Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Podsumowanie

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Streszczenie

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Wprowadzenie

The hippocampus plays an important role in memory and learning as well as emotional behavior. One main function consists of the consolidation of short-term memory into long-term memory, which requires high plasticity of the nervous system. The dentate gyrus of the hippocampus acts as the primary gateway for input information and is also one of two brain regions with ongoing neurogenesis throughout adulthood^1,2. The development of the hippocampal structure occurs during late embryogenesis and particularly during the first 3 to 4 weeks postnatal³. During early development of the dentate gyrus a stem cell pool is established required for postnatal as well as adult neurogenesis⁴. Developing neurons pass through various stages, from the stem cell through several stages of progenitor cells to the immature and finally the mature neuron during postnatal as well as adult neurogenesis. At different stages of neurogenesis the expression of specific genes is required to allow the maturation and integration of new neurons into the hippocampal circuitry^5,6.

Using mouse genetics and phenotype analysis by immunohistochemistry as well as molecular methods allowed defining the expression pattern and function of many of these genes. In addition microarray analysis as well as chromatin immunoprecipitation (ChIP) provided information about potential direct and indirect target genes^7,8. However, there are still many open questions concerning the regulatory mechanisms of hippocampal development, in particular the development of the dentate gyrus. To gain further insight how specific genes are regulated a system is required allowing the manipulation of a small number of cells by down- or up-regulation of the gene of interest and/or its target genes and follow their fate during development. In utero electroporation of shRNAs, cDNA of genes of interest or Cre recombinase provides such a tool. To ensure the presence of the desired DNA or small RNAs expression plasmids should be used for electroporation. This approach is very successfully implemented in studying cortical development^9,10, but is a more challenging approach examining the development of the dentate gyrus due to the position of the hippocampal structures in deeper brain layers.

Ex utero electroporation followed by organotypic slice culture is one approach to circumvent this problem^11,12. In contrast to in utero electroporation not the whole embryo but only the head is used allowing therefore to place the electrodes in a more favorable way to direct the shRNA/DNA towards the hippocampus and dentate gyrus. Our group successfully employed ex utero electroporation to study the role of the transcription factor Bcl11b during dentate gyrus development⁸. Bcl11b has a dual role in dentate gyrus development by regulating progenitor cell proliferation as well as differentiation as was demonstrated by immunohistochemistry. To further define a mechanism for Bcl11b involvement in these processes, protocols of the Polleux group^11,12 were adjusted to study the dentate gyrus as described below in the protocol section. In a first approach the question was addressed whether Bcl11b is regulating neuronal cell differentiation cell autonomously. A second approach examined whether Desmoplakin, a direct target gene of Bcl11b, is sufficient to rescue the Bcl11b phenotype.

Protokół

NOTE: All animal experiments were carried out in accordance with the German law and were approved by the government offices in Tübingen.

1. Preparation of Micropipettes, Solutions and Membranes

1. Preparation of Micropipettes
   1. Pull glass micropipettes using a micropipette puller with the following program: Heat: 540, Pull: 125, Velocity: 20 and Delay: 140. The needle length amounts to 5.5 cm.
   2. Bevel needles using a microgrinder to obtain a suitable tip size of 4 mm. Store the needles in a box or 15 cm Petri dish to prevent damaging of the tips.
2. Preparation of Solutions
   1. Plasmid DNA Solution
      1. Prepare plasmid DNA containing the desired cDNA construct using an Endotoxin free Maxi-prep kit according to manufacturer’s protocol.
      2. Adjust plasmid DNA solution to a final concentration of 3 µg/µl (without GFP spike vector) or 4 µg/µl (with 1 µg of GFP spike vector) in endotoxin free Tris-EDTA buffer containing Fast Green (final concentration 0.05%).
   2. Laminin Stock solution
      1. Dissolve 1 mg of laminin in sterile water to a final volume of 1 ml. Prepare 100 µl aliquots and store at -80 °C.
   3. Poly-L-Lysine Stock Solution
      1. Dissolve 50 mg of poly-L-lysine in 50 ml of sterile water to a final concentration of 1 mg/ml. Prepare 1 ml aliquots and store in -20 °C.
   4. Complete Hank’s Balanced Salt Solution (Complete HBSS)
      1. Prepare Complete HBSS by combining 100 ml of 10x HBSS, 2.5 ml of 1 M HEPES buffer (pH 7.4), 30 ml of 1 M D-glucose, 10 ml of 100 mM CaCl₂, 10 ml of 100 mM MgSO₄, and 4 ml of 1 M NaHCO₃. Add sterile water up to 1 L and store at 4 °C.
         NOTE: Autoclave all solutions expect 1 M HEPES buffer and 1 M D-glucose, which are filter sterilized.
   5. Slice Culture Medium
      1. Prepare slice culture medium by adding 35 ml of Basal Medium Eagle, 12.9 ml of complete HBSS (1.2.4), 1.35 ml of 1 M D-glucose, 250 µl of 200 mM L-glutamine, and 500 µl of penicillin-streptomycin to obtain a final volume of 50 ml. Add horse serum to a final concentration of 5% and store at 4 °C.
   6. Low-Melting Point (LMP) Agarose
      1. Prepare a 4% LMP agarose solution by adding 2 g of LMP agarose to 50 ml of complete HBSS (1.2.4) followed by heating in a microwave for 1-2 min at high power. Keep this solution in a water bath at 37 – 39 °C. Store the solution at 4 °C and reuse.
   7. Paraformaldehyde Solution
      1. In a fume hood prepare a 4% paraformaldehyde (PFA) solution by adding 4 g of paraformaldehyde to 100 ml of 1x PBS. Heat the solution to 60 °C and add a few drops of 1 N NaOH until the solution becomes clear.
   8. Permeabilization Solution
      1. Dissolve 9 g of BSA in 300 ml of 1x PBS containing 0.3% Trition X-100 and store at 4 °C. Add 10% sodium azide for long term storage.
3. Coating of Membrane Inserts
   1. Dilute one aliquot of laminin stock solution (1.2.2) and one aliquot of poly-L-lysine stock solution (1.2.3) in sterile water to a final volume of 12 ml.
   2. Place membrane inserts into 6 well plates with each well containing 2 ml of sterile water. Add 1 ml of coating solution on top of the membrane and incubate O/N at 37 °C in a 5% CO₂ incubator.
   3. After incubation wash the membrane inserts three times with 1 ml of sterile water and dry. Use coated membrane inserts on the same day or store at 4 °C for up to four weeks in a dry 6 well plate.

2. DNA Injection and Electroporation of E15.5 and E18.5 Embryos

1. Anesthetize a time mated female mouse by placing it into an anesthetizing chamber saturated with 5% isoflurane and connected to a vaporizer. Circulate isoflurane and oxygen at a rate of 1 L/min. Keep the animal in the box for 2-4 min or until unconscious which is tested by pinching between the paws of the mouse.
2. Euthanize the unconscious mouse by cervical dislocation on embryonic day (E) 15.5 or 18.5. Dissect the uterus containing the embryos¹³ and place it into a Petri dish containing 15-20 ml of cold complete HBSS.
   NOTE: From this point onwards, keep the embryos and tissues on ice.
3. Use a pair of scissors to separate each embryo from the uterine horn and place into a second Petri dish containing cold complete HBSS.
4. Under a dissecting microscope, sever the uterine muscle wall and the placenta using a pair of fine forceps (#55) and scissors. Carefully release the embryo from the yolk sac.
5. Use a pair of Bonn scissors to decapitate the embryos just above the forelimbs at a 60° angle. If the experiment requires genotyping of the embryos, collect a tissue sample for genomic DNA isolation (a small piece of tail).
6. Transfer the head to a clean and dry Petri dish. Because the head had been decapitated in a 60° angle, the head should tilt to one side when placed dorsal side up.
7. Place a needle carefully into the middle of the hemisphere close to the bregma (Figure 1A, B). Inject approximately 2-3 µl (at 3 or 4 µg/µl) of DNA solution by stepping on the pedal of the picospritzer III using 30 pounds of pressure for the duration of 10-15 msec per pulse, applying 5-8 pulses. The duration and number of the pulses depends on the diameter of the needle opening with smaller openings requiring more time. The interval between each pulse amounts to 1 sec.
8. Before placing the electrodes, apply a few drops of complete HBSS on the head of the embryo. Place the electrodes in such a way that the ‘negative’ terminal is on the same side as the injected ventricle and the ‘positive’ electrode on the opposite side of the injected ventricle below the ear of the embryo’s head (Figure 1C, D). Apply 5 pulses of 50 V.
   1. Use 3 mm electrodes for E 15.5 and 5 mm electrodes for E 18.5.

3. Dissection of the Brain

1. After the electroporation, peel off the skin from the head with the help of a pair of fine forceps. Using a pair of spring scissors make a small incision in the middle of the cerebellum at the midline of the skull.
2. Insert the spring scissors into the incision and cut longitudinally along the sagittal suture. Peel off the skull and detach the brain from the skull by using fine forceps. Transfer the whole brain into 15-20 ml cold complete HBSS solution.
3. Meanwhile, pour 4% LMP agarose, kept at 37-39 °C in a water bath, into a peel-away mold.
4. Take the brain out of the complete HBSS by a small scoop spatula and drain excess of HBSS by using fine tissue paper or Kimwipes.
5. Place the whole brain gently into the agarose and adjust its position with a fine needle. Keep the mold on ice until the agarose is solidified and the block is sectioned (for coronal sections the olfactory bulbs point up).

4. Vibratome Sectioning and Slice Culture

1. Trim the LMP agarose blocks and glue them to the specimen stage using ‘super glue’. After the glue is dry transfer the specimen stage to the buffer tray of the vibratome and fill with ice cold complete HBSS until the block is immersed in the solution.
   NOTE: Sterilize all instrument and equipment surfaces with 70% ethanol before sectioning.
2. Prepare 250 µm thick vibratome sections using a new blade as followed.
   1. Trim the block with the following settings; frequency – 60 Hz, amplitude – 0.7 µm, speed 16-18 mm/sec. Cut the sections containing the desired tissue with the above settings at slow speed (9 mm/sec). Starting the sectioning from the hindbrain, collect 5-7 sections from the cerebrum.
   2. Transfer the sections to a clean 6 well culture dish containing 5 ml of ice cold complete HBSS with the help of a bent spatula and keep on ice till all the sections are collected (Figure 1E).
3. Moisten the membrane in a cross fashion with 100 µl of complete HBSS before placing the sections on the membrane, to facilitate the orientation of the sections.
4. Transfer the sections using a bent spatula onto the membrane (pick up a corner of the section with forceps and pull onto the spatula and then use the forceps to push the section onto the membrane). Place up to five sections on one membrane and arrange by using forceps (Figure 1F). Do not overlap the sections with each other.
   1. Take the excess of HBSS off the membrane using a pipette. The specific membranes used here are attached to a frame and inserted into the tissue culture plate, which allows the tissue to be in contact but not covered by the medium.
5. Place the membrane inserts into a 6 well plate containing 1.8 ml of slice culture medium (1.2.5) (Figure 1G, H). Incubate the culture dish at 37 °C with 5% CO₂ for 11 DIV or 14 DIV. Change half the medium (0.9 ml) every second day.
   NOTE: At this stage, add reagents like Bromodeoxyuridine (BrdU; 10 µM final concentration) for labeling proliferating cells to the media for the first 20 hr of the culture time.

