Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Summary

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Abstract

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2^fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

Introduction

Modern studies of the structure and function of the brain usually require genetic manipulation of specific genes in neuronal cells. To probe the functions of different genes, transgenic mice carrying mutant alleles, including knockout and knock-in alleles have been routinely generated. Stereotaxic brain surgery for adult rodents is a standard method to locally deliver drugs, viruses, tracers and other reagents to specific regions of rodent brains^1,2. Applying the stereotaxic brain surgery to transgenic mice permits one to genetically manipulate the gene function and neuronal activity in specific neuronal populations of the mouse brain. The cell type-specific manipulation provides a powerful approach to decipher neuronal functions in complex neural circuits of the brain^3,4,5.

Neural development of the nervous system begins at early embryonic stages, and the developmental processes continue after birth until the juvenile period. Postnatal maturation of the nervous system includes the precise synaptic wiring of neural circuits, which is essential for physiological and cognitive functions of the brain⁶. Therefore, studying developmental events that occur in neonatal time windows is important not only for understanding normal neural development, but it may also provide insights into the pathogenesis of neurodevelopmental and neuropsychiatric disorders^7,8. Although the methods of stereotaxic brain surgery for adult rodents are readily available^2,9, few protocols are available on the internet for stereotaxic brain surgery in neonatal mice^10,11. In fact, stereotaxic microinjections of reagents into the brains of neonatal mouse pups are difficult, because the head of neonatal pup is too fragile to be fixed in the standard stereotaxic apparatus. Nonetheless, the application of stereotaxic brain surgery to transgenic mice is feasible for neonatal mice¹². Here, we describe a simple method with a homemade setup to perform stereotaxic brain surgery in newborn mouse pups. We demonstrate that this technique allows one to conditionally delete floxed genes by microinjecting AAV-expressing Cre DNA recombinase into the striatum of reporter gene mice and conditionally floxed transgenic mice. This technique is also applicable to deliver reagents into the neonatal striatum of wild-type mice.

Protocol

The animal protocols described here have been approved by the Animal Care and Use Committees of National Yang-Ming University.

1. Preparation of The Holder for Neonatal Pups in The Stereotaxic Apparatus

1. Make the head tray: cut the bottom of a 1.5 mL centrifuge tube (15 mm long) into the shape that fits the head of neonatal pups by removing 1/5 of the wall of the tube.
2. Take a pipette tip box with the right size that fits with the pedestal of stereotaxic apparatus, and remove the top cover. Affix the head tray prepared in step 1.1 and a tissue embedding cassette onto the base of tip tray with hot-melting adhesive. The height of the tissue embedding cassette is 7 mm, which is used to support the pup's neck and body in the head-fixed position.
3. Place the whole setup in a standard stereotaxic apparatus.

2. Preparation of 30G Injection Stainless Steel Needles

1. Pre-clean 30G syringe needles by soaking them in chloroform for 3 days.
2. Use forceps to carefully and slowly pull the needle out from its polypropylene hub in a chemical hood.
3. Wash the needles with absolute ethanol for 20 min followed by 70% ethanol rinses for 3 × 10 min on a shaker at 50 rpm. Let the needles air dry, and store the cleaned needles in a clean box at room temperature (RT) until use.

3. Prepare an Adapter for Microinjection Tubing

1. Connect the microliter syringe to a 30G injection needle with a PE10 polyethylene tube (PE10 tube). Prepare a PE20 polyethylene tubing (PE20 tube) adaptor for bridging the injection needle and the microliter syringe. The use of the adaptor is necessary, because the diameter of PE10 tube is much smaller than that of the needle of the 26G microliter syringe.
2. Prepare the tubing for the microliter syringe: connect the pre-cleaned 30G injection needle with 5 cm of PE20 tube, and seal the junction with instant glue. This tubing adaptor is reusable.
   NOTE: If the needle of microinjection syringe is 30G, the preparation (step 3.1) and usage (step 3.3) of this adaptor is not necessary.
3. Connect the tubing adaptor (prepared in step 3.1) with a microliter syringe (10 µL).
4. Connect one end of PE10 tube (no longer than 60 cm) onto the 30G needle of the PE20 tubing adaptor. Mount a new 30G injection needle onto the other end of the PE10 tube.

4. Load the Microinjection Tube with Autoclaved Distilled Water, Dye and Viruses

1. Remove the plunger of the microliter syringe. Use a 25G syringe to load the microliter syringe and its connected PE tube with autoclaved distilled water to remove air from the tubing.
2. Place the plunger back to the microliter syringe, and push the plunger until 2 µL of distilled water remains in the barrel. Do not let the volume of distilled water become lower than 2 µL.
3. Mount carefully the microliter syringe onto the micro flow rate syringe pump.
4. Pipette small amounts of 0.1% fast green dye (prepared in 0.9% saline and filtered with 0.22 µm filter) and the virus liquids to a piece of parafilm.
5. Withdraw a small amount of air to make an air bubble visible at the junction between the 30G injection needle and PE10 tube followed by loading 0.7 µL of filtered fast green into the tube to test the flow of fluids in the microinjection tubing.
6. Withdraw another small amount of air to make a second air bubble followed by loading the virus liquids into the microinjection tubing.
7. Attach and secure the 30G microinjection needle to the arm of stereotaxic apparatus.

5. Anesthesia of Neonatal Mice by Hypothermia

1. Place the pup in a latex glove sleeve and immerse it in crushed ice up to the neck for 5 min.
2. Pinch the pup's feet with forceps to make sure no withdrawal response of its feet.
3. Place the pup with latex glove sleeve in the head tray and put some crushed ice around the latex sleeve to keep it cold for hypothermia anesthesia.
4. Apply vet ointment on pup's eyes to prevent dryness while under anesthesia if possible.

6. Microinjection

1. Prepare sterile surgery by wiping the stereotaxic instrument thoroughly with 70% ethanol. Sterilize the surgical instrument by immersing them in 70% ethanol.
2. Scrub the pup's head with 70% ethanol. Locate the landmark lambda on the skull and mark the lambda with a marker pen. Aim the needle tip to the lambda, and set the anterior-posterior (AP) and medial-lateral (ML) coordinates as zero.
3. Move the injection arm to the target site according to the X and Y coordinates of the target site. For the striatum of postnatal day (P) 2 pups, the coordinates are: AP, +2.4 mm anterior to the lambda; ML, ±1.0 mm lateral from the midline; dorsal-ventral (DV), −1.7 mm from the skull. Mark the position of fast green dye in PE10 tube with a pen.
4. Bring down the 30G injection needle slowly to penetrate through the skin and skull, and then bring up the needle tip until it stops at the surface of the skull. Set the DV coordinate as zero.
5. Bring down the 30G injection needle slowly until it reaches the DV coordinate of the target site. Wait for 1 min to allow the parenchyma resuming its normal shape. Run the microinjection program (100 nL/min).
6. Make sure that the mark of fast green dye is moving in the PE tube to ensure the virus liquid is injected into the brain.
7. Wait for 1 min after the termination of the microinjection, and then slowly and progressively bring up the needle to 1/2 height of DV depth within 30 s. After 30 s, slowly withdraw the needle from the pup's head.
8. Repeat step 6.2 to 6.7 until the microinjections of all targeted sites are completed.

7. Post-surgical Recovery of The Pup

1. Warm up the pup for 20 min in a 33 °C incubator. Check the recovery of the pup from hypothermia anesthesia every 5 min until the pup has regained sufficient consciousness to maintain sternal recumbency.
2. Return the pup back to the dam after the pup is fully recovered.

Results

For the first set of experiment, we microinjected 200 nL of AAV9.hSynapsin.HI.eGFP-Cre.WPRE.SV40 viruses (AAV-eGFP-Cre, 1/10 dilution in Dulbecco's phosphate buffered saline) that express the Cre DNA recombinase fused with GFP into P2 striatum of Ai14 mice. The Ai14 mice express tdTomato reporter gene upon Cre-mediated deletion of loxP-flanked (floxed) STOP cassette (Figure 2F). The brains were harvested at P14 for immunostaining of GFP and tdTomato. Many AAV...

Discussion

In the present study, we demonstrate a simple and reliable stereotaxic method for injecting AAV viruses into the striatum of neonatal mouse brains. We microinjected AAV-eGFP-Cre viruses into the striatum of Ai14 reporter mice at P2 and then analyzed the reporter gene expression at P14. We found AAV transduced GFP-positive cells throughout the striatum at rostrocaudal levels. Moreover, nearly all GFP-positive cells co-expressed the tdTomato reporter gene in striatal cells, suggesting a successful Cre-loxP DNA recombinatio...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
30G PrecisionGlide Needle	Becton Dickinson	REF 305106
Chloroform	JT Baker	9180-03
Hamilton MICROLITER Syringe	Hamilton	80300	30G needle fit for PE10 tube; 26G needle needs a PE20 adaptor
Polyethylene tubing PE20	Becton Dickinson	427406
Polyethylene tubing PE10	Becton Dickinson	427401
Micro Flow Rate Syringe Pump	Longer Precision Pump Co.	TJ-2A (Controller) and L0107-2A (Drive Unit)
25G syringe	Becton Dickinson	REF 302105
Fast green	Sigma-Aldrich	F-7252	0.1%
Standard Stereotaxic Instruments	RWD Life Science	68037	Without using 68030 Mouse/Neonatal Rat Adaptor
Anti-FOXP2 antibody	Abcam	ab16046	Rabbit polyclonal to FOXP2, 1:4,000
Anti-RFP antibody	Abcam	ab65856	Mouse monoclonal to RFP, 1:1,000
BX63 microscope	Olympus	BX63
LSM 880 confocal microscope	Zeiss	LSM 880
Goat anti-rabbit conjugated Alexa fluor594	Jackson lmmunoReserch Laboratories Inc.	111-585-003
AAV9.hSynapsin.HI.eGFP-Cre.WPRE.SV40	Penn Vector Core	AV-9-PV1848	Lot # CS0987, 5.506x1013 (GC/mL)
AAV9.chicken actin-eGFP	AAV core, Institute of Biomedical Sciences, Academia Sinica, Taiwan	N/A	1x1014 (GC/ml)
B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J	The Jackson Labtorary	007914	Ai14
B6(Cg)-Foxp2tm1.1Sfis/CfreJ	The Jackson Labtorary	026259	Foxp2fl/fl
Dulbecco’s phosphate buffered saline	Corning cellgro	21-030-CVR