This work was supported by NIH Grant HL-113905

All animal procedures carried out for this study were approved by the Institutional Animal Care and Use Committee of Nova Southeastern University in accord with the Guide for the Care and Use of Laboratory Animals, 8th Edition (The National Academies Press, Washington, DC, 2011).
1. Preparing Tissue Sections for Receptor Autoradiography
<ol>
	<li>Upon sacrifice, harvest fresh brain tissues, and wrap in aluminum foil and place in a -20 &#176;C freezer as soon as possible. To maintain the correct shape, place brains in a brain mold that simulates the inside of the skull, wrap in aluminum foil and place in a - 20 &#176;C freezer. After 30 min, place tissues in a sealable freezer storage bag and move to a -80 &#176;C freezer for long-term storage.
	<ol>
		<li>Obtain the fresh frozen tissue specimen of interest from the -80 &#176;C freezer, and transfer to a cryostat set to a minimum of -10 &#176;C and maximum of -18 &#176;C, to avoid thawing.</li>
		<li>Place the specimen onto the tissue mount with a glycol and resin-based embedding medium, only embedding a small part of the specimen into the medium. For brain, the brain is mounted vertically to enable sectioning in the coronal plane.</li>
	</ol>
	</li>
	<li>Place the tissue mount onto the microtome within the cryostat and firmly tighten in place. Ensure the left and right side of the brain are in the same antero-postero coordinates, and that the dorso-ventral axis is perpendicular to commonly used brain atlases.</li>
	<li>Begin cutting at the desired thickness (20 &#181;m recommended) and thaw mount the sections onto microscope slides in a vertical direction to have a greater surface area of space in the slide. Collect sections onto slides in sequential sets of five, i.e. the first set of slides are labelled 1-1, 1-2, 1-3, 1-4, and 1-5 (Figure 3).</li>
	<li>After filling a set of slides with sections, allow slides to air dry for up to 1 hr, then place the slides in a plastic slide box in a self-sealing freezer storage bag, and store at -20 &#176;C.</li>
</ol>
2. Receptor Autoradiography
CAUTION: Radioactivity. Use protective attire to handle radioactivity. Disposal is depended upon establishment, and must follow guidelines to properly decay (half-life 60 days) or be picked up by a certified company.&#160;
<ol>
	<li>Remove the &#34;-1&#34; and &#34;-2&#34; slides of interest from the -20 &#176;C freezer and mount into slide grips (Figure 5). Mount the &#34;-1&#34; slides together for &#39;non-specific&#39; treatment, and the &#34;-2&#34; slides for &#39;total&#39; treatment. &#39;Non-specific&#39; jars will contain 10 &#181;M final concentrations of PD123319 an AT2R antagonist, and losartan an AT1R antagonist, in assay medium buffer (AM5) (Table 1), the &#39;total&#39; binding jars will only contain 10 &#181;M PD123319 in AM5.</li>
	<li>Invert the slide grips to place the slides into the pre-incubation Coplin jars filled with 35-40 ml of AM5, and respective inhibitors for 30 min at room temperature. Start subsequent sets in their pre-incubation bath at 4 min intervals.</li>
	<li>Immediately transfer slides from the pre incubation solution to the incubation slide mailers, containing 10 ml of AM5, plus a predetermined concentration of 125I-SI-Ang II (calculated in Figure 4) with the respective inhibitors, for 60-90 min at room temperature. If there is sufficient radioligand, 10 slides can be placed (back to back) in modified slide grips and incubated with 125I-SI-Ang II in Coplin jars.</li>
	<li>Place slides back into the slide grips (if needed), blot, and rinse by gently swirling for 1-2 sec in 400 ml of distilled water in two separate containers.</li>
	<li>Transfer the slides sequentially into four Coplin jars containing 35-40 ml of AM5 for exactly 1 min each (as illustrated in Figure 4).</li>
	<li>After the last 1 min rinse, gently swirl the sections for 1-2 sec in four changes of ice cold distilled water.</li>
	<li>Blow-dry the slides with cool air (Caution: hot air volatizes the radioactive compound) using four hair dryers set up from different angles for 4 min (Figure 5), until all the sections are dry, then place on a paper towel.</li>
	<li>Mount slides with tissues facing up onto a cardboard for apposition to X-ray film using double sided tape.</li>
	<li>Mount at least one, 125 Iodine calibration standard slide onto each cardboard (Figure 6). 
	Note: Calibration standards consist of brain paste thoroughly mixed with 125Iodine bound to a compound containing a phenol ring, compacted by centrifugation in a 1 ml tuberculin syringe, that are cryostat sectioned at the same thickness as the brain sections and thaw-mounted onto microscope slides. Alternatively, 125I calibration standards in plastic resin sectioned at 20 &#181;m thickness can be obtained commercially. The plastic resin in these standards partially shields the film from the radiation such that a tissue equivalency of ~40% should be factored into the calibration.</li>
</ol>
3. Film Exposure and Development
<ol>
	<li>Proceed to a darkroom with a strap-back X-ray cassette and autoradiography film. Open the cassette and place the cardboard with slides inside (Figure 6).
	<ol>
		<li>Turn the lights off and turn on the safelight. Carefully open the box of X-Ray film, remove one film, and place the film shiny side up (with the jagged edge on the bottom right corner) on top of the slides in the cassette. Carefully close the cassette, and twist the locking bars to seal out light (Figure 6). Expose the slides for several days to several weeks at -20 &#176;C.</li>
	</ol>
	</li>
	<li>In the darkroom, open the cassette and proceed with the film developing process.
	<ol>
		<li>Place the slides into the trays consecutively; developer for 2 min, water containing 5% glacial acetic acid for 30 sec, and fixer for 5 min. The films are the placed into a tray with running water for 20 min, then placed into Photoflo for no more than 10 sec and hung. 
		Note: The exposure time is determined empirically and can involve multiple films with different exposure times: long enough to obtain measurable signals from areas with low binding, but not so long as to saturate the film by areas with high binding. Once acceptable exposures are obtained, then proceed to the next step.</li>
	</ol>
	</li>
</ol>
4. Histology
<ol>
	<li>Prepare the thionin stain and staining reagents (Table 2, Figure 7).
	<ol>
		<li>Place the &#34;-3&#34; slides in the slide rack, and transfer in sequential order beginning with deionized water for 1 min, then thionin stain solution for 10 min followed by three dips into deionized waters, and one 30 sec wash in deionized water.</li>
		<li>Following the water, place the slide rack into ethanol washes as follows; 50%, 70%, and 90% for 30 sec, followed by two ethanol washes at 100% for 1 min each. Lastly, place the slide rack into xylene for 3 min, then transfer over to a second xylene solution for 5 min.</li>
	</ol>
	</li>
	<li>Remove one slide at a time from the last xylene bath, and cover the upper edge of the slide with a resin base in organic solvent mounting medium, and place a 24 mm x 60 mm coverslip onto the slide. Allow slides to sufficiently dry for ~48 hr and then scan into the computer at 2,400 dpi grayscale.</li>
</ol>
5. Densitometric Image Analysis
<ol>
	<li>Place the film shiny side down, with the jagged edge on the bottom left corner and scan using a proprietary scanner capable of transmitting the film density information without any distortion in the imaging system computer.</li>
	<li>Open the proprietary imaging system and utilize the calibration bars to establish calibration standards for future densitometric analysis of the images on the film (Figures 9-12).</li>
	<li>Measure areas of interest by either establishing a template or empirically outlining the areas of interest. Data is the collected and separated based on film, either control or wild type (section), and region. Make sure to include density (fmol/g), scan area, and total target area in measurements (Figure 9).
	<ol>
		<li>Adjust scan area bars by assuring that region of interest falls between parameters of highlighting (Figure 10).</li>
		<li>Export data into a spreadsheet (Table 4). Multiply the density times the total target area, then divide by the scan area in order to obtain the value for the binding of that specific area. Once this is done to all values measured, substrate the established non-specific from total to yield the specific binding present. Averages may also be performed for these values.</li>
	</ol>
	</li>
</ol>

Robert Speth has licensed antibodies to angiotensin II receptors to a commercial vendor and receives royalties from the sale of said antibodies. The other authors have nothing to disclose.

The protocol described identifies the visualization of &#39;total&#39; and &#39;non-specific&#39; binding of the radioligand in adjacent sections of coronal sections of a rodent brain previously harvested and stored at -80 &#176;C , and can be readily applicable to virtually every tissue that has anatomically resolved substructures which display differential amounts of receptors or radioligand binding sites. The procedures described within the protocol are simple and the analysis is critical for correctly interpreting re...

Cardiovascular disease continues to be the leading cause of death and disability in the United States, causing more than 30% of deaths in the U.S. in 20111. The most recent statistics from the American Heart Association indicate that more than one person in three has one or more type of cardiovascular disease. Cardiovascular research continues to make strides against understanding this disease, but as generations begin getting older it is imperative to continue these efforts. The Renin-angiotensin System (RAS) plays a central role in the regulation of the cardiovascular system primarily by promoting atherosclerosis, inflammation, systemic vasoconstriction, and activation of the sympathetic nervous system (Figure 1)2-8.
The RAS is a hormonal system that is activated when juxtaglomerular cells of the kidney secrete renin into the bloodstream in response to decreased blood pressure, increased sympathetic stimulation, or decreased sodium flow by the macula densa. Renin metabolizes angiotensinogen (synthesized in the liver) to form angiotensin I (Ang I). Ang I is then metabolized by angiotensin-converting enzyme (ACE), an ectoenzyme on the luminal side of vascular endothelial cells, primarily in the lungs and kidneys, to form angiotensin II (Ang II), the main effector peptide of the RAS. Ang II is capable of activating two receptor subtypes; the type 1 receptor (AT1R) and the type 2 receptor (AT2R), both which regulate the cardiovascular system, maintain fluid and electrolyte homeostasis and are now considered to affect cognitive function and neurodegenerative disease processes8,9. A local, brain-specific RAS is reported to independently synthesize Ang II. In the brain, the precursor protein angiotensinogen is synthesized in astroglia10 converted to Ang I by a renin-like enzyme3, possibly prorenin bound to the prorenin receptor11, and subsequently converted to Ang II by angiotensin-converting enzyme which is abundantly expressed on the extracellular surface of neurons in the brain12. This intrabrain generated Ang II is the agonist for the brain AT1 and AT2 receptors that are isolated from blood-borne Ang II.
While the AT1R plays an important physiological role, it is better known for its pathophysiological effects throughout the body, primarily affecting the cardiovascular system and kidneys (Figure 2). When Ang II binds to the AT1R, it causes vasoconstriction; increasing resistance to blood flow and raising blood pressure. It also promotes the synthesis and secretion of aldosterone and vasopressin, leading to increased sodium and water retention. These effects can also induce ischemic brain damage and cognitive impairments and is linked to Parkinson&#39;s disease, Alzheimer&#39;s disease, and diabetes, as well as being recently identified to affect learning and memory13-15. There is a feedback loop in the RAS in that AT1R on the juxtaglomerular cells in the kidney inhibits renin secretion. Interestingly, the AT2R generally counter-regulates the action of AT1R, causing vasodilation, neurite outgrowth, axonal regeneration, anti-proliferation, and cerebroprotection among many others16-20. The AT2R has also been identified as a target for anti-hypertension and recently, anti-cancer drugs21. Determining the localization and density of Ang II receptors within various tissues and how they are impacted by various treatments and disease states using quantitative densitometric receptor autoradiography will help uncover the role the RAS plays in specific diseases.
Receptor autoradiography has been used for over 30 years as an effective method for indicating the presence of angiotensin II receptors and other components of the RAS in the brain and other tissues of the rat, mouse, guinea pig, dog and human under a variety of experimental conditions22-34. The importance of locating Ang II receptors within the brain is that one can apply functional neuroanatomy to the actions of Ang II in the brain, e.g., the presence of AT1R in the paraventricular nucleus of the hypothalamus (PVN) suggests a function of Ang II in the brain to stimulate vasopressin, oxytocin or corticotropin releasing hormone (CRH) release, or activation of the sympathetic nervous system. Thus, drugs that block the AT1R might decrease some of these PVN-mediated effects associated with over activity of the brain RAS. Work in progress suggests that the use of AT1R antagonists can decrease Post-traumatic Stress Disorder (PTSD)-induced release of CRH and ameliorate the symptoms of PTSD (Hurt et al., submitted for publication). The PVN, subfornical organ (SFO), and amygdala are known for regulating homeostasis, appetite/thirst, sleep, memory, emotional reactions, and are the target areas of this demonstration study. These regions were examined by collecting coronal sections of a brain on microscope slides, and treating the sections with specific inhibitors along with a specific radioligand for Ang II receptors. In this study, all materials and reagents along with suggested vendors are listed, Iodine-125 was used to radiolabel an Ang II receptor antagonist, sarcosine1, isoleucine8 Ang II (SI Ang II), which was then purified as the mono125 I-SI Ang II using HPLC methods as described previously35. The use of this high specific activity radioligand allows the visualization of areas of low, moderate and high receptor density after exposure of the radiolabeled sections to x-ray film. By calibrating the film with brain paste standards containing known amounts of Iodine-125, the specific amount of Ang II receptor binding in an area can be quantified. In experimental studies, the Ang II receptor binding in the brains of experimental subjects can be compared to that in the brains of control subjects. This can indicate whether the actions of Ang II are altered in response to a genetic condition, phenotypic abnormality, disease state or drug treatment. This knowledge can then be applied to the development of therapies to treat diseases associated with dysregulation of the RAS. Alternative techniques that identify receptor binding sites, but with reduced anatomical resolution, are binding assays that use tissue membrane preparations derived from tissue homogenates, which are incubated with the radioligand over a range of concentrations to assess radioligand binding affinity as the dissociation constant (KD) and maximal binding capacity (Bmax) of the tissue of interest.
The protocol described here can be broken down into 5 major components: Preparing Tissue Sections for Receptor Autoradiography; Receptor Autoradiography; Film Exposure and Development; Histology; and Densitometric Image Analysis.

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			<td>500ml Plastic Beakers</td>
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			<td>Commercial hair dryers</td>
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			<td>Model SD6X</td>
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			<td>14-961-26</td>
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			<td>USB Corporation</td>
			<td>15-699</td>
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			<td>Ethanol</td>
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			<td>16-100-210&#160;</td>
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			<td>Formulary Substitute for D-19 Developer</td>
			<td>Photographers Formulary, Inc.&#160;</td>
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			<td>A38 SI-212</td>
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			<td>Fisher</td>
			<td>SH75-125D</td>
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			<td>Kodak</td>
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			<td>MCID</td>
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			<td>Peel-A-Way slide grips</td>
			<td>VWR</td>
			<td>48440-003</td>
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			<td>Permount</td>
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			<td>SP15-100</td>
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			<td>PD123319</td>
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			<td>Premium Charged Slides , Fine Ground Edge</td>
			<td>Premiere Microscope Slides</td>
			<td>9308W</td>
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			<td>125I Ligands</td>
			<td>Perkin Elmer</td>
			<td>NEX- 248</td>
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			<td>125SI-Ang II&#160;</td>
			<td>George Washington University</td>
			<td>Radioiodinated by Dr. Speth</td>
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			<td>Slide Mailers</td>
			<td>Fisher Scientific</td>
			<td>HS15986</td>
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			<td>Sodium Dibasic Phosphate Anhydrous (Na2PO4)</td>
			<td>Fisher</td>
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			<td>Sodium Acetate (Anhydrous)</td>
			<td>Fisher</td>
			<td>BP333-500</td>
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			<td>Thionin&#160;</td>
			<td>Fisher</td>
			<td>T409-25</td>
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			<td>Spectronics Corporation</td>
			<td>Four Square</td>
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			<td>Xylene</td>
			<td>Fisher&#160;</td>
			<td>X3P-1GAL</td>
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receptor autoradiography protocol for the localized visualization of angiotensin ii receptors

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to perform receptor autoradiographic mapping.
Tissue specimens are harvested and stored at -80 &#176;C. A cryostat is used to coronally section the tissue (brain) and thaw-mount the sections onto charged slides. The slide-mounted tissue sections are incubated in 125I-SI-Ang II to radiolabel Ang II receptors. Adjacent slides are separated into two sets: &#39;non-specific binding&#39; (NSP) in the presence of a receptor saturating concentration of non-radiolabeled Ang II, or an AT1 Ang II receptor subtype (AT1R) selective Ang II receptor antagonist, and &#39;total binding&#39; with no AT1R antagonist. A saturating concentration of AT2 Ang II receptor subtype (AT2R) antagonist (PD123319, 10 &#181;M) is also present in the incubation buffer to limit 125I-SI-Ang II binding to the AT1R subtype. During a 30 min pre-incubation at ~22 &#176;C, NSP slides are exposed to 10 &#181;M PD123319 and losartan, while &#39;total binding&#39; slides are exposed to 10 &#181;M PD123319. Slides are then incubated with 125I-SI-Ang II in the presence of PD123319 for &#39;total binding&#39;, and PD123319 and losartan for NSP in assay buffer, followed by several &#39;washes&#39; in buffer, and water to remove salt and non-specifically bound radioligand. The slides are dried using blow-dryers, then exposed to autoradiography film using a specialized film and cassette. The film is developed and the images are scanned into a computer for visual and quantitative densitometry using a proprietary imaging system and a spreadsheet. An additional set of slides are thionin-stained for histological comparisons.
The advantage of using receptor autoradiography is the ability to visualize Ang II receptors in situ, within a section of a tissue specimen, and anatomically identify the region of the tissue by comparing it to an adjacent histological reference section.

The overview of the metabolic pathway of the Renin-Angiotensin System is shown in Figure 1 and the direct focus on the Angiotensin II receptor subtypes (AT1R and AT2R) is described in Figure 2. Figure 3 displays the transfer of coronal brain sections onto microscope slides, which are then run through a receptor autoradiography procedure using a predetermined 125I-SIAng II concentration as seen in F...

Watch this Scientific Journal Video about Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors at JoVE.com

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II.

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to ...

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17.8K Views.  Nova Southeastern University. Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II. 

Video: Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

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, ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

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 ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors&#8212;an essential step toward a model of odor coding in the mammalian olfactory system.

Olfactory receptor activation patterns encode odor identity, but the lack of published data identifying odorant ligands for mammalian olfactory receptors hinders the development of a comprehensive model of odor coding. This protocol describes a method to facilitate high-throughput identification of olfactory receptor ligands and quantification of receptor activation.

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. Few studies have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem, which contains second order neurons of the auditory pathway, is a potential site for gene transfer. In this protocol, a technique for direct and maximal exposure of the murine DCN via a posterior fossa approach is demonstrated. This approach allows for either acute or survival surgery. Following direct visualization of the DCN, a host of experiments are possible, including injection of opsins into the cochlear nucleus and subsequent stimulation by an optical fiber coupled to a blue light laser.&#160;Other neurophysiology experiments, such as electrical stimulation and neural injector tracings are also feasible.&#160;The level of visualization and the duration of stimulation achievable make this approach applicable to a wide range of experiments.

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

Protocol for Isolating the Mouse Circle of Willis

The cerebral arterial circle (circulus arteriosus cerebri) or circle of Willis (CoW) is a circulatory anastomosis surrounding the optic chiasma and hypothalamus that supplies blood to the brain and surrounding structures. It has been implicated in several cerebrovascular disorders, including cerebral amyloid angiopathy (CAA)-associated vasculopathies, intracranial atherosclerosis and intracranial aneurysms. Studies of the molecular mechanisms underlying these diseases for the identification of novel drug targets for their prevention require animal models. Some of these models may be transgenic, whereas others will involve isolation of the cerebro-vasculature, including the CoW.The method described here is suitable for CoW isolation in any mouse lineage and has considerable potential for screening (expression of genes, protein production, posttranslational protein modifications, secretome analysis, etc.) studies on the large vessels of the mouse cerebro-vasculature. It can also be used for ex vivo studies, by adapting the organ bath system developed for isolated mouse olfactory arteries.

We describe here a reproducible protocol for isolating the mouse circle of Willis.

The cerebral arterial circle (circulus arteriosus cerebri) or circle of Willis (CoW) is a circulatory anastomosis surrounding the optic chiasma and ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...