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Jan 2018

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Studying the Mechanisms of Developmental Vocal Learning and Adult Vocal Performance in Zebra Finches through Lentiviral Injection
通过慢病毒注射研究斑马雀的发育声乐学习和成年声乐表演的机制   

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Abstract

Here we provide a detailed step-by-step protocol for using lentivirus to manipulate miRNA expression in Area X of juvenile zebra finches and for analyzing the consequences on song learning and song performance. This protocol has four parts: 1) making the lentiviral construct to overexpress miRNA miR-9; 2) packaging the lentiviral vector; 3) stereotaxic injection of the lentivirus into Area X of juvenile zebra finches; 4) analysis of song learning and song performance in juvenile and adult zebra finches. These methods complement the methods employed in recent works that showed changing FoxP2 gene expression in Area X with lentivirus or adeno-associated virus leads to impairments in song behavior.

Keywords: Zebra finch (斑马雀), Area X (X区), miR-9 (miR-9), Lentivirus (慢病毒), Song learning (声乐学习), Song performance (声乐表演)

Background

The zebra finch, with its well-characterized song behavior and the underlying neural circuitry, provides a unique animal model to study neural mechanisms underlying vocal communication and related sensory-motor learning. In recent years, several laboratories began using viral vectors to manipulate gene expression in the zebra finch brain and to study the functional consequences. These efforts are best illustrated by studies of the FoxP2 gene, which encodes the forkhead box p2 transcription factor. The FoxP2 protein controls the expression of hundreds of downstream genes that have important roles in nervous system development. Mutations in the human FoxP2 gene cause speech and language impairments (Lai et al., 2001). In songbirds, knockdown or overexpression of the FoxP2 gene in Area X of zebra finches, a basal ganglia nucleus critical for vocal learning, profoundly impairs song behavior (Haesler et al., 2007; Murugan et al., 2013; Heston and White, 2015). These studies significantly extended the usage of the zebra finch model to study gene functions in neural circuit development, vocal communication behavior, as well as in speech and language-related neural developmental disorders. We recently reported that overexpression of miRNA miR-9 in Area X of juvenile zebra finches impairs song learning and performance (Shi et al., 2018). Hoping others might benefit from this study, here we provide step-by-step protocols for lentivirus cloning and production, stereotaxic injection of the virus into Area X of juveniles, and analysis of the impact of miR-9 overexpression on song learning and performance using the software Sound Analysis Pro (Tchernichovski et al., 2000). With minor modifications, these methods can be tailored to study other miRNAs or genes in vocal learning and performance in songbirds.

