Supported by NIH/NIDCD R01 DC 011582. Imaging experiments were performed in the University of Colorado Anschutz Medical Campus Advanced Light Microscopy Core supported in part by NIH/NCRR Colorado CTSI Grant Number UL1 RR025780 and the Rocky Mountain Neurlogical Disorders Core Center Grant NIH P30NS048154. Dr. Sascha du Lac from the Salk Institute provided us with the GlyT2-GFP mice.

1. Optical Genotyping
1. Optical Genotyping of Mouse Pups
<ol>
	<li>Check for expression of the respective fluorescent marker using a laser pointer of the appropriate excitation wavelength (405 nm in the experiments described here) and corresponding filter goggles blocking the excitation wavelength but passing the emission wavelength (450 - 700 nm in the experiments described here). Point the laser pointer at the back of the head or the spinal cord of the mouse pup (see Figure&#160;1). Avoid shining the laser into the eyes and lengthy exposure of the skin to laser light.</li>
</ol>
2. Optical Genotyping of Older Animals
<ol>
	<li>Deeply anesthetize the mouse (aged p14 to p138 in the experiments described here) with an overdose of pentobarbital through an intraperitoneal injection (120 mg/kg bodyweight). Confirm proper anesthesia by checking the animal&#39;s reflexes (briefly pinch the tip of the ear or one of the hind legs to elicit the retraction reflex of the ear or leg).&#160;</li>
	<li>Note: Despite the use of an overdose of pentobarbital (120 mg/kg bodyweight) we refer to the injection procedure as &#34;deep anesthesia&#34; instead of euthanasia, because the animal is ideally still alive (i.e. its heart is still beating), when the surgery and the perfusion begin. This ensures a more efficient perfusion of the inner organs including the brain.&#160;</li>
	<li>Once the animal does not show any reflexes, carefully remove the skin overlying the skull (Figure&#160;2A) by making an incision in the skin covering the back of the head and cutting to the midsection of the skull. Expose the uncovered skull to laser light and observe the fluorescence through the filter goggles as described above. If a positive GFP signal is observed, proceed to step 2, if not, sacrifice the animal through decapitation&#160;(second/ensuring form of euthanasia following our IACUC regulations).&#160;</li>
</ol>
Note: In even older mice ( &#62;1 month) the fluorescence can be observed through the eyes of the animal.
2. Transcardial Perfusion and Brain Preparation
<ol>
	<li>Perfuse transcardially with ice-cold phosphate buffered saline (PBS; NaCl: 137 mM, KCl: 2.7 mM, KH2PO4: 1.76 mM, Na2HPO4: 10 mM) using a 30 G syringe needle to largely remove the blood from the animal.
	<ol>
		<li>First, carefully open the rib cage with sharp scissors and then push the needle into the left ventricle of the heart. Start pumping the PBS into the heart and the aorta and immediately following this step open the right atrium of the heart with the scissors to allow for the blood to exit the body. Note: Perfusion takes 5 - 10 min&#160;depending on the size of the animal.</li>
	</ol>
	</li>
	<li>After exsanguination through perfusion, decapitate the animal, secure its head with pin needles (or 30 G syringe needles) by piercing them through the eye sockets in a preparation dish and remove the brain from the skull.
	<ol>
		<li>First, use sharp scissors to cut off the skin overlaying the skull: make a small incision in the back of the head, then cut along the midline on the dorsal side of the head, cut to the sides just caudally of the eyes of the animal and lastly cut in the caudal direction to completely remove the flaps of skin at the back of the head.</li>
		<li>Next, repeat the same cutting pattern for the skull itself, make sure to remove both cochleas in the process, because this will significantly simplify the removal of the brain stem in the end. After this procedure the caudal half of the brain should lie completely exposed.</li>
		<li>In the next step, use a sharp razor blade or a scalpel to cut through the whole forebrain at an angle of about 45&#176; and about 2 mm&#160;rostral from the cerebellum. Scoop out the separated front half of the brain with a bent spatula.</li>
		<li>Use the spatula to elevate the caudal half of the brain at its rostral end and subsequently cut through the cranial nerves that are still connected to the brain stem on its ventral side. As an end result, the caudal half of the brain including the brainstem should fall freely off the rest of the prepared animal head.</li>
		<li>Flip the removed caudal half of the brain to expose its ventral side and cut along the coronal plane just rostral (for the anterograde injections) or just caudal (for the retrograde injections) of the ventrally located &#34;bulbs&#34; containing the trapezoid body (see step 2.3).
		<ol>
			<li>Always use a sharp razor blade or a scalpel for the cutting procedure to minimize cell death caused by excessive mechanical stress on the tissue.&#160;As an end result, a brain explant spanning about 1 cm in length and containing the complete superior olivary complex along other brain regions should have been obtained.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
Note: This protocol demonstrates the procedure for a tracer injection into the trapezoid body located in the auditory brainstem. Adapt the location and orientation of the cutting planes and injection sites as needed. If the fluorescing brain regions of interest lie close enough to the brain surface, so that they can be identified and injected without cutting the brain (see Figure&#160;2B), then the coronal cutting step can be omitted.
