Laser-guided Neuronal Tracing In Brain Explants

Summary

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.

Abstract

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.

Introduction

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'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 mortem^2-4, others rely on the active transport of the dye in live neurons, as discovered in 1971^5-6. 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 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 technique^7-8 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 new^9-13 (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.

Protocol

1. Optical Genotyping

1. Optical Genotyping of Mouse Pups

1. 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 1). Avoid shining the laser into the eyes and lengthy exposure of the skin to laser light.

2. Optical Genotyping of Older Animals

1. 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'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). 
2. Note: Despite the use of an overdose of pentobarbital (120 mg/kg bodyweight) we refer to the injection procedure as "deep anesthesia" 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. 
3. Once the animal does not show any reflexes, carefully remove the skin overlying the skull (Figure 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 (second/ensuring form of euthanasia following our IACUC regulations). 

Note: In even older mice ( >1 month) the fluorescence can be observed through the eyes of the animal.

2. Transcardial Perfusion and Brain Preparation

1. 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.
   1. 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 depending on the size of the animal.
2. 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.
   1. 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.
   2. 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.
   3. In the next step, use a sharp razor blade or a scalpel to cut through the whole forebrain at an angle of about 45° and about 2 mm rostral from the cerebellum. Scoop out the separated front half of the brain with a bent spatula.
   4. 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.
   5. 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 "bulbs" containing the trapezoid body (see step 2.3).
      1. 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. 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.

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 2B), then the coronal cutting step can be omitted.

3. Identify the trapezoid body as two ventral prominent "bulbs" containing the ventral nucleus of the trapezoid body (VNTB).

Note: For further anatomical reference regarding the approximate stereotactic location of these cutting planes, see Franklin & Paxinos, 2008. Panel 78 of the atlas, Bregma ~ -5.7 mm shows the medial nucleus of the trapezoid body (=MNTB) labeled as "Tz". Panel 69 shows the ventral nucleus of the trapezoid body (VNTB) labeled as "MVPO" (medioventral periolivary nucleus)¹⁹.

3. Anterograde/Retrograde Tracing

Note: Perform all following procedures at RT (25° C).

1. 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.
2. 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 PBS_^12-13 (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).
   1. 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.
3. Illuminate the brain explant using a statically mounted laser pointer of the appropriate wavelength (item No. 2 in Figure 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.
4. 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.
5. 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.
6. 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).
   1. 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 3B). Use 10 - 20 repetitions of this protocol, administered over several minutes^7,8. Make sure to ground the electrode properly - the grounding wire should be connected to the bath solution!
7. 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.

4. Incubation

1. 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, 25° C) for 1 - 4 hr, while the dye is being transported. Bubble the solution during the entire incubation period.
2. 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° C to allow post-fixation overnight (O/N).

5. Tissue Slicing and Mounting

1. 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 μm thick slices on a vibratome. Mount slices using a mounting medium on a glass slide, and coverslip.
2. Optionally, label the sections with fluorescent Nissl for 25 min (for example blue Nissl at a 1:100 dilution in AB media).
   1. 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.
   2. Precede and succeed the Nissl labeling step by 3 washing steps in PBS (10 min each).

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.

Results

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's brains through the skull and overlying skin (Figure 1A - D). Emitted fluorescence can be seen through the skin and the skull of mouse pups at least up to postnatal day 3 (Figure 1C). The procedure is shown in Fig...

Discussion

A general strength of in vitro tracer electroporation, as opposed to in 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...

Materials

List of materials used in this article

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