Materials and Reagents

  1. Pipette tips and Eppendorf tubes
  2. 10 cm cell culture plates (Corning, catalog number: 430167 )
  3. 24-well cell culture plates (Corning, Costar®, catalog number: 3524 )
  4. 0.45 μm filter (Merck, catalog number: SCHVU01RE )
  5. 30 ml Polyallomer conical centrifuge tube (Beckman Coulter, catalog number: 358126 )
  6. Insulin syringe (Smiths Medical, catalog number: 4429-1 )
  7. 25 G syringe needles
  8. Betadine Surgical Scrub (Purdue Products)
  9. Zebra finch tissue (e.g., the brain)
  10. XL10 Gold Ultracompetent cells (Agilent Technologies, catalog number: 200314 )
  11. Oneshot Stbl3 competent E. Coli (Thermo Fisher Scientific, InvitrogenTM, catalog number: C737303 )
  12. 293LTV Cells (Cell Biolabs, catalog number: LTV-100 )
  13. A lentiviral vector that contains the human ubiquitin promoter driving the expression of the mCherry fluorescent marker (Edbauer et al., 2010)
  14. Lentivirus packaging plasmids psPAX2 and VSVG (Addgene, catalog numbers: 12260 and 35616 )
  15. PCR primers for miR-9 precursor amplification (Integrated DNA Technologies)
    Forward primer: 5'-GATGCTAGC TGTGTGTGTGGTTCCCGGTGGCAGCT-3'
    Reverse primer: 5'-CATGGCGCGCC GGACCCGCAGCCCTTACCTGGAGCCC-3'
    Note: The forward primer contains a NheI site and the reverse primer contains an AscI site (underlined).
  16. PfuUltraII Fusion HS DNA polymerase (Agilent Technologies, catalog number: 600670 )
  17. Restriction enzymes AscI and NheI (New England BioLabs, catalog numbers: R0558S , R0131S )
  18. T4 DNA ligase (New England BioLabs, catalog number: M0202 )
  19. LB broth (Thermo Fisher Scientific, catalog number: 12780052 )
  20. Agar (Thermo Fisher Scientific, catalog number: 22700025 )
  21. Ampicillin (Sigma-Aldrich, catalog number: A0166-5G )
  22. Genomic DNA isolation kit (QIAGEN, catalog number: 69504 )
  23. Gel extraction kit (QIAGEN, catalog number: 28704 )
  24. PCR purification kit (QIAGEN, catalog number: 28004 )
  25. EndoFree Plasmid Maxi Kit (QIAGEN, catalog number: 12362 )
  26. Agarose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 16500-500 )
  27. 50x TAE buffer (QIAGEN, catalog number: 129237 )
  28. IMDM Glutamax cell culture medium (Thermo Fisher Scientific, catalog number: 31980097 )
  29. Fetal bovine serum (FBS) (Thermo Fisher Scientific, catalog number: 10437028 )
  30. 2 M Calcium Solution
  31. Penicillin-Streptomycin 5,000 U/ml (Thermo Fisher Scientific, catalog number: 15070063 )
  32. Cell culture medium IMDM supplemented with 10% FBS and 50 U/ml Penicillin-Streptomycin unless otherwise indicated
  33. CalPhos Mammalian Transfection Kit (Takara Bio, catalog number: 631312 )
  34. Phosphate buffered saline (PBS, PH 7.4, Thermo Fisher Scientific, catalog number: 10010-023 )
  35. Ketamine (Henry Schein Ketathesia)
  36. Xylazine (Henry Schein Vet)
  37. Metacam (Boehringer Ingelheim Vetmedica)
  38. Fluorescent dye (Thermo Fisher Scientific, catalog number: C34775 )
  39. Ethanol, 200 proof (Koptec)
  40. Vetbond (3M)
  41. Solution A (see Recipes)
  42. Solution B (see Recipes)

Equipment

  1. Pipettes
  2. Gel electrophoresis apparatus (Bio-Rad)
  3. Water bath (37 °C and 42 °C, Precision)
  4. Incubator with shaker (32 °C or 37 °C for growing bacteria)
  5. Tissue culture hood
  6. Tissue culture incubator temperature at 37 °C
  7. Ultracentrifuge and SW28 rotor (Beckman Coulter, Optima, model: LE-80K )
  8. Bench top centrifuge (Eppendorf, model: 5804 R )
  9. Bench top centrifuge (Eppendorf, model: 5414 R )
  10. ND-1000 Spectrophotometer (Thermo Fisher Scientific, model: NanoDropTM 1000, catalog number: ND-1000 )
  11. Thermocycler (Bio-Rad)
  12. Stereotaxic head holder (MyNeurolab)
  13. Oil hydraulic micromanipulator (NARISHIGE, model: MO-10 )
  14. Glass needle puller (NARISHIGE, model: PC-10 )
  15. Glass Capillary (WIRETROL 1-5 μl) (Drummond Scientific, Wiretrol®, catalog number: 5-000-1001 )
  16. Track light (Motic, model: MLC-150C )
  17. Thermal pat (Kent Scientific, model: DCT-15
  18. Scanning microscope with fluorescent light
  19. Surgery tools: scissors and forceps (Fine Science Tools)
  20. Microphone (Audio-Technica, catalog number: AT803b )
  21. Amplifier (M-Audio, model: 2626 )
  22. Window Computer
  23. Sound prove chamber (constructed following Sound Analysis Pro User Manual)
  24. LED Light (Super Bright LEDs, catalog number: RLBN-NW30SMD )

Software

  1. Sound Analysis Program (SAP) Version 1.02 (Tchernichovski et al., 2000), http://soundanalysispro.com

Procedure

Experiments involving lentivirus and animals should be approved by the Institutional Animal Care and Use Committee and the Institutional Biosafety Committee and follow institutional or national regulations. When working with bacteria or virus, all glassware, pipet tips, tubes, and solutions should be autoclaved when applicable before use. All surgery tools should be autoclaved before use, and surgical procedures should be performed under aseptic conditions.


  1. Clone the zebra finch miR-9 gene into a lentiviral vector
    1. Isolate genomic DNA from any zebra finch tissue (e.g., the brain) using the QIAGEN genomic DNA isolation kit.
    2. Amplify the zebra finch miR-9 gene from the genomic DNA using PCR (denaturing: 95 °C/10 sec; annealing: 54 °C/25 sec; and extension: 72 °C/25 sec; 40 cycles).
    3. Separate the PCR product by electrophoresis on 1.5% agarose gel.
    4. Cut out the 290 bp band from the gel, and purify the DNA fragment using the QIAGEN Gel Extraction kit.
    5. Digest the PCR product with the restriction enzymes NheI and AscI at 37 °C for 2-3 h.
    6. Digest the lentiviral vector with restriction enzymes NheI and AscI at 37 °C for 2-3 h.
    7. Purify the digested lentiviral vector by gel electrophoresis followed by gel extraction.
    8. Ligate the miR-9 fragment to the lentiviral vector (molar ratio: 5:1) with T4 DNA ligase in 20 μl ligation buffer at 4 °C overnight.
    9. Transform the Stbl3 cells with DNA ligation mix.
    10. Plate the transformed Stbl3 cells onto LB agar plate with Ampicillin (100 mg/ml).
    11. Grow the bacteria at 32 °C for 20 h.
    12. Pick a single colony and grow in 250 ml LB broth/ampicillin at 32 °C for 15-20 h.
    13. Purify the plasmid DNA using the QIAGEN EndoFree Plasmid Maxi Kit.
    14. Re-suspend the plasmid DNA in 10 mM Tris buffer (pH 7.5).
    15. Quantify the plasmid DNA with Nanodrop.
    16. Validate the plasmid DNA by sequencing and/or digestion with restriction enzymes NheI and AscI (see the plasmid map in Figure 1).
    17. Prepare the packaging plasmids psPAX2 and VSVG similarly as described in Steps A9-A15, except XL10 Gold Ultracompetent cells are used and the bacteria are grown at 37 °C.


      Figure 1. The plasmid map of the Lenti-miR-9 vector

  2. Production of lentivirus
    1. Seed 293LTV cells 3 x 106/10 cm plates (typically 6 plates) in IMDM medium, supplemented with antibiotics (unless otherwise indicated) and 10% FBS the day before transfection.
    2. Replace 75% of the medium with IMDM (no FBS) next day, 2 h before transfection (cells are about 70% confluent).
    3. Prepare solution A and solution B in separate tubes (see Recipes below).
    4. Add solution B to solution A dropwise while gently shake the transfection mix (A + B).
    5. Let the transfection mix sit at room temperature for 15 min.
    6. Gently add transfection mix dropwise to cell culture plate (1.4 ml transfection solution per 10 cm plate).
    7. Incubate the transfected cells at 37 °C for 8-10 h.
    8. Remove and discard the calcium phosphate-containing medium and replace with 8 ml IMDM containing 2% FBS.
    9. Collect the virus-containing cell culture medium at 48 h after transfection (store at 4 °C until Step B11) and replace the medium with 8 ml IMDM containing 2% FBS.
    10. Collect the virus-containing cell culture medium at 72 h after transfection (cells can be discarded afterward).
    11. Combine the collected cell culture medium and spin at 720 x g (2,000 rpm, Eppendorf centrifuge, 5804 R )/10 min at 4 °C.
    12. Save the supernatant and filter it with a sterile 0.45 μm filter.
    13. Spin the supernatant at 82,700 (rav) x g (25,000 rpm, ultracentrifuge) for 2 h at 4 °C.
    14. Discard supernatant and rinse the pellet briefly with PBS.
    15. Re-suspend the pellet in 50-60 μl PBS at 4 °C overnight.
    16. Bleach all waste medium and plastic wares before throwing them away.

    Titer the virus
    1. Seed 293LTV cells in a 24-well plate to 2 x 104 cells per well in IMDM w/10% FBS.
    2. Twenty-four hours later, change medium to IMDM w/2% FBS.
    3. Make serial viral dilutions with IMDM medium: 10-1, 10-2, 10-3, 10-4, 10-5, and 10-6.
    4. Add 1 μl of each viral dilution to cells in each well, three wells per viral dilution.
    5. Seventy-two hours later, count the number of fluorescent cells per well starting from the 10-5 or 10-6 dilution and average the cell counts from the triplicate wells.
    6. The titer is the number of fluorescent cells per well times the dilution factor.
      e.g., if the cell count is 3/well at 10-6 dilution, the titer would be 3 x 106 IU.
      Typically we obtain a titer about 2-3 x 106 IU/μl (IU = infection unit).

  3. Injection of the lentivirus into juvenile Area X
    1. Prepare the male juvenile finches for injection by removing their father at day 10, and keeping them with their mothers in a sound attenuated chamber until day 30. Viral injection is performed at 25 ± 1 days of age.
    2. Weigh and anesthetize the animal by intramuscular injection with 24 μg/Ketamine-12 μg/Xylazine per g of body weight.
    3. Mount the animal onto the stereotaxic head holder platform with the tail up by 10 degree, and tighten the mouth bar and the ear bars.
    4. Disinfect the scalp with iodine and pluck the feather away from the top of head.
    5. Open the scalp along the middle line about 1-1.2 cm using a pair of scissors.
    6. Pull a glass injection needle using a needle puller. The heating temperature can be adjusted by turning the dial so that the inner diameter at the needle tip is 25-30 μm (can be done beforehand).
    7. Briefly spin the virus solution before injection for 5 min at 9,300 x g (10,000 rpm, Eppendorf centrifuge, 5414 R) at 4 °C.
    8. Fill the injection needle with 1 μl mineral oil, 1-2 μl viral solution, and 0.5 μl mineral oil.
    9. Install the injection needle onto the stereotaxic manipulator.
    10. Move the injection needle to the bregma point using the stereotaxic manipulator and record the anterior/posterior and medial/lateral coordinates (this is the reference point for the injection coordinates).
    11. Move the injection needle to above Area X (middle point of A/P and M/L injection coordinates) and make a mark.
    12. Open a small window 1-1.5 mm2 on the skull at the marked site using a 25 G syringe needle. 
    13. Make an opening in the dura with a 25 G needle to facilitate entry of the glass needle.
    14. Inject each Area X at 6 or 8 sites at the following coordinates (Figure 2B): anterior/posterior, 2.8 and 3.2 mm; medial/lateral, 1.3 and 1.5 mm; dorsal/ventral, 4.2 and 4.4 mm (from the surface of the skull). For behavioral experiments, virus is injected bilaterally.
    15. Inject 120 nl viral solution at each site over a period of 2 min using the hydraulic pressure device.
    16. Let the injection needle remain at the site for 2 min before injection and 5 min after injection before removal to facilitate diffusion of viral solution.
    17. Put back the skull bone to the opening and close the skin (one side slightly over another side) and apply Vetbond to seal the scalp.
    18. Put the animal on a thermal pat at temperature 30 °C until it wakes up (takes about 30 min).
    19. Return the animal to the home cage.
    20. Disinfect the surgery area with 70% ethanol, bleach the injection needles and throw them into a sharp waste container, wash and autoclave surgical tools.
    21. Record the following information: injection date, animal ID, injection agents, coordinates and volume of injection.


      Figure 2. Injecting the lentivirus into Area X of the zebra finch brain. A. Stereotaxic setup for surgical procedures. B. Schematic illustration showing the coordinates for viral injection into Area X. C. Exemplar brain section showing virally expressed mCherry signal in Area X. D. mCherry-labeled neurons in Area X.