<ol start="3">
	<li>Identify the trapezoid body as two ventral prominent &#34;bulbs&#34; containing the ventral nucleus of the trapezoid body (VNTB).</li>
</ol>
Note: For further anatomical reference regarding the approximate stereotactic location of these cutting planes, see Franklin &#38; Paxinos, 2008. Panel 78 of the atlas, Bregma ~ -5.7 mm shows the medial nucleus of the trapezoid body (=MNTB) labeled as &#34;Tz&#34;. Panel 69 shows the ventral nucleus of the trapezoid body (VNTB) labeled as &#34;MVPO&#34; (medioventral periolivary nucleus)19.
3. Anterograde/Retrograde Tracing
Note: Perform all following procedures at RT&#160;(25&#176; C).
<ol>
	<li>After cutting, place the brainstem explant in a preparation dish containing oxygenated (95% O2, 5% CO2) dissecting solution (NaCl: 125 mM, KCl: 2.5 mM, MgCl2: 1 mM, CaCl2: 0.1 mM, glucose: 25 mM, NaH2PO4: 1.25 mM, NaHCO3: 25 mM, ascorbic acid: 0.4 mM, myo-inositol: 3 mM, pyruvic acid: 2 mM), securing it with 30 G syringe needles. Ensure that the area of injection is facing upwards.</li>
	<li>Pull injection pipettes from borosilicate glass and fill them with one of the following three solutions: a) a 1.0 mg/ml solution of choleratoxin subunit-b conjugated to an Alexa fluorophore 555 in PBS12-13 (mainly used for retrograde transport), b) a 1%-dilution in 0.9% saline of dextran tetramethylrhodamine 555 (3,000 MW, mainly used for retrograde transport) or c) a 1%-dilution in 0.9% saline of 10,000 MW dextran-TRITC 555 (mainly used for anterograde transport).
	<ol>
		<li>Ensure that injection pipette resistances range from 2.5 to 3.5 MOhm and that they are manufactured in 3 - 4 pulling cycles. For the experiments described here, achieve this by selecting a pre-programmed pulling algorithm on the pipette puller.</li>
	</ol>
	</li>
	<li>Illuminate the brain explant using a statically mounted laser pointer of the appropriate wavelength (item No. 2 in Figure&#160;3A; 405 nm wavelength in the experiments described here). Use the laser pointer as a guide for the injections, and aim the laser beam at the fluorescently labeled brain nucleus or cells of interest that will be injected.</li>
	<li>Find and observe the illuminated target area through a binocular microscope while wearing the compatible filter goggles (450 - 700 nm band-pass filter in the experiments described here). Insert the injection electrode via the micromanipulator into the area of interest.</li>
	<li>Inject the tracer substance. The injections consist of 2 - 10 pressure pulses at 15 psi for 50 msec each, using a pressure injection device, directed perpendicular into the region of interest (ROI) within the brain section. Administer each pressure pulse at intervals of 10 - 15 sec to allow for the dye to spread.</li>
	<li>In the case of the tetramethylrhodamine (TRITC) injections, additionally stimulate electrically to enhance the dye uptake through electroporation with the electrode placed in the brain area of interest (MNTB and VNTB in the experiments described here).
	<ol>
		<li>Use 8 TTL pulses of 8 Volts for 50 msec with 50 msec interpulse intervals, driven by a stimulator and amplified to 55 V with a stimulation isolation unit (items No. 7 and 9 in Figure&#160;3B). Use 10 - 20 repetitions of this protocol, administered over several minutes7,8. Make sure to ground the electrode properly - the grounding wire should be connected to the bath solution!</li>
	</ol>
	</li>
	<li>Check for success of the injection. Since a fluorescent tracer with a longer emission wavelength is also emitting a fluorescent signal upon laser excitation with shorter wavelengths, visually check for the dye uptake/spread in the injected target area while illuminating the area with the laser and observing through the filter goggles.</li>
</ol>
4. Incubation
<ol>
	<li>After the injection, incubate the brainstems in oxygenated artificial cerebrospinal fluid (ACSF; NaCl: 125 mM, KCl: 2.5 mM, MgCl2: 1 mM, CaCl2: 2 mM, glucose: 25 mM, NaH2PO4: 1.25 mM, NaHCO3: 25 mM, ascorbic acid: 0.4 mM, myo-inositol: 3 mM, pyruvic acid: 2 mM, bubbled with 95% O2 - 5% CO2) at room temperature (RT,&#160;25&#176; C) for 1 - 4 hr, while the dye is being transported. Bubble the solution during the entire incubation period.</li>
	<li>Subsequently, transfer the brainstems in 4% paraformaldehyde (PFA) dissolved in PBS (approx. 100 ml; pH 7 for the PFA/PBS mix) and incubate them at 4&#176; C to allow post-fixation overnight (O/N).</li>
</ol>
5. Tissue Slicing and Mounting
<ol>
	<li>The next day, wash the brainstem three times in PBS (5 min for each washing step), cover it in 4% agar and then cut into 50 - 80 &#956;m thick slices on a vibratome. Mount slices using a mounting medium on a glass slide, and coverslip.</li>
	<li>Optionally, label the sections with fluorescent Nissl for 25 min (for example blue Nissl at a 1:100 dilution in AB media).
	<ol>
		<li>To prepare AB media, mix 250 ml of 0.2 M phosphate buffer (PB; 51 mM KH2PO4, 150 mM Na2HPO4), 15 ml 5 M NaCl, 15 ml 10% Triton-X, 220 ml ddH2O and 5 g bovine serum albumine.</li>
		<li>Precede and succeed the Nissl labeling step by 3 washing steps in PBS (10 min each).</li>
	</ol>
	</li>
</ol>
Note: In cases where thicker sections need to be mounted and analyzed (in experiments described here such slices were 100 - 500 microns thick to preserve axonal connections over bigger distances), the technique can be combined with tissue clearing. We found the ClearT212 to be successful20. When a tissue clearing step is included, perform it before mounting the sections.

