The Slice Overlay Assay: A Versatile Tool to Study the Influence of Extracellular Signals on Neuronal Development

See allHide authors and affiliations

Science's STKE  11 Jun 2002:
Vol. 2002, Issue 136, pp. pl9
DOI: 10.1126/stke.2002.136.pl9


We have developed a technique that allows characterization and identification of extracellular signals that regulate various aspects of neuronal differentiation. In this in vitro assay, dissociated cells isolated from the developing cerebral wall are labeled and cultured over organotypic cortical slices. We have used this slice overlay assay to identify some of the extracellular cues that regulate differentiation and patterning of axons and dendrites in the cerebral cortex. This assay can be easily adapted to identify factors that control other aspects of neuronal development, such as proliferation and survival, and can also be used to evaluate the role of extracellular signals in the development of nonneural tissues.


Neuronal differentiation is greatly influenced by extracellular signals present in the local environment. The experimental strategies that have been used to examine this influence fall into two categories: (i) in vitro culture of dissociated neurons on artificial two-dimensional substrates such as glass or plastic, and (ii) in vivo transplantation of labeled dissociated neurons. Although these techniques have led to important insights, they each have major limitations. Although the in vitro cultures allow the cellular environment to be manipulated, the substrate over which the cells grow has little in common with the in vivo environment. The in vivo transplantation approach is not suitable for biochemical and molecular perturbation experiments. To overcome these limitations, we developed the Slice Overlay Assay, an in vitro assay in which cells from the developing cerebral wall are cultured over cortical slices. The advantage of this approach is that it allows unrestricted cellular access while cells can grow in an environment resembling that in vivo. This technique and related methods represent powerful approaches to the identification of factors that control neuronal adhesion, acquisition of cell-specific phenotypes, and regulation of axonal and dendritic patterning (1-6).


Preparation of Coated Slides

Cell culture inserts for six-well plates (Becton Dickinson #353102)

Laminin [Sigma-Aldrich #L2020 (]

Poly-L-lysine (Sigma-Aldrich #P5899)

Six-well culture plates [Becton Dickinson #353502 (]

Preparation of Cortical Slices

0.2-μm filter mounted on 500-ml bottles

Basal Medium Eagle without L-glutamine (Sigma-Aldrich #B-1522)

CaCl2 (Sigma-Aldrich, #C-3881)

Cell Culture inserts for six-well plates (Becton Dickinson #353102)


Fine forceps [FST Instruments (]

Hepes-free acid (Sigma Aldrich #H4034)

Hepes-buffered saline solution (HBSS) 10× without calcium and magnesium [Gibco #14180-046 (]

Horse serum, heat-inactivated (Gibco #26050-047)

L-glutamine (Gibco #25030-081)

Low-melting point agarose, molecular biology grade; gelling temperature ~30 to 32°C (Qbiogene #AGAL0050, Carlsbad, CA)

MgSO4 (Sigma-Aldrich #M-7506)

N2 supplement (Gibco #17502-048)

Micro-dissecting knife (FST Instruments)

NaHCO3 (Sigma-Aldrich #S-6297)

Penicillin (10,000 units/ml)-streptomycin (10 mg/ml) (Gibco #15140-122)

Plastic tissue-embedding molds (PolySciences, Warrington, MA)

Six-well plates for tissue culture (Becton-Dickinson #353502)

Preparation of Dissociated Cortical Neurons

0. 2-μm syringe filter

Bovine serum albumin (BSA) (Sigma-Aldrich #A-7906)

1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) [Molecular Probes #D-282 (]



L-cysteine (Sigma-Aldrich #C-1276)



Phenol red, 0.5%

Papain [Roche #108014 (]

Trypan blue

Trypsin inhibitor (Sigma-Aldrich #T-6522)

Immunofluorescent Labeling

BSA (Sigma-Aldrich #A-7906)

Cy2-conjugated goat anti-rabbit IgG [Jackson ImmunoResearch (] or any green fluorochrome-conjugated anti-rabbit IgG

Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) or any red fluorochrome-conjugated anti-rabbit IgG

Ethanol, 100%

Hoechst 33258 (bis-benzimide), 10 mg/ml solution in water (Molecular Probes #H-3569)


Mouse antibody against microtubule-associated protein 2 (MAP2) (Sigma-Aldrich #M-4403)



Normal goat serum


Rabbit polyclonal antibody against green fluorescent protein (GFP) (Molecular Probes #A-6455)

Triton X-100 (Sigma-Aldrich #X-100)


37°C water bath

Binocular loop with back illumination


Incubator, 5% CO2, humidified

Inverted microscope with long-distance working objectives (10× and 20×) with numerical aperture of about 0.6.

Microwave oven



Recipe 1: Laminin Working Solution
Dilute 1 mg of laminin with sterile H2O to a final volume of 1 ml to make a 1 mg/ml stock solution. Prepare 100-μl aliquots and freeze at –80°C.
Recipe 2: Poly-L-lysine Working Solution
In a 50-ml tube, dilute 20 mg of poly-L-lysine with sterile H2O to a final volume of 20 ml to make a 1 mg/ml stock solution. Prepare 1-ml aliquots and freeze at –20°C.
Recipe 3: Complete Hank's Balanced Salt Solution (Complete HBSS)
Stock Solution Volume Final Concentration
10× HBSS50 ml
1 M Hepes (pH 7.4)1.25 ml2.5 mM
1 M D-glucose 15 ml30 mM
100 mM CaCl25 ml1 mM
100 mM MgSO45 ml1 mM
1 M NaHCO3 2 ml4 mM
Add double distilled water (ddH2O) to a total volume of 500 ml. Sterile filter with a 0.2-μm filter. Store at 4°C.
Recipe 4: Slice Culture Medium
Stock Solution Volume Final Concentration
Basal Medium Eagle35 ml
Complete HBSS (Recipe 3)12.9 ml
1 M D-glucose1.35 ml20 mM
200 mM L-glutamine0.25 ml1 mM
Penicillin-streptomycin0.5 ml100 units/ml of penicillin and 0.1 mg/ml of streptomycin
Sterile filter with a 0.2-μm filter, then add heat-inactivated horse serum to a final concentration of 5%.
Recipe 5: Low-Melting Point (LMP) Agarose
1. Dilute 0.75 g of LMP agarose in 25 ml of Complete HBSS (Recipe 3) in a 50-ml sterile conical tube. Mix well.
2. Microwave this solution about 2 min at intermediate power. Monitor the heating constantly to prevent overflow during boiling.
3. When the agarose is completely dissolved, place the solution in the water bath at 37°C until ready for use.
Note: This solution can be stored at 4°C and reused once.
Recipe 6: Dissociation Medium (DM)
Stock Solution Volume Final Concentration
1 M Na2SO420.44 ml98 mM
0.5 M K2SO415 ml30 mM
1 M MgCl21.45 ml5.8 mM
100 mM CaCl20.63 ml0.25 mM
1 M Hepes (pH 7.4) 250 μl1 mM
1 M glucose 5 ml20 mM
Phenol red (0.5%)0.5 ml0.001%
0.1 N NaOH0.5 ml0.125 mN
Add sterile ddH2O to a total volume of 250 ml. Store at 4°C. Sterile filter with a 0.2-µm bottle filter.
Note: Keep the stock solutions of 1 M Na2SO4 and 0.5 M K2SO4 at room temperature, or a precipitate will form.
Recipe 7: Enzyme Solution
DM (Recipe 6)20 ml
Cysteine6.4 mg
Papain400 units
Mix and let dissolve for 15 min in a 37°C water bath. Mix and adjust the pH with 0.1 N NaOH to about 7.4. This usually requires about 6 drops of 0.1 N NaOH. The pH is monitored by the color of the solution: Pink is too basic and yellow is too acidic. Filter through a 0.2-μm syringe filter.
Note: This solution should be prepared immediately before use.
Recipe 8: Heavy Inhibitory Solution (HI)
DM (Recipe 6)6 ml
BSA60 mg
Trypsin inhibitor60 mg
Warm the DM to 37°C, then add BSA and trypsin inhibitor and mix to dissolve. Adjust the pH with 0.1 N NaOH to about 7.4. This usually requires about 12 drops of 0.1 N NaOH. The pH is monitored by the color of the solution: Pink is too basic and yellow is too acidic. Filter through a 0.2-μm syringe filter. Warm the solution in water bath at 37°C before use.
Note: This solution should be prepared immediately before use.
Recipe 9: Light Inhibitory Solution (LI)
DM (Recipe 6)9 ml
HI (Recipe 8)1 ml
Prewarm the DM to 37°C, then add the HI. Filter through a 0.2-μm syringe filter. Warm the solution in a 37°C water bath before use.
Note: This solution should be prepared immediately before use.
Recipe 10: Serum-Free Medium
Basal Medium Eagle100 ml
N2 supplement1 ml
L-glutamine (200 mM)0.5 ml
Penicillin (10,000 units/ml)-streptomycin (10 mg/ml)1 ml
Prepare under sterile hood immediately before use. Warm the solution in water bath at 37°C before use.
Recipe 11: DiI Stock Solution
Dilute to a final concentration of 10 mg/ml in 100% ethanol. Store at 4°C.
Note: The ethanol should not contain any trace of water; otherwise, the DiI will not go into solution because of its hydrophobicity.
Recipe 12: Phosphate-Buffered Saline
NaCl80 g
KCl2 g
Na2HPO4•7H2O11.5 g
KH2PO42 g
Dissolve in 1 liter of ddH2O for 10× PBS. Adjust pH to 7.5. To make 1× PBS, dilute 10× PBS in ddH2O at 1:9 ratio.
Recipe 13: 4% Paraformaldehyde
1. Under a fume hood, dissolve 16 g of paraformaldehyde in 360 ml of ddH2O.
2. Heat the solution to 60°C while stirring and add 400 μl of 2N HCl. The solution should be clear.
3. Allow the solution to cool to 20°C, then add 400 μl of 2N NaOH and 40 ml of 10× PBS (Recipe 12).
4. Using a pH meter, adjust the pH to 7.4.
Note: This solution must be prepared in a chemical fume hood, especially while heating.
Recipe 14: Permeabilization Solution
PBS, 10× (Recipe 12)50 ml
H2O450 ml
BSA15 g
Triton X-1001.5 ml
NaN3 (10% solution in ddH2O)5 ml
Note: BSA can be difficult to dissolve. To dissolve, warm to 37°C while stirring
Recipe 15: Hoechst 33258 Stock Solution
Dilute the commercial solution (10mg/ml) at 1:10000 in 1× PBS (Recipe 12) to prepare a stock solution at 1 μg/ml. Store at 4°C. This solution can be reused twice.


