Exclusive labeling of direct and indirect pathway neurons in the mouse neostriatum by an adeno-associated virus vector with Cre/lox system

Summary We developed an adeno-associated virus (AAV) vector-based technique to label mouse neostriatal neurons comprising direct and indirect pathways with different fluorescent proteins and analyze their axonal projections. The AAV vector expresses GFP or RFP in the presence or absence of Cre recombinase and should be useful for labeling two cell populations exclusively dependent on its expression. Here, we describe the AAV vector design, stereotaxic injection of the AAV vector, and a highly sensitive immunoperoxidase method for axon visualization. For complete details on the use and execution of this protocol, please refer to Okamoto et al. (2020).


SUMMARY
We developed an adeno-associated virus (AAV) vector-based technique to label mouse neostriatal neurons comprising direct and indirect pathways with different fluorescent proteins and analyze their axonal projections. The AAV vector expresses GFP or RFP in the presence or absence of Cre recombinase and should be useful for labeling two cell populations exclusively dependent on its expression. Here, we describe the AAV vector design, stereotaxic injection of the AAV vector, and a highly sensitive immunoperoxidase method for axon visualization. For complete details on the use and execution of this protocol, please refer to Okamoto et al. (2020).

BEFORE YOU BEGIN
We developed a method using an adeno-associated virus (AAV) vector system to label two groups of neurons with different fluorescent proteins. The vector contains a flip-excision (FLEX) switch (Schnutgen et al., 2003), and expresses GFP or RFP based on the expression, or the lack, of Cre recombinase ( Figures 1A and 1B). This labeling method offers a great advantage for the simultaneous labeling of mutually exclusive systems in the nervous system, such as excitatory and inhibitory neurons.
After preparation of the AAV vector, we injected the vector into the mouse neostriatum (caudate-putamen, CPu), and then analyzed the axonal projections of direct and indirect pathway medium-sized spiny neurons (dMSNs and iMSNs) . The production and purification methods of AAV vectors have been described in previous studies (Hamamoto et al., 2017;Ito et al., 2015;Iwano et al., 2018;Kataoka et al., 2014;Sohn et al., 2016;Sohn et al., 2017;Suzuki et al., 2015). We have also recently established a method to stably obtain a high-titer virus solution by recovering virus particles from cell pellets and the supernatant (medium) . For a detailed protocol of the AAV vector preparation, refer to the literature.
Here, we describe step-by-step procedures for stereotaxic injection of a virus solution into the mouse CPu, signal enhancement via peroxidase activity, and axon tracing. In particular, the signal enhancement method, namely the biotinylated tyramine-glucose oxidase (BT-GO) reaction, is cost-effective and straightforward Kuramoto et al., 2009) and should be useful for experiments that require specific staining with a high signal-to-noise ratio. Figure 1. Exclusive labeling of Cre-positive and -negative cells by an AAV vector (A) Construct of the AAV vector. The vector expresses RFP or GFP in the absence or presence of Cre recombinase by a flipexcision (FLEX) switch. Both GFP and RFP are tagged with a membrane-targeting protein, palmitoylation signal (pal). (B) Schematic diagram of the exclusive labeling method. Two cell populations are selectively labeled with different fluorescent proteins based on the expression of Cre recombinase. BGHpA, a polyadenylation signal derived from bovine growth hormone gene; E/SYN, enhanced human synapsin I promoter (Hioki et al., 2007); ITR, inverted terminal repeat. Filter the solution through filter paper to remove any debris. The solution may be preserved at 20 C-25 C for up to 1 year.

Reagent
Final concentration Amount  Note: When all biotin-NHS reacts with tyramine hydrochloride, 128 mM of BT solution will be obtained.

Reagent Final concentration Amount
Sodium azide 20% (w/v) 2 g ddH 2 O N/A up to 10 mL The solution may be preserved at 20 C-25 C for up to 1 year.

Reagent
Final concentration Amount

STEP-BY-STEP METHOD DETAILS Virus injection
Timing:~1 h Here, we describe the stereotaxic injection of an AAV vector solution (200 nL) into the mouse CPu.
1. Disinfect the instruments for use in the injection experiment with 70% ethanol. Please refer to institutional guidelines for specific instructions on proper aseptic technique. 2. Fabricate a glass micropipette using a PC-100 puller with a single pulling mode. Set the heating value to 73.5 and pulling force to 125 g ( Figures 3A and 3B).