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We have nothing to disclose.

A general strength of in vitro tracer electroporation, as opposed to in&#160;vivo tracing studies, is that it gives researchers better access to the brain area of interest and hence, does not involve expensive stereotactic (and often electrophysiological) equipment. In addition, the survival period required for the brain explants spans only a few hours (1 - 4) instead of days or even weeks in the case of in vivo tracer injections (see a detailed review on the use of dextran amines and other tra...

In order to define a certain neuronal (micro)circuit, one must start by finding the various participants of said circuit, and their connection pattern. Ever since Waller&#39;s publication about neurofiber tracing through lesioning1 a large variety of neuroanatomical tracing techniques has been established. Some of these techniques can be applied in fixed tissue post mortem2-4, others rely on the active transport of the dye in live neurons, as discovered in 19715-6. The latter can be further subdivided in two groups discriminating between methods taking advantage of active retrograde (from the injected area to the source of a given projection, i.e. the somata of neurons that are projecting to said area) and active anterograde (from the injected area to the target of a given projection, i.e. the axonal projections and the axonal terminals of the labeled neurons) transport. Also, in some cases tracer material is injected into live animals which then survive the injection by several days or weeks (in&#160;vivo tracer injections), while in other cases explanted brains are injected and incubate for several hours after the injection in artificial cerebrospinal fluid (in vitro tracer injections).
In this protocol we modified an existing in vitro electroporation technique7-8 to label neuronal somata and processes in anterograde and retrograde tracing experiments using choleratoxin subunit-b and tetramethylrhodamine dextran as the tracing substances. The overall goal of this protocol is to provide neuroscientists with an efficient tool to trace neuronal connectivity patterns between different brain nuclei, while taking advantage of available transgenic mouse lines and basic optical equipment in order to increase targeting accuracy during tracer injections. Although the method of anterograde and retrograde tracing using choleratoxin and dextran amine and their respective fluorescently labeled conjugates is not new9-13 (as is the method of electroporation, e.g. Haas et al.14), the combination of tracer injections with electroporation in an in vitro preparation involving blocks of brain tissue is a more recent development7. Its main advantage over neuronal tracing techniques using the same type of tracer dyes in live animals is the increased labeling intensity, because of the higher efficiency with which the electroporated dye is being taken up by neurons. An additional advantage is the shortened incubation period (required for the dye transport) and its increased target accuracy during the tracer injection, because the experimenter has visual control over the target area of the injection. The latter also means that no expensive stereotactic equipment is required to find the nucleus or brain area of interest.
To additionally increase targeting accuracy, we took advantage of a transgenic mouse line, which expresses GFP in its glycinergic subpopulation of neurons15 and basic optical equipment consisting of a hand-held laser pointer of 405 nm wavelength and matching band-pass filtering goggles (450 - 700 nm). Thus, we achieved a significant further increase in targeting accuracy by identifying the injection area through its fluorescent signal and by providing a finer way to control for the dye spread within the injection area through the observation of the interaction between the indigenous GFP signal and the tracer fluorescence. Our technique also allows to uncover the functionality of a circuit along with its connectivity by identifying GFP-positive inhibitory neurons (or excitatory in other mouse lines) that were filled with the tracer.
In summary, we further enhanced a powerful neuroscientific tool to study the connectome of the vertebrate brain and assess the different neuroanatomical features of a given neurocircuit. By using transgenic mice along with inexpensive and widely available optical equipment we were able to significantly increase the targeting accuracy of our injections. Furthermore, the transgenic mice allowed us to identify the type of the traced connections, which helped uncovering the functionality of an inhibitory microcircuit in the auditory brainstem.

<table><tbody><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S7653</td><td>All chemicals are from Sigma-Aldrich, unless noted otherwise.</td></tr><tr><td>Potassium chloride&amp;nbsp;</td><td></td><td>P9333</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td></td><td>P5655</td><td></td></tr><tr><td>Sodium phosphate di-basic</td><td></td><td>S907</td><td></td></tr><tr><td>Magnesium chloride</td><td></td><td>M2670</td><td></td></tr><tr><td>Calcium chloride</td><td></td><td>C5080</td><td></td></tr><tr><td>Glucose</td><td></td><td>G7528</td><td></td></tr><tr><td>Sodium bicarbonate</td><td></td><td>S6297</td><td></td></tr><tr><td>Ascorbic acid</td><td></td><td>A4544</td><td></td></tr><tr><td>Myo-inositol</td><td></td><td>I5125</td><td></td></tr><tr><td>Sodium pyruvate</td><td></td><td>P2256</td><td></td></tr><tr><td>Bovine serum albumine</td><td></td><td>A2153</td><td>optional, for additional (immuno)histochemistry</td></tr><tr><td>Triton-X-100</td><td></td><td>X100</td><td>optional, for additional (immuno)histochemistry</td></tr><tr><td>Poly(ethylene glycol), 8000 MW</td><td></td><td>P2139</td><td>optional, for brain clearing</td></tr><tr><td>Formamide</td><td>Fisher Scientific</td><td>F84</td><td>optional, for brain clearing</td></tr><tr><td>Choleratoxin subunit-b</td><td>Molecular Probes</td><td>C-34776 (Alexa 555)&amp;nbsp;</td><td></td></tr><tr><td>Dextrane tetramethyl-rhodamine</td><td>Molecular Probes</td><td>D-7162 (Alexa 555)</td><td></td></tr><tr><td>Fluorescent Nissl</td><td>Invitrogen</td><td>N-21479 (blue)</td><td>optional</td></tr><tr><td>Paraformaldehyde</td><td>Fisher Scientific</td><td>SF93</td><td></td></tr><tr><td>Agarose</td><td>Invitrogen</td><td>16520</td><td></td></tr><tr><td>Fluoromount-G</td><td>Southern Biotech</td><td>0100-01</td><td></td></tr><tr><td>Pentobarbi-tal</td><td>Vortech Pharmaceuticals</td><td>Fatal-Plus&amp;nbsp;</td><td></td></tr><tr><td>Borosilicate glass filaments</td><td>Harvard Apparatus</td><td>G150F-10</td><td></td></tr><tr><td>Pipette puller</td><td>Zeitz Instruments, Germany</td><td>DMZ Universal Puller</td><td></td></tr><tr><td>Perfusion setup</td><td>Custom-made</td><td></td><td></td></tr><tr><td>Laser pointer&amp;nbsp;</td><td>laserpointerpro.com, Hong Kong</td><td>HK-88007294 (405 nm)</td><td></td></tr><tr><td>Filter/safety goggles</td><td>Dragon Lasers, China</td><td>LSG09 (band-pass 450-700 nm)</td><td></td></tr><tr><td>Bionocular microscope&amp;nbsp;</td><td>Wild Heerbrugg, Switzerland</td><td>Wild M3</td><td>Equipped with high-intensity illuminator (MI-150; Dolan-Jenner Inc.)&amp;nbsp;</td></tr><tr><td>Picospritzer</td><td>Parker Instruments</td><td>Picospritzer III</td><td></td></tr><tr><td>PC with installed MC Stimulus software</td><td>Multi Channel Sys-tems, Germany (software)</td><td></td><td></td></tr><tr><td>2-channel stimulator</td><td>Multi Channel Sys-tems, Germany</td><td>STG-1002</td><td></td></tr><tr><td>Stimulation isolation unit</td><td>A.M.P.I., Israel</td><td>Iso-Flex</td><td></td></tr><tr><td>Micromanipulator</td><td>Narishige, Japan</td><td>YOU-1</td><td></td></tr><tr><td>Vibratome</td><td>Leica, Germany</td><td>VT1000S</td><td></td></tr></tbody></table>