5. Fixation of the Sections Followed by Immunofluorescence Staining

1. Use a clean and sharp scalpel blade to cut and trim the membranes depending on the orientation of the sections.
2. Transfer the sections along with the membrane to a 24 well plate containing 1 ml of 4% PFA (1.2.7). Incubate the sections for 1 hr at RT followed by 3 washes with 1x PBS for 15 min each. Incubate the sections O/N with permeabilization solution at 4 °C with gentle agitation.
3. The following day, incubate the sections with appropriate primary antibodies, diluted in permeabilization solution, O/N or for 48 hr at 4 °C with gentle agitation.
4. Wash the sections 3 times for 15 min with 1x PBS and incubate O/N at 4 °C with the appropriate secondary antibodies diluted in permeabilization solution.
5. After the incubation with secondary antibodies, wash the sections once with 1x PBS for 15 min followed by DAPI staining for 10 min.
6. Wash the sections 3 times with 1x PBS for 15 min each and transfer to microscope slides. Add ImmunoMount and gently place a coverslip on top of the sections. Dry the slides O/N at 4 °C and seal with nail polish.
   NOTE: Keep the slides always at 4 °C.
   1. Analyze the slice cultures by confocal microscopy (Figure 1I).

Wyniki

Ablation of the transcription factor Bcl11b causes the impairment of progenitor cell proliferation and neuronal differentiation resulting in a reduced dentate gyrus size and cell number. Furthermore mutant neurons fail to integrate into the hippocampal circuitry causing learning and memory impairment⁸. To answer questions concerning the regulatory mechanism(s) of Bcl11b in these processes ex utero electroporation was employed.

Addressing the question whether Bcl11b cell-aut...

Dyskusje

The hippocampus has an important function in learning and memory. The dentate gyrus is also one of two brain regions where neurogenesis occurs not only during development but also throughout adulthood. Postnatal and adult hippocampal neurogenesis proceeds in a similar way involving many common factors. Defining the regulatory mechanisms of these factors will be very helpful in understanding neurodegenerative diseases which in turn will lead to new therapies and preventive measures. To obtain this information one requires...

Materiały

Name	Company	Catalog Number	Comments
Flaming/ Brown Micropipette Puller	Sutter Instruments Company (USA)	P-97
Fine Glass Pipettes	Warner Instruments	G100F-4
Microgrinder	Narishige, Japan	EG-44
Anesthetic Bracket unit	Harvard Apparatus	PY2 34-0412
Halovet Vaporizer	Harvard Apparatus	PY2 34-0398
Fluovac System	Harvard Apparatus	PY2 34-0387
IMS Fluosorber	Harvard Apparatus	PY2 34-0415
Anesthetizing Chamber	Harvard Apparatus	PY2 34-0460
Electroporator	BEX Company	CUY21 EDIT
Tweezers with disk electrodes	BEX Company	LF650P3	3 mm electrodes for E15.5
Tweezers with disk electrodes	BEX Company	LF650P5	5 mm electrodes for E18.5
Picospritzer III	Parker Hannifin Corporation	P/N 052-0500-900
HM 650 V Vibrating Blade Microtome, 230 V	Thermo Scientific	920120
Dissection Microscope	Carl Zeiss Microscopy Gmbh	Stemi SV8
Inverted Microscope	Leica	Leica DM IL LED
Confocal Microscope	Leica	Sp5II
6 well dish	BD Falcon	#353502
6 well dish	CELLSTAR	#657160
Tissue culture inserts	BD Falcon	#353090
Fast Green	Sigma	F7252
Laminin	Sigma	#L2020
Poly-L-lysine	Sigma	#P5899
Spring scissors	Fine Science Tools	15003-08
Extra Fine Bonn Scissors	Fine Science Tools	14084-08
Forceps	Dumont #55	11255-20 Inox
HBSS 10x	Life Technology	14180-046
BME	Life Technology	41010-26