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&#160;</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)&#160;</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&#160;</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&#160;</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&#160;</td>
			<td>Wild Heerbrugg, Switzerland</td>
			<td>Wild M3</td>
			<td>Equipped with high-intensity illuminator (MI-150; Dolan-Jenner Inc.)&#160;</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

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Closed-loop Neuro-robotic Experiments to Test Computational Properties of Neuronal Networks

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation of a given stimulus might be dependent on context and history. If this is actually the case, bi-directional interactions between the brain (or if need be a reduced model of it) and sensory-motor system can shed a light on how encoding and decoding of information is performed. Here an experimental system is introduced and described in which the activity of a neuronal element (i.e., a network of neurons extracted from embryonic mammalian hippocampi) is given context and used to control the movement of an artificial agent, while environmental information is fed back to the culture as a sequence of electrical stimuli. This architecture allows a quick selection of diverse encoding, decoding, and learning algorithms to test different hypotheses on the computational properties of neuronal networks.

In this paper, an experimental framework to perform closed-loop experiments is presented, in which information processing (i.e., coding and decoding) and learning of neuronal assemblies are studied during the continuous interaction with a robotic body.

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation ...

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 ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...