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.

Figures 1 and 2 show how the laser pointer and laser goggles can be used to quickly and inexpensively genotype GFP positive animals from a litter. In cases of young mouse pups, the technique can be used to noninvasively identify GFP label in the animal&#39;s brains through the skull and overlying skin (Figure&#160;1A - D). Emitted fluorescence can be seen through the skin and the skull of mouse pups at least up to postnatal day 3 (Figure&#160;1C). The procedure is shown ...

Watch this Scientific Journal Video about Laser-guided Neuronal Tracing In Brain Explants at JoVE.com

Laser-guided Neuronal Tracing In Brain Explants

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 ...

laser-guided-neuronal-tracing-in-brain-explants

Research

JoVE Journal

Neuroscience

8.8K Views.  University of Colorado School of Medicine. 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.

Video: Laser-guided Neuronal Tracing In Brain Explants

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

Genetics

Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult retinal neurons in vitro. Procedures for preparing retinal cell cultures start with the dissection, digestion and trituration of the retina, and end with the plating of isolated retinal cells on dishes made especially for use with laser tweezers. These dishes are divided into a cell adhesive half and a cell repellant half. The cell adhesive side is coated with a layer of Sal-1 antibodies, which provide a substrate upon which our cells grow. Other adhesive substrates could be used for other cell types. The cell repellant side is coated with a thin layer of poly-HEMA. The cells plated on the poly-HEMA side of the dish are trapped with the laser tweezers, transported and then placed adjacent to a cell on the Sal-1 side to create a pair. Formation of cell groups of any size should be possible with this technique. "Laser-tweezers-controlled micromanipulation" means that the investigator can choose which cells to move, and the desired distance between the cells can be standardized. Because the laser beam goes through transparent surfaces of the culture dish, cell selection and placement are done in an enclosed, sterile environment. Cells can be monitored by video time-lapse and used with any cell biological technique required. This technique may help investigations of cell-cell interactions.

This video describes the manipulation of cultured neurons using laser tweezers in vitro.

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult ...

Biology

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits&#160;in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations&#160;in vitro.

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the ...

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 ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point measurements and can therefore only evaluate longitudinal changes of cell migration and survival to a limited extent. This paper provides a reliable, minimally invasive protocol to transplant and longitudinally track neural progenitor cells (NPCs) in the adult mouse brain. Before transplantation, cells are transduced with a lentiviral vector comprising a bioluminescent (firefly-luciferase) and fluorescent (green fluorescent protein [GFP]) reporter. The NPCs are transplanted into the right cortical hemisphere using stereotaxic injections in the sensorimotor cortex. Following transplantation, grafted cells were detected through the intact skull for up to five weeks (at days 0, 3, 14, 21, 35) with a resolution limit of 6,000 cells using in vivo bioluminescence imaging. Subsequently, the transplanted cells are identified in histological brain sections and further characterized with immunofluorescence. Thus, this protocol provides a valuable tool to transplant, track, quantify, and characterize cells in the mouse brain.

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

We describe the intraparenchymal transplantation of human neural progenitor cells transduced with a dual reporter vector expressing luciferase-green fluorescent protein (GFP) in the mouse brain. After transplantation, the luciferase signal is repeatedly measured using in vivo bioluminescence and GFP-expressing grafted cells identified in brain sections using fluorescence microscopy.

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point ...

Laser-guided Neuronal Tracing In Brain Explants

Summary

Explore More Videos

Chapters in this video

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

Neural Explant Cultures from Xenopus laevis

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

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

Laser Nanosurgery of Cerebellar Axons In Vivo

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain