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...

Watch this Scientific Journal Video about Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat at JoVE.com

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 ...

improving-application-high-molecular-weight-biotinylated-dextran

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

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

Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

It is well known that anesthesia alters neural response properties in various regions of the brain.13. In the auditory system, fundamental response properties of brainstem neurons including threshold, frequency specificity, and inhibitory sidebands are altered in significant ways under anesthesia1-2. These observations prompted physiologists to seek ways to record from single neurons without the contaminating effects of anesthesia. One result was a decerebrate preparation, where the brainstem was completely transected at the level of the midbrain4. The drawbacks of this preparation are a formidable surgery, the elimination of descending projections from the forebrain, and an inability to use sensory stimulation to examine structures above the midbrain. A different strategy has been to implant electrode arrays chronically to record from single neurons and multiunit clusters while the animal is awake and/or behaving5,6. These techniques however are not compatible with injecting tracer dyes after first electrophysiologically characterizing a brain structure. To avoid altering neural response properties with anesthetics while recording electrophysiological response properties from single neurons, we have adapted a head restraint technique long used in bats7-9 to mouse10-12. Using this method, we are able to conduct electrophysiological recordings over several days in the unanesthetized mouse. At the end of the recording sessions, we can then inject a dye to reconstruct electrode positions and recording sites or inject a tracer so that pathways to and from the recording loci can be determined. This method allows for well isolated single neuron recordings over multiple days without the use anesthetics.

Electrophysiological characterization of neuronal responses is important for understanding brain function and for guiding the placement of dyes for pathway tracing. However, many studies are performed in anesthetized animals. To understand brain function without anesthetics, we developed a method to record neuronal response properties and inject dyes in awake mouse.

It is well known that anesthesia alters neural response properties in various regions of the brain.13.  In the auditory system, fundamental response ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, direction5-7, ocular dominance8,9 and binocular disparity9. Orientation selectivity is the most widely studied feature and a continuous map with a quasi-periodic layout for preferred orientation is present across the entire primary visual cortex10,11. Integrating the synaptic, cellular and network contributions that lead to stimulus selective responses in these functional maps requires the hybridization of imaging techniques that span sub-micron to millimeter spatial scales. With conventional intrinsic signal optical imaging, the overall layout of functional maps across the entire surface of the visual cortex can be determined12. The development of in vivo two-photon microscopy using calcium sensitive dyes enables one to determine the synaptic input arriving at individual dendritic spines13 or record activity simultaneously from hundreds of individual neuronal cell bodies6,14. Consequently, combining intrinsic signal imaging with the sub-micron spatial resolution of two-photon microscopy offers the possibility of determining exactly which dendritic segments and cells contribute to the micro-domain of any functional map in the neocortex. Here we demonstrate a high-yield method for rapidly obtaining a cortical orientation map and targeting a specific micro-domain in this functional map for labeling neurons with fluorescent dyes in a non-rodent mammal. With the same microscope used for two-photon imaging, we first generate an orientation map using intrinsic signal optical imaging. Then we show how to target a micro-domain of interest using a micropipette loaded with dye to either label a population of neuronal cell bodies or label a single neuron such that dendrites, spines and axons are visible in vivo. Our refinements over previous methods facilitate an examination of neuronal structure-function relationships with sub-cellular resolution in the framework of neocortical functional architectures.

A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

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 ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive functions including decision making, sensory-guided behavior or memory. To understand how such intricate neuronal networks perform such tasks, a crucial step is to determine the function (or electrical activity) of individual cell types within the network, preferentially when the animal is performing a relevant cognitive task. Additionally, it is equally important to determine the anatomical structure of the network and the morphological architecture of the individual neurons to allow reverse engineering the cortical network. Technical breakthroughs available today allow recording cellular activity in awake, behaving animals with the valuable option of post&#160;hoc identifying the recorded neurons. Here, we demonstrate the juxtasomal biocytin labeling technique, which involves recording action potential spiking in the extracellular (or loose-patch) configuration using conventional patch pipettes. The juxtasomal recording configuration is relatively stable and applicable across behavioral conditions, including anesthetized, sedated, awake head-fixed, and even in the freely moving animal. Thus, this method allows linking cell-type specific action potential spiking during animal behavior to reconstruction of the individual neurons and ultimately, the entire cortical microcircuit. In this video manuscript, we show how individual neurons in the juxtasomal configuration can be labeled with biocytin in the urethane-anaesthetized rat for post&#160;hoc identification and morphological reconstruction.

To understand the structure of neuronal networks, functional and morphological characterization of individual neurons is a necessity. Here, we demonstrate juxtasomal biocytin labeling, which allows electrophysiological recordings in the extracellular configuration, yet maintaining the ability to intracellularly label the neuron for post&#160;hoc reconstruction of dendritic and axonal architecture.

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a premise for determining the role of neuron types within a neural circuit, and their significance in information encoding relative to learning. Likewise, the method can be used to test the physiological significance of two or more connected brain regions in awake and anesthetized animals. The current study demonstrates how VTA glutamate neurons modulate the firing rate of putative pyramidal neurons in the CA1 (hippocampus) of anesthetized mice. This protocol employs adeno-associated virus (AAV)-dependent labeling of VTA glutamate neurons for the tracing of VTA presynaptic glutamate terminals in the layers of the hippocampus. Expression of light-controlled opsin (channelrhodopsin; hChR2) and fluorescence protein (eYFP) harbored by the AAV vector permitted anterograde tracing of VTA glutamate terminals, and photostimulation of VTA glutamate neuron cell bodies (in the VTA). High-impedance acute silicon electrodes were positioned in the CA1 to detect multi-unit and single-unit responses to VTA photostimulation in vivo. The results of this study demonstrate the&#160;layer-dependent distribution of presynaptic VTA glutamate terminals in the hippocampus (CA1, CA3, and DG). Also, the photostimulation of VTA glutamate neurons increased the firing and burst rate of putative CA1 pyramidal units in vivo.

The current protocol demonstrates a simple method for tracing of ventral tegmental area (VTA) glutamate projections to the hippocampus. Photostimulation of VTA glutamate neurons was combined with CA1 recording to demonstrate how VTA glutamate terminals modulate CA1 putative pyramidal firing rate in vivo.

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...

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

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Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

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

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

Laser-guided Neuronal Tracing In Brain Explants

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus