This study was funded by the National Natural Science Foundation of China (Project Code no. 81373557; no. 81403327).

This study was approved by the ethics committee at the China Academy of Chinese Medical Sciences (reference number 20160014). All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, D.C., 1996). Four adult male rats (weight 250-280 g) were used in this study. All animals were housed in a 12 h light/dark cycle with controlled temperature and humidity, and allowed free access to food and water. The instruments and materials used in the present study were showed in Figure 1.&#160;&#160;Before the surgery, all instruments, such as stereotaxic frame and glass pipette, were cleaned using 70% ethanol.
1. Surgical Procedures
<ol>
	<li>Determine the coordinate area of interest in VPM using a stereotaxic atlas9 (Figure 2A).</li>
	<li>Prepare a 1 &#181;L micro-syringe equipped with a glass micropipette (with a tip diameter of approximately 10-20 &#181;m) (Figure 1D) and test it with liquid paraffin.</li>
	<li>Anesthetize the rats with 7% chloral hydrate&#160;(0.7&#160;mL/100 g) by intraperitoneal injection.</li>
	<li>Once deep anesthesia is confirmed with tail pinch and pedal withdrawal reflex, shave the top of the animal&#39;s head with an electric razor and scrub the surgical site 3 times&#160;with 10% povidone iodine followed by 70% ethanol respectively.</li>
	<li>Place the rat into the stereotaxic device by placing blunt ear bars into the ears and place the rat&#39;s upper incisors into the mouth holder (Figure 1E), and then apply ophthalmic ointment on the eyes.</li>
	<li>Clean the head skin of the surgical site again using 70% ethanol. Use sterile surgical gloves and towels to maintain the surgery under the sterile conditions.</li>
	<li>Make a sagittal incision in the skin with a scalpel along the sagittal suture (Figure 1F).</li>
	<li>Scrape the muscle and periosteum away from the skull using sterile cotton-tipped applicators throughout the surgery to control bleeding (Figure 1F).</li>
	<li>Using predefined coordinates from an atlas (Figure 2A), determine the location (-3.3 mm Bregma point, 2.6 mm right to midline) of craniotomy (Figure 1G).</li>
	<li>Perform a craniotomy using a burr drill with a round-tip bit (#106) (Figure 1H), and continue drilling to about a 1 mm depth within a few minutes until reaching the meninges (Figure 1I).</li>
	<li>Excise the dura mater using microforceps to expose the cerebral cortex over the injection site (Figure 1I).</li>
	<li>Change liquid paraffin in the micro-syringe with 10% BDA (10,000 molecular weight, in distilled water) solution (Figure 1J).</li>
	<li>Mount the syringe into the microinjection apparatus and connect with a micro-pump (Figure 1K). 
	NOTE: The volume injected is dependent on the speed of the micro-pump. Here it was adjusted to 30 nL/min (Figure 1L).</li>
	<li>Under a stereomicroscope, insert a glass micropipette manually with the microinjection apparatus into the VPM through the cortical surface of the brain at the depth of 5.8 mm (Figure 1M).</li>
	<li>Pressure-inject a 100 nL of 10% BDA into the VPM over a period of 3 min (35 nL/min) with a micro-pump (Figure 1L, M).</li>
	<li>After injection, keep the pipette in place for an additional 5 min and then withdraw slowly.</li>
	<li>Suture the wound with sterile thread (Figure 1N). Follow your local animal care committee guidelines for pre- and&#160;post-surgical analgesia.&#160;</li>
	<li>Place the rat in a warm recovery area until it regains consciousness and is fully recovered.&#160;</li>
	<li>Return the recovered rat back to its cage.</li>
</ol>
2. Perfusions and Sections
<ol>
	<li>After a survival time of typically 10 days, inject the animal with an overdose of 10% urethane (2 mL/100 g) by intraperitoneal injection to induce euthanasia.</li>
	<li>Perfuse the experimental rats in the hood (Figure 1O).
	<ol>
		<li>Once the breath stops, using scissors and forceps, open the thoracic cavity of the rat to access the heart. Insert an intravenous catheter into the left ventricle toward the aorta, and then open the right auricle.</li>
		<li>First perfuse with 0.9% phosphate-buffered saline (PBS) at physiological temperature (37 &#176;C) about 1-2 min until the blood exiting from the heart is clear, and then continue with 250-300 mL 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4).</li>
	</ol>
	</li>
	<li>After the perfusion, incise the head skin and open the skull, and then dissect out the rat brain. Post-fix the dissected brain in 4% paraformaldehyde for 2 h at room temperature (26 &#176;C), then cryoprotect in 30% sucrose in 0.1 M PBS (pH 7.4) for 3 days at 4 &#176;C until the brain is immersed in the solution (Figure 1P).</li>
	<li>Once the brain is immersed in the solution, divide the brain into three blocks in the coronal direction on the brain matrices (Figure 1Q). The central block contains the VPM and S1.</li>
	<li>Cut the central block of the brain at 40 &#181;m on a freezing stage sliding microtome system in the coronal direction. Collect these sections orderly in a 6-well dish with 0.1 M PBS (pH 7.4) (Figures 1R, S).</li>
</ol>
3. Standard ABC Staining
NOTE: Free floating sections from every third coronal section of the brain were used for visualizing the BDA labeling with standard ABC procedure10.
<ol>
	<li>Rinse the sections in 0.1 M PBS for about 1 min.</li>
	<li>Incubate the sections in 1% ABC solution in 0.1 M PB (pH 7.4) containing 0.3% Triton X-100 for 1 h at room temperature.</li>
	<li>Wash the sections three times in 50 mM Tris buffer (pH 7.4).</li>
	<li>Stain the sections in a solution containing 0.02% 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) and 0.01% H2O2 in 50 mM Tris buffer for about 2-5 min at room temperature.</li>
	<li>Wash the sections three times in 50 mM Tris buffer (pH 7.4).</li>
	<li>Mount the sections on microscope slides using standard histochemical techniques (Figure 1T; see Supplemental Video File III, perfusion and sections).</li>
	<li>Dry the sections in the air overnight at room temperature.</li>
	<li>Dehydrate the sections briefly in a series of alcohol (50%, 70%, 95%, 100%) solutions. Submerge the slides in each solution for about 15 s. Do not allow the slides to dry out between each step.</li>
	<li>Clear the sections in xylene three times, about 20 min.</li>
	<li>Put 2 or 3 drops of balsam on the slices then place coverslips on the sections.</li>
</ol>
4. Double Fluorescent Staining for BDA and Basic Neural Elements in Cerebral Cortex
NOTE: In contrast, double fluorescent staining was carried out for observing the correlation of BDA labeling and basic neural elements on the adjacent sections to the above used with streptavidin-AF594 and counterstained with fluorescent Nissl stain AF500/525.
<ol>
	<li>Rinse the sections in 0.1 M PBS for about 1 min.</li>
	<li>Incubate the sections in a mixed solution of streptavidin-AF594 (1:500) and AF500/525 green fluorescent Nissl stain (1:1,000) in 0.1 M PBS (pH 7.4) containing 0.3% Triton X-100 for 2 h at room temperature.</li>
	<li>Wash the sections three times in 0.1 M PB (pH 7.4).</li>
	<li>Mount the sections on microscope slides using standard histochemical techniques. Dry the sections in the air for about 1 h.</li>
	<li>Apply coverslips to the fluorescent sections with 50% glycerin in distilled water before observation.</li>
</ol>
5. Double Fluorescent Staining for BDA and Interneurons in Cerebral Cortex
NOTE: Double fluorescent staining was carried out for observing the correlation of BDA labeling and interneurons on the representative sections in the cortical target with streptavidin-AF594 and PV-immunochemistry.
<ol>
	<li>Rinse the representative sections in 0.1 M PBS (pH 7.4) for about 1 min.</li>
	<li>Incubate the sections in a blocking solution containing 3% normal goat serum and 0.3% Triton X-100 in 0.1 M PBS for 30 min.</li>
	<li>Transfer the sections into a solution of mouse monoclonal anti-PV IgG (1:1,000) in 0.1 M PBS (pH 7.4) containing 1% normal goat serum and 0.3% Triton X-100 for overnight at 4 &#176;C.</li>
	<li>On the following day, wash the sections three times in 0.1 M PBS (pH 7.4).</li>
	<li>Expose the sections to a mixed solution of goat anti-mouse-AF488 secondary antibody (1:500), streptavidin-AF594 (1:500), and 4&#39;,6-diamidino-2-phenylindole dihydrochloride (DAPI, 1:40,000) in 0.1 M PBS (pH 7.4) containing 1% normal goat serum and 0.3% Triton X-100 for 1 h.</li>
	<li>Repeat steps 4.3 to 4.5.</li>
</ol>
6. Observation
<ol>
	<li>Take images of the VPM, thalamocortical axons, and corticothalamic neurons.
	<ol>
		<li>Observe the fluorescent samples with a confocal imaging system equipped with objectives lenses (4x, NA: 0.13; 10x, NA: 0.40; and 40x, NA: 0.95). Use excitation and emission wavelengths of 405 (blue), 488 (green) and 559 (red) nm. 
		NOTE: Here, the confocal pinhole is 152 &#181;m (4x, 10x) and 105 &#181;m (40x).The spatial resolution of image capture is 1024 &#215; 1024 pixel (4x, 10x) and 640 &#215; 640 pixel (40x).</li>
		<li>Take twenty images in successive frames of 2 &#181;m from each section at the thickness of 40 &#181;m (Z series).</li>
		<li>Integrate the images into a single in-focus image with the confocal image processing software system for three-dimensional analyzing as follows: set start focal plane &#8594; set end focal plane &#8594; set step size &#8594; choose depth pattern &#8594; image capture &#8594; Z series.</li>
	</ol>
	</li>
	<li>Take brightfield images by a light microscope equipped with a digital camera (4x, NA: 0.13; 10x, NA: 0.40; and 40x, NA: 0.95 lenses). Use an exposure time of 500 ms. Use photo editing software to adjust the brightness and contrast of images and to add labels.</li>
</ol>

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The authors have nothing to disclose.

Selecting a proper tracer is a critical step for a successful neural tracing experiment. In the family of BDA, high molecular weight BDA (10,000 molecular weight) was recommended to be preferentially transported through the anterograde neural pathway in contrast to low molecular weight BDA (3,000 molecular weight)2,3,11,12,13. However, many studies also sugges...

High molecular weight BDA (10,000 molecular weight), a highly sensitive tracer, has been used for tracing neural pathways in the central nervous system for over 20 years1. Although the use of the BDA is a common neural tract tracing technique, the quality of BDA labeling can be affected in animals by various factors1,2,3. Our recent study indicated that the optimal structure of BDA labeling is not only associated with a proper post-injection survival time, but also correlated with the staining method4. Until now, conventional avidin-biotin-peroxidase complex (ABC), streptavidin-fluorescein isothiocyanate, and streptavidin-AF594 staining methods were used for revealing the BDA labeling in previous studies2,3,4,5. In comparison, fluorescent staining for BDA can be easily performed.
In order to extend the application of high molecular weight BDA, a refined protocol was introduced in the present study. Following the injection of BDA into the VPM of the thalamus in the rat brain, BDA labeling was revealed by the regular method of standard ABC staining as well as by double fluorescent staining, which was carried out for observing the correlation of BDA labeling and basic neural elements or interneurons in the cortical target with streptavidin-AF594 and fluorescent Nissl histochemistry or PV-immunochemistry, respectively. Through the reciprocal neural pathways between VPM and the primary somatosensory cortex (S1)6,7,8, we focused our observation on BDA labeling in the thalamocortical projected axons and corticothalamic projected cell somas in the S1. By this process, we expected to provide not only a detailed protocol for obtaining the high quality of neural labeling with high molecular weight BDA, but also a refined protocol on the combination of fluorescent BDA labeling and other fluorescent neural markers with histochemistry or immunochemistry. This approach is preferable to study the local neural circuits and their chemical characteristics under a confocal laser scanning microscopy.

<table>
	<tbody>
		<tr>
			<td>Biotinylated dextran amine (BDA)</td>
			<td>Molecular Probes</td>
			<td>D1956</td>
			<td>10,000 molecular weight</td>
		</tr>
		<tr>
			<td>Streptavidin-Alexa Fluor 594</td>
			<td>Molecular Probes</td>
			<td>S32356</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>500/525 green fluorescent Nissl stain</td>
			<td>Molecular Probes</td>
			<td>N21480</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>Brain stereotaxis instrument</td>
			<td>Narishige</td>
			<td>SR-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing microtome</td>
			<td>Thermo</td>
			<td>Microm International GmbH</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal imaging</td>
			<td>Olympus</td>
			<td>FV1200</td>
			<td></td>
		</tr>
		<tr>
			<td>system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Drill</td>
			<td>Saeyang Microtech</td>
			<td>Marathon-N7</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague Dawley</td>
			<td>Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences</td>
			<td>SCKX (JUN) 2012-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectastain ABC Kit</td>
			<td>Vector Laboratories</td>
			<td>PK-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>superfrost plus microscope slides</td>
			<td>Thermo</td>
			<td>#4951PLUS-001</td>
			<td>25x75x1mm</td>
		</tr>
		<tr>
			<td>Photoshop and Illustration</td>
			<td>Adobe</td>
			<td>CS5</td>
			<td></td>
		</tr>
	</tbody>
</table>

improving the application of high molecular weight biotinylated dextran amine for thalamocortical projection tracing in the rat

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of its labeling was affected by various factors, here, we provide a refined protocol for the application of high molecular weight BDA for studying optimal neural labeling in the central nervous system. After stereotactic injection of BDA into the ventral posteromedial nucleus (VPM) of the thalamus in the rat through a delicate glass pipette, BDA was stained with fluorescent streptavidin-Alexa (AF) 594 and counterstained with fluorescent Nissl stain AF500/525. On the background of green Nissl staining, the red BDA labeling, including neuronal cell bodies and axonal terminals, was more distinctly demonstrated in the somatosensory cortex. Furthermore, double fluorescent staining for BDA and the calcium-binding protein parvalbumin (PV) was carried out to observe the correlation of BDA labeling and PV-positive interneurons in the cortical target, providing the opportunity to study the local neural circuits and their chemical characteristics. Thus, this refined method is not only suitable for visualizing high quality neural labeling with the high molecular weight BDA through reciprocal neural pathways between the thalamus and cerebral cortex, but also will permit the simultaneous demonstration of other neural markers with fluorescent histochemistry or immunochemistry.

Survival of 10 days post injection of BDA into the VPM was sufficient for producing intense neural labeling on the corresponding cortical areas ipsilateral to the injection side (Figure 2). Both conventional ABC and fluorescent staining procedures for BDA revealed the similar pattern of neural labeling on the S1, including anterogradely labeled thalamocortical axons and retrogradely labeled corticothalamic neurons (Figure 2C<stro...

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Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat

Here, we present a refined protocol to effectively reveal biotinylated dextran amine (BDA) labeling with a fluorescent staining method through a reciprocal neural pathway. It is suitable for analyzing the fine structure of BDA labeling and distinguishing it from other neural elements under a confocal laser scanning microscope.

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of ...

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8.9K Views.  China Academy of Chinese Medical Sciences. Here, we present a refined protocol to effectively reveal biotinylated dextran amine (BDA) labeling with a fluorescent staining method through a reciprocal neural pathway. It is suitable for analyzing the fine structure of BDA labeling and distinguishing it from other neural elements under a confocal laser scanning microscope.

Video: Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat

Progressive-ratio Responding for Palatable High-fat and High-sugar Food in Mice

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can produce a rewarding effect that strengthens action-outcome associations and reinforces future behavior directed at obtaining these foods. Increasing evidence that the rewarding effects of energy-dense foods play a profound role in overeating and the development of obesity has heightened interest in studying the genes, molecules and neural circuitry that modulate food reward1,2. The rewarding impact of different stimuli can be studied by measuring the willingness to work to obtain them, such as in operant conditioning tasks3. Operant models of food reward measure acquired and voluntary behavioral responses that are directed at obtaining food. A commonly used measure of reward strength is an operant procedure known as the progressive ratio (PR) schedule of reinforcement.4,5 In the PR task, the subject is required to make an increasing number of operant responses for each successive reward. The pioneering study of Hodos (1961) demonstrated that the number of responses made to obtain the last reward, termed the breakpoint, serves as an index of reward strength4. While operant procedures that measure changes in response rate alone cannot separate changes in reward strength from alterations in performance capacity, the breakpoint derived from the PR schedule is a well-validated measure of the rewarding effects of food. The PR task has been used extensively to assess the rewarding impact of drugs of abuse and food in rats (e.g.,6-8), but to a lesser extent in mice9. The increased use of genetically engineered mice and diet-induced obese mouse models has heightened demands for behavioral measures of food reward in mice. In the present article we detail the materials and procedures used to train mice to respond (lever-press) for a high-fat and high-sugar food pellets on a PR schedule of reinforcement. We show that breakpoint response thresholds increase following acute food deprivation and decrease with peripheral administration of the anorectic hormone leptin and thereby validate the use of this food-operant paradigm in mice.

The present report details the protocol employed to measure the rewarding effects of high-fat food in mice using a progressive ratio operant conditioning task.

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can ...

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 ...

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the brain by utilizing the swine pathogen pseudorabies virus (PRV)1. PRV does not infect great apes, including humans, so it is most commonly used in studies on small mammals, especially rodents. The pseudorabies strain PRV152 expresses the enhanced green fluorescent protein (eGFP) reporter gene and only crosses functional synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site2,3. Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)4,5&#160;.&#160;This protocol will deal exclusively with PRV152. By delivering the virus at a peripheral site, such as muscle, it is possible to limit the entry of the virus into the brain through a specific set of neurons. The resulting pattern of eGFP signal throughout the brain then resolves the neurons that are connected to the initially infected cells. As the distributed nature of transsynaptic tracing with pseudorabies virus makes interpreting specific connections within an identified network difficult, we present a sensitive and reliable method employing biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb) for confirming the connections between cells identified using PRV152. Immunochemical detection of BDA and CTb with peroxidase and DAB (3, 3&#39;-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites6-11.

Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This ...

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures.
A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

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 ...

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) experiments. Determining the appropriate protocol to follow throughout the sample preparation process can be difficult as each step must be optimized to comply with the unique characteristics of the analytes of interest. This process involves not only finding a compatible matrix that can desorb and ionize the molecules of interest efficiently, but also selecting the appropriate matrix deposition technique. For example, a wet matrix deposition technique, which entails dissolving a matrix in solvent, is superior for desorption of most proteins and peptides, whereas dry matrix deposition techniques are particularly effective for ionization of lipids. Sublimation has been reported as a highly efficient method of dry matrix deposition for the detection of lipids in tissue by MALDI IMS due to the homogeneity of matrix crystal deposition and minimal analyte delocalization as compared to many wet deposition methods 1,2. Broadly, it involves placing a sample and powdered matrix in a vacuum-sealed chamber with the samples pressed against a cold surface. The apparatus is then lowered into a heated bath (sand or oil), resulting in sublimation of the powdered matrix onto the cooled tissue sample surface. Here we describe a sublimation protocol using 1,5-diaminonaphthalene (DAN) matrix for the detection and visualization of gangliosides in the rat brain using MALDI IMS.

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass ...

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.