Coating Plates with Laminin and Poly-L-lysine

1. Add one aliquot of Laminin Working Solution (Recipe 1) and one aliquot of Poly-L-Lysine Working Solution (Recipe 2) to 12 ml of sterile ddH2O to make a coating solution (enough for 12 inserts).

2. Place one culture insert into each well of two six-well plates.

3. Add 2 ml of sterile ddH2O into the bottom of each well of the plate underneath the membrane of the insert.

4. Add 1 ml of the coating solution on top of the membrane.

5. Place in a humidified incubator at 37°C 5% CO2 overnight.

Note: Coated plates can be stored if not used the same day. Wrap the plates with Parafilm and store at 4°C for up to 4 weeks.

The Overlay Assay

Preparing Slices from Developing Cortex

This procedure can be applied to rat or mouse cortex from animals between embryonic day 15 (E15) through postnatal day 7 (P7) (Fig. 1).

Fig. 1.

Diagrammatic representation of the slice overlay assay. Cortical cells from a GFP-expressing transgenic mouse are harvested from E15 embryos and plated onto cortical slices prepared from embryonic or neonatal rat cerebral cortex. The plated cells respond to cues provided by the slice and differentiate into neurons of appropriate morphology after 2 to 5 days in culture.

1. Place precoated inserts in 35-mm plates over 1.8 ml of Slice Culture Medium (Recipe 4). This volume is important to provide the appropriate air-medium interface for the slices.

2. Prepare LMP Agarose (Recipe 5) and place in water bath.

3. Euthanize the animal.

3a. To isolate embryonic brains, euthanize a pregnant female rat with CO2 (5 min). Remove the uterus and place in 15 ml of Complete HBSS (Recipe 3) in a 10-cm petri dish on ice.

3b. To isolate brains from postnatal animals, euthanize the animal with an overdose of pentobarbital by intraperitoneal injection.

4. Quickly remove the brain from the skull and place the brain in a dish containing ice-cold Complete HBSS (Recipe 3) on ice.

5. Pour the LMP Agarose into individual plastic tissue embedding molds on ice. Monitor the temperature with a thermometer. When the temperature approaches 38°C, blot as much of the Complete HBSS as possible from around the brain with a clean "Kimwipes"-type tissue, and embed the brain in the LMP Agarose.

6. Leave the mold on ice for 5 min to allow the agarose gel to harden.

7. Place the embedded brains in a vibratome containing cold HBSS in the slicing chamber.

Note: Usually, we do not remove the pia from embryonic brains. Remove the pia from early postnatal brains, because it presents a strong mechanical resistance that can impair slicing.

8. Slice coronal sections from the brains at 250 mμ (for embryonic brains) or 300 mμ (for postnatal brains) with the vibratome.

Note: Always use an intermediate to low speed of blade progression, but the maximal amplitude of blade vibration.

9. Transfer the slices as they are being cut to a new dish containing ice-cold Complete HBSS (Recipe 3) using a large-opening flamed glass Pasteur pipette.

Notes: Avoid using a paintbrush to transfer the slices, because this can very easily damage the tissue. The other advantage of using a flamed large-opening Pasteur pipette is that the tissue is never in contact with air, reducing the probability of contamination. A potential drawback of transferring the slices with a Pasteur pipette is that the tissue may detach from the agarose.

10. For postnatal tissue, microdissect the cortical regions of interest using a microdissecting knife and fine forceps.

Note: For embryonic slices, we found that leaving the agarose around the slices favors the maintenance of the histotypic aspect of the tissue during long-term co-culture (for example, a week in vitro).

11. Carefully transfer the dissected tissues onto the coated membranes of the culture inserts and remove excess Complete HBSS with a pipette. In six-well plate inserts, up to eight embryonic slices or five postnatal slices can be cultured simultaneously. Place in the 37°C incubator.

Note: We use a fine spatula to transfer the slice and its surrounding agarose onto the membrane.

Dissociation of Cortical Neurons

This method can be applied to E16 to E19 rat embryos and to E14 to E17 mouse embryos.

1. Prepare Enzyme Solution (Recipe 7), HI (Recipe 8), LI (Recipe 9), and Serum-Free Medium (Recipe 10). Place at 37°C.

2. Prepare the area for dissection by spraying with 70% ethanol and wiping dry.

3. Sterilize dissection instruments by immersion in 70% ethanol for 10 min. Air dry dissection instruments by propping on petri dish covers.

4. Place two 100-mm petri dishes on ice and add 15 ml of Complete HBSS (Recipe 3) to each.

5. Euthanize a pregnant rat with CO2.

6. Remove the embryos from the uterus and place in the petri dish containing Complete HBSS on ice.

7. Use fine scissors or forceps to remove bone (cranium) overlying the brain.

8. Transfer brains to the second 100-mm petri dish containing Complete HBSS.

9. Remove the pia, starting at the bottom of the brain.

10. Cut the brain along the midline. Pinch off thalamus at the ventral medial surface.

11. With the medial surface of the brain facing you, remove the hippocampus by pinching with a pair of forceps.

12. Pinch off the basal ganglia and lateral pyriform cortex (roughly the ventral third of the brain). You should now be left with a "bowl" of brain tissue representing the neocortex.

13. Transfer the cortex into a new petri dish containing fresh Complete HBSS (Recipe 3) and cut it with fine forceps into pieces of about 1 mm.

14. Use a cut and flamed Pasteur pipette to transfer the pieces of cortex to half (10 ml) of the Enzyme Solution (Recipe 7) in a 50-ml tube. Minimize the amount of HBSS during the transfer of the pieces of tissue to prevent dilution of the Enzyme Solution.

15. Incubate the tissue in Enzyme Solution at 37°C for 20 min.

16. Add remaining 10 ml of Enzyme Solution (Recipe 7) and continue incubating at 37°C for an additional 20 min. Rock the tube occasionally.

17. Move the tissue in the Enzyme Solution and the HI (Recipe 8), LI (Recipe 9), and Serum-Free Medium (Recipe 10) to a tissue culture hood.

18. Gently remove the Enzyme Solution by pipetting, leaving the tissue at the bottom of the tube.

19. Rinse tissue once with 10 ml of LI (Recipe 9).

20. Gently remove the LI.

21. Add 5 ml of HI (Recipe 8) and incubate the tissue for 2 min at 37°C.

22. Gently remove the HI.

23. Rinse the tissue once with 5 ml of Serum-Free Medium (Recipe 10).

24. Gently remove the Serum-Free Medium.

25. Add 5 ml of fresh Serum-Free Medium (Recipe 10) and triturate the tissue gently about 10 to 20 times with a 5-ml pipette.

Note: This should result in virtually complete dissociation of the tissue into a single-cell suspension.

26. Allow any debris to settle to the bottom of the tube, and transfer the cell suspension to a new 50-ml conical tube.

27. Add 15 μl of Trypan blue to a 15-μl aliquot of the cells, and count viable cells using a hemacytometer.

Note: Remember to take into account this dilution factor of 2 when calculating the actual cell concentration of your solution.

Labeling of Wild-Type Neurons by DiI

For the short-term co-culture assay, cortical cells from wild-type animals are labeled with the carbocyanine fluorescent dye DiI. This produces bright cellular labeling, which we found particularly well suited for studying very early aspects of neuronal differentiation, such as the initial orientation of axon outgrowth within the first hours in culture (1). However, this procedure is not suited for more long-term aspects of neuronal differentiation, such as dendritic development, because DiI is internalized after about a day in culture. For long-term co-cultures, cortical neurons are isolated from transgenic mice expressing enhanced green fluorescent protein (EGFP) under the control of a ubiquitous β-actin promoter [(7); see above, Dissociation of Cortical Neurons]. The volumes recommended can be adjusted if larger numbers of cells are required.

1. Sonicate the DiI solution for 30 s at maximum power before use to ensure that macroscopic DiI crystals are not present in the solution.

Note: DiI crystals can produce a strong contaminating labeling once added to the cortical slices.

2. Add 10 μl of DiI Stock Solution (Recipe 11) to 1 ml of dissociated cell suspension (5 × 106 to 5 × 107 cells per ml) in 15-ml conical tubes.

3. Incubate for 10 min in a 37°C water bath.

4. Centrifuge the cell suspension at 1000 rpm for 5 min at room temperature in a table-top centrifuge.

5. Discard the supernatant and resuspend the cell pellet gently in 5 ml of fresh, prewarmed Serum-Free Medium (Recipe 10).

6. Wash the cells three more times in 5 ml of prewarmed Serum-Free Medium (Recipe 10), centrifuging to pellet the cells after each wash.

Note: This step removes microscopic DiI crystals.

7. Resuspend the cells at a final concentration of 5 × 105 viable cells per ml in Serum-Free Medium (Recipe 10). These labeled cells will be plated onto the cortical slices.

Note: The cortical slices should be prepared about 1 hour before the cells are added.

Plating Labeled Neurons onto Cortical Slices

1. Triturate with a pipette 300 μl of the labeled cell suspension around the cortical slices, not directly onto the slices. The exact volume of dissociated cells depends on the number of slices and the surface of the membrane they occupy. The tip of the pipette should almost touch the membrane. Remove at least 300 μl of the medium from the well underneath the membrane when triturating.

Notes: Do not try to drop the solution onto the slices, because this results in avoidance of the slices by the dissociated cells. In fact, the slices should float in the dissociated cell solution; they will then settle rapidly back onto the membrane by gravity. In 5 to 10 min, the solution of dissociated cells will filter through the membrane and become evenly distributed on the slices.

2. Shake the plate containing the cortical slices and the dissociated cells gently three or four times in the x-y axis (never in circles).

3. Place the plates with the cortical slices and the dissociated cells in the incubator at 37°C and 5% CO2, and do not touch or move for at least 20 min.

4. Place the solution of dissociated cells back onto ice.

5. After 20 min, check with the microscope to determine if the cells are present evenly on the slices. If they are not, repeat the above procedure.

6. Place the slices with cells in incubator at 37°C and 5% CO2.

Multiple Immunofluorescence Protocol to Detect GFP and Other Antigens

For short-term co-cultures, in which DiI-labeled neurons are cultured with the slices, the observation and collection of results should be performed on unfixed tissue, using an inverted microscope equipped with a long distance-working objective.

For long-term co-cultures in which EGFP-expresssing neurons are cultured with the slices, the following procedure improves the level of EGFP fluorescence and can be used to perform double or triple staining. To observe dendritic development of EGFP-expressing neurons, we routinely perform double immunofluorescence labeling of GFP and MAP2.

1. Incubate the slices for 4 to 6 hours in Permeabilization Solution (Recipe 14) at 4°C for at least 6 hours (or overnight) with gentle agitation.

2. Dilute rabbit anti-GFP and mouse anti-MAP2 1:2000 each in the appropriate volume of Permeabilization Solution (Recipe 14) (1 ml per well in six-well plates).

3. Incubate the slices in the diluted primary antibody solution overnight at 4°C with gentle agitation.

4. Wash the slices eight times with 3 ml of 1× PBS (Recipe 12) for 15 min with gentle agitation at room temperature. Remove each wash with a pipette.

5. Dilute the secondary antibodies (Cy2-conjugated goat anti-rabbit IgG and Cy3-conjugated goat anti-mouse IgG) 1:600 each in the appropriate volume of Permeabilization Solution (Recipe 14) (1 ml per well in six-well plates, supplemented with 5% normal goat serum to block nonspecific binding.)

6. Incubate the slices overnight in the secondary antibody solution at 4°C with gentle agitation.

7. Wash the slices four times in 3 ml of 1× PBS for 15 min at 4°C with gentle agitation.

8. Add 2 ml of the diluted Hoechst 33258 (Recipe 15) to the slices and incubate for 4 hours at room temperature with gentle agitation.

9. Wash the slices twice in 3 ml of 1× PBS for 15 min at room temperature with gentle agitation.

10. Mount the slices on microscopic slides with an appropriate mounting medium for fluorescence, place a coverslip over the slices, and seal with nail polish.

Notes and Remarks

This procedure will give reproducible results if performed in a minimal amount of time. Typically, the slices should be plated onto the membrane within 1.5 to 2 hours of euthanizing the animal. The tissue should be kept on ice and always covered with Complete HBSS. The dissection of the cortices used to obtain the dissociated cells should also be performed as quickly as possible, within 1 hour of euthanizing the animal, for optimal results. The time elapsed between the plating of the slices onto the membrane and the plating of the cells onto the slices should not exceed 4 hours.

The main advantages of this method are: (i) the ease of implementation, (ii) the ease of screening for factors controlling specific aspects of neuronal differentiation using function-blocking antibodies and pharmacological approaches, and (iii) the ability to determine the cell-autonomous or cell nonautonomous role of a factors by performing the co-culture using a combination of dissociated neurons, slices, or both, isolated from wild-type or transgenic animals (1, 4).

The predecessor of the Slice Overlay Assay is a technique developed in the late 1980s in which fluorescently labeled dissociated neurons were cultured on frozen sections of central or peripheral nervous tissue (8-12). These early studies were very useful for initial characterization of the molecules that inhibit axon outgrowth in adult myelinated white matter. These studies demonstrated that central nervous system white matter, but not gray matter, is nonpermissive for neuronal cell adhesion and fiber outgrowth. More recently, such an assay has been used to investigate the spatial and temporal aspects of the adhesion of thalamic neurons in the cortex (2, 3). The authors showed that the adhesion pattern of dissociated thalamic neurons on cortical slices in vitro reflects the pattern of outgrowth of their axons in vivo. However, one of the limitations of these studies concerned the difficulty of studying long-term aspects of neuronal or glial differentiation, because of the type of fluorescent dyes (such as DiI) used to visualize neuronal morphology. We overcame this problem through the use of neurons isolated from transgenic mice expressing EGFP under the control of a ubiquitous β-actin promoter and cytomegalovirus enhancer (7). Since we published our results using this assay to identify factors that regulate patterning of axons and dendrites in the developing cortex (1, 4), several researchers have used it to identify factors that control the differentiation of neurons and glia in the cortex (6), and to identify factors that control outgrowth of granule cells from the dentate gyrus (5). This method is a powerful tool for the characterization and identification of factors that regulate virtually any aspect of neuronal development, including proliferation, survival, differentiation, and axonal and dendritic patterning.


  1. 1.
  2. 2.
  3. 3.
  4. 4.
  5. 5.
  6. 6.
  7. 7.
  8. 8.
  9. 9.
  10. 10.
  11. 11.
  12. 12.
View Abstract

Navigate This Article