP or self-administration methods alone.

Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in research to model various components of drug reinforcement, consumption, and addiction in humans. In this report, we combined traditional CPP and self-administration methods as a novel approach to studying drug reinforcement and addiction in rats.

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction.  Operant drug ...

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

Automated Interactive Video Playback for Studies of Animal Communication

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, parameter-based computer animation offers the opportunity to independently manipulate any number of behavioral, morphological, or spectral characteristics in the context of realistic, moving images of animals on screen. A major limitation of conventional playback, however, is that the visual stimulus lacks the ability to interact with the live animal. Borrowing from video-game technology, we have created an automated, interactive system for video playback that controls animations in response to real-time signals from a video tracking system. We demonstrated this method by conducting mate-choice trials on female swordtail fish, Xiphophorus birchmanni. Females were given a simultaneous choice between a courting male conspecific and a courting male heterospecific (X. malinche) on opposite sides of an aquarium. The virtual male stimulus was programmed to track the horizontal position of the female, as courting males do in the wild. Mate-choice trials on wild-caught X. birchmanni females were used to validate the prototype's ability to effectively generate a realistic visual stimulus.

Video playback is a widely used technique in animal behavior. We created and evaluated a program that applies rules-based, interactive playback of 3-D computer animations in response to real-time, automated data on subject behavior.

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...