Note:
The setting values will vary depending on the filament. Find the best conditions for your purpose.
Note: The solution may be preserved at 4 C for up to 1 month.
6. Aspirate 500 nL of an AAV vector solution on a Parafilm sheet through the tip of a glass micropipette using capillary action, and mark the top surface of the solution with a permanent marker.

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(e.g., 500 nL) and mark the level with a permanent marker so that you can inject the exact amount of the virus solution ( Figure 3D).  and butorphanol tartrate (Vetorphale; 5 mg/kg) in saline (0.9% NaCl). Please refer to institutional guidelines for proper anesthesia. 8. Mount the anesthetized mouse into a stereotaxic instrument using an auxiliary ear bar. Adjust the position of the mouth and nose fixing clamp (incisor bar) until the lambda and bregma are equal in height (flat-skull position) ( Figure 3E). 9. Make an incision in the scalp with a scalpel along the midline to expose the skull. 10. After marking the skull above the CPu with a surgical marker-pen (NESCO DERMARK R), thin the skull above the marked site using a hand drill with a steel hole cutter ( Figure 3F). The injection coordinates for the CPu are as follows: 0.8 mm anterior to bregma, 2.0 mm lateral to the midline, and 2.5 mm ventral to the brain surface.
CRITICAL: While thinning the skull by a hand drill, wet with saline appropriately to avoid damage to the cortices by frictional heat.
11. Carefully remove the remaining bone using fine-tip tweezers to expose the surface of the brain. 12. Set the Picospritzer III to 40 psi injection pressure and 5 ms injection duration ( Figures 3G and 3H).
CRITICAL: Eject a drop of virus solution from the tip of the glass micropipette to make sure it is not clogged, before inserting it into the brain. Troubleshooting 1 13. Move the glass micropipette to the brain surface and slowly insert it into the target ( Figure 3I). Pull the micropipette upward approximately 0.05 mm to make space for the virus solution to diffuse. Troubleshooting 2, 3, and 4 14. Inject the virus solution into the mouse CPu by pressure pulses through a glass micropipette attached to Picospritzer III for 5 min. Troubleshooting 5 15. Leave the micropipette in place for 5 min, and then remove it slowly. 16. After closing and sterilizing the wound, administer antibiotics (e.g., gentamicin ointment) locally. 17. Recover the mouse from anesthesia by intraperitoneal injection of atipamezole (Antisedan; 1.5 mg/kg) in saline after approximately 15 min. Please refer to institutional guidelines for anesthesia management. 18. Maintain the mouse in specific pathogen-free conditions under a 12 h light/dark cycle with ad libitum access to food and water for 1 week after the AAV injection.

Tissue preparation
Timing: 2-3 days Note: Filtrate PBS and the fixative solution through filter paper before perfusion.
Here, we introduce the preparation of sections for free-floating immunostaining.
19. Anesthetize the mouse deeply by intraperitoneal injection of sodium pentobarbital (Somnopentyl; 200 mg/kg) in saline. Please refer to institutional guidelines for proper anesthesia. 20. Open the thoracic cavity with surgical scissors, cut the right atrial appendage to bleed, and immediately insert a 22-gauge needle from the apex into the left ventricle of the heart. 21. Perfuse with 20 mL of PBS using a syringe for 3 min to remove the blood from the circulatory system. 22. Perfuse with 20 mL of a fixative solution using another syringe at the same speed.
CRITICAL: Be careful not to allow any small bubbles to enter during the perfusion. The inclusion of bubbles results in incomplete fixation.
23. Remove the brain from the skull and place it in the same fixative solution for 12-24 h at 4 C.

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24. Replace the fixative solution with 30% (w/v) sucrose in 0.1 M PB and gently shake it for 1-2 days at 4 C to cryoprotect the brain tissue. 25. Approximately 30 min before sectioning, turn on the low temperature circulator and set it to 4 C ( Figure 4A). 26. Mount the brain tissue onto the stage of a freezing microtome with OCT compound, and adjust the stage angle to keep the sagittal plane of the brain horizontal ( Figure 4C). 27. Turn on the electro-freezing component and set it to À25 C. 28. Quickly freeze the brain tissue using dry ice powder and leave it in place for at least 5 min (Figure 4D).
Note: We recommend dry ice powder prepared by a snow horn for rapid tissue freezing ( Figure 4B).
29. Cut the brain tissue into 20-mm-thick parasagittal sections ( Figure 4E), collect the sections with a paintbrush, and store them in 6 vials containing 0.02% sodium azide in 0.1 M PB ( Figure 4F). Each vial will contain around 20 sections.
Note: For long-term storage (e.g., 5 years), increase the concentration of sodium azide to 0.2%.

Immunofluorescence staining
Timing: 2 days Here, we explain the procedure for double immunofluorescence staining for GFP and RFP. All the incubations are performed at 20 C-25 C with a gentle shake.
30. Wash the sections with 8 mL of PBS for 10 min twice, and then with PBS-X for 30 min. 31. Incubate the sections for 12-24 h with a mixture of 20 mg/mL of chicken polyclonal anti-GFP antibody and 1 mg/mL of affinity-purified rabbit antibody against mRFP1 in 500 mL of PBS-XCD. Note: The sections can be kept for about 2 years at À20 C. Figures 5A-5F). Troubleshooting 6 and 7

Immunoperoxidase staining
Timing: 4 days CRITICAL: Before incubation with the sections, mix solutions A and B of ABC Elite kit at 1:100 dilution each in 500 mL of PBS-X and incubate the mixture for at least 30 min at 20 C-25 C with a gentle shake to form the avidin-biotinylated peroxidase complex (ABC).
We developed a cost-effective and straightforward signal amplification method called BT-GO reaction. Here, we describe the protocol in detail. All the incubations are performed at 20 C-25 C with a gentle shake.
38. Wash the sections with 8 mL of PBS for 10 min twice. Note: The optimal concentration of the BT solution used in the reaction should be determined by the end user. We recommend a dilution of the solution from 1:5,000 to 1:500,000 for the trial.
49. Add 10 mL of b-D-glucose solution to a final concentration of 2 mg/mL and mix the solution and sections well. 50. Incubate the sections for 30 min ( Figure 6A). 51. Wash the sections with 8 mL of 0.1 M PB for 10 min. 52. Wash the sections with 8 mL of PBS-X for 10 min twice. 53. Incubate the sections for 1 h with 1/100-diluted ABC in 500 mL of PBS-X. 54. Wash the sections with 8 mL of PBS-X for 10 min twice. 55. Wash the sections with 8 mL of PBS for 10 min twice. 56. Incubate the sections for 30-60 min with 0.02% (w/v) DAB and 0.0001% (v/v) H 2 O 2 in 8 mL of 50 mM Tris-HCl (pH 7.6).
CRITICAL: Check the progress of DAB deposition under the microscope and stop the reaction at a high signal-to-noise ratio.  (Figures 6B and 6C) and capture images using a Nikon D-700 camera. Troubleshooting 8 Note: Specimens can be stored for at least 10 years at 20 C-25 C with protection from light.

Axon tracing
Timing:~4 days By using the virtual slide system, the efficiency of the two-dimensional reconstruction is greatly increased. We briefly describe the reconstruction method using a TOCO virtual slide scanner.
65. Acquire a color image of the entire section with a spatial resolution of 1.038 mm/pixel using the digital slide scanner TOCO with a 103 objective lens. 66. Trace and digitize the axons of DAB-stained neurons on the images with a digital pen tablet and a graphic software ADOBE ILLUSTRATOR CS3.
Alternatives: Virtual slide systems from other manufacturers are also available.
Note: Axons are sometimes out of focus, where multiple axon fibers cross one another. Additional observations with a bright field microscope may also be required to reconstruct the axons accurately.

EXPECTED OUTCOMES
The stereotaxic injection method allows us to deliver the transgene to the restricted area of the mouse CPu (Figure 3). The range of the infection area will be isotropic, with a radius of 0.5 to 0.8 mm. If the infection area is further confined, it may be more effective to use iontophoresis than pressure injection Wang et al., 2014). Moreover, section preparation with a freezing microtome not only completes the process in a short time but may also lead to the improvement of antibody penetration and a signal-to-noise ratio due to freeze-thaw treatment ( Figure 4).
By injecting an AAV vector solution into the CPu of a Drd1-Cre mouse, we successfully visualized dMSNs and iMSNs with different fluorescent proteins, GFP and RFP. While the projection of RFP-ll OPEN ACCESS positive axon fibers was restricted to the external segment of the globus pallidus (GPe), GFP-positive axon fibers extended caudally beyond the region ( Figure 5). We then amplified the signals of GFPpositive and RFP-positive axon fibers in the GPe using the BT-GO reaction ( Figure 6). The reaction remarkably enhanced the signals while leaving the noise unaltered, thus contributing to the increase in the signal-to-noise ratio.

LIMITATIONS
The present exclusive labeling method allows us to visualize two cell populations with different fluorescent proteins based on the expression of Cre recombinase. However, the Cre-negative population may contain multiple cell types. Indeed, in addition to iMSNs, interneurons in the CPu not expressing Cre recombinase were labeled with RFP in the present study. Nonetheless, they can be distinguished by their morphology, such as the cell body size and the presence of spines on the dendrites . Given that interneurons do not send axons outside of the CPu, the distributions of dMSN and iMSN axon fibers, which are the focus of our study, can be quantitatively analyzed within the GPe. When using this exclusive labeling method, careful attention should be paid to the characteristics of the Cre-negative cell population.
Direct delivery of the virus vectors with stereotaxic injections has the advantage of being suitable for any specific region of the animal brain at any time. On the other hand, a disadvantage is that the infection area is limited. When a wide-area gene transfer is required, an AAV serotype that efficiently crosses the blood-brain barrier, such as AAV-PHP.eB (Chan et al., 2017), should be administrated intravenously.
The BT-GO reaction efficiently increases the signal-to-noise ratio; we Kuramoto et al., 2009) and others (Igarashi et al., 2012;Sun et al., 2020) have applied this method to axonal projection analysis. However, bright-field staining by DAB deposition via the BT-GO reaction has the drawback that the GFP and RFP signals cannot be observed simultaneously in the same section. The development of signal amplification methods that enable fluorescent multi-color labeling is expected in future studies.

TROUBLESHOOTING Problem 1
No virus solution is released from the glass micropipette (step 12).

Potential solution
Set the injection duration of Picospritzer III to a longer time. If the solution is still not ejected, check the shape of the glass micropipette tip under a stereomicroscope.

Problem 2
The glass micropipette cannot be inserted into the brain (step 13).

Potential solution
Perform a small incision in the dura mater with fine-tip tweezers or a needle.

Problem 3
The infection site is out of the target position (step 13).

Potential solution
In stereotactic injection, it is critical to mount animals into a stereotaxic apparatus properly. In particular, ensure that the ear bars are inserted properly.

Problem 4
Infected cells are frequently observed in the area of passage of the glass micropipette (step 13).

Potential solution
The following points should be verified: 1) the glass micropipette is thin, 2) there is no bubble inside the glass micropipette, 3) slow injection of the virus solution, and 4) slow removal of the glass micropipette.
Problem 5 Excessive numbers of cells are labeled with the virus vectors (step 14).

Potential solution
The concentration of the solution should be determined according to the purpose of the experiment. Lower concentrations label fewer neurons.

Problem 6
The expression of fluorescent proteins is low (step 37).

Potential solution
The survival time of animals after virus injection should be determined based on the purpose of the experiments. In general, longer survival times result in higher expression.

Potential solution
It is recommended to evaporate as much water as possible in the mounting medium for fluorescence microscopy.

Potential solution
Higher concentrations of primary antibodies may result in higher background immunostaining, especially in immunoperoxidase staining. First, the concentration of the primary antibody and then the concentration of the BT solution should be reduced (primary antibodies, a factor of 5; BT solution, a factor of 10-100).

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Hiroyuki Hioki (h-hioki@juntendo.ac.jp).

Materials availability
All plasmids used in this study are available from the Lead Contact upon request.

Data and code availability
This study did not generate any new data or code.