  4. Song recording and analysis
    1. Keep the injected juveniles with their mothers until day 30, give an adult male tutor to each injected juvenile, and keep the pair in a sound-attenuated chamber from day 30 to day 70.
    2. Record undirected songs for each juvenile pupil at specified age for two days in the absence of the tutor.
    3. Sort manually all song files recorded in one day from 8 AM to 12 PM and eliminate files representing cage noise (this step can be done automatically using the SAP).
    4. Select 20 song files approximately evenly spread across the entire set of sorted song files (e.g., select the first, 11th, 21st, 31st, etc. if there are 200 song files) for each pupil.
    5. Counting the average number of syllables per motif
      Count manually the total number of syllables and the total number of motifs in 20 pupil song files (50-80 motif renditions) and 10 tutor song files (25-40 motif renditions). In cases when a pupil or a tutor song has multiple versions of motifs, include all versions in counting and exclude partial motifs typically appearing at the beginning or the end of a song file. Divide the total number of syllables by the total number of motifs for both the pupil and its tutor. Compare the number of syllables per motif for each pupil to that of its tutor.
    6. Counting the number of missing syllable
      Count manually the number of syllable types (A, B, C, D, etc.) in 20 pupil song files (50-80 motif renditions) and 10 tutor song files (25-40 motif renditions). If a syllable type occurs only in the tutor’s song, but not in the pupil’s song, or if the frequency of a syllable in a pupil’s song is less than 10% of its frequency in the tutor’s song, it is defined as a missing syllable.
    7. Motif similarity analysis
      Compare 20 pupil motifs with 10 tutor motifs and obtain a motif similarity score for each comparison using the default asymmetric time course mode of SAP. Average % similarity of the 200 pairwise comparisons to obtain a motif similarity score.
    8. Maximum motif similarity analysis
      Rank the 200 motif similarity measurements (20 pupil motifs x 10 tutor motifs) for each pupil and average the 10 highest values (top 5%) to obtain the maximum motif similarity score.
    9. Syllable accuracy analysis
      Measure the accuracy score for each syllable of a pupil’s song motif in 20 renditions using the default asymmetric mode of SAP. Average the accuracy scores of all syllables in a pupil’s motif to obtain a syllable accuracy score for that pupil.
    10. Syllable feature analysis
      Measure each syllable feature (duration, mean frequency, goodness of pitch, frequency modulation, and Wiener entropy) for each syllable in 20 pupil motif renditions and 10 tutor motif renditions using the SAP, and average the measurements for all renditions.
      Calculate the difference from the tutor (%) for each acoustic feature and for each syllable:
      (pupil’s measurement - tutor’s measurement)/tutor’s measurement.
      Average the percentage difference values of all syllables for each syllable feature.
    11. Syllable feature variation
      Calculate a coefficient of variation for each acoustic feature for 20 renditions of a syllable, and average the coefficients of variation for all syllable types for each acoustic feature.
    12. Syllable transition entropy analysis
      1. Segment all songs recorded in two days from 8 AM to 12 PM using the auto-segmentation function of SAP (Typically 10,000-19,000 syllables can be obtained).
      2. Classify these syllables into types (clusters) using the clustering module of SAP.
      3. Visually validate the clusters by matching clusters with syllable types in the sonograms, and manually correct obvious cases of false classification (e.g., due to segmentation inconsistency).
      4. Calculate the transition frequencies between all pairs of syllable types, which results in a matrix. For example, for a song motif containing five syllable types (A, B, C, D, and E), calculate syllable transition frequencies for A to A, A to B, A to C, A to D, A to E; B to A, B to B, B to C, and so on. 
      5. For each syllable type t (each row in the matrix), calculate the relative transition probability: pt = transition frequency between a syllable pair divided by the sum of transition frequencies of all syllable pairs in a row.
      6. Compute transition entropy for each syllable type t: Entropyt = sum [pt x log(pt)].
      7. Compute a weighted transition entropy for each syllable type:
        Entropytw = Entropyt x syllable weight, so as to give higher weight to the more frequent syllable types. A syllable weight is defined as the transition frequencies of a given syllable type (sum of a row in the matrix) divided by the sum of transition frequencies of all syllable types (sum of the entire matrix). 
      8. Finally, calculate the overall transition entropy for a song by averaging transition entropies of all its syllable types.
    13. Quantifying the amount of singing
      Segment all song files recorded for each bird between 8 AM to 12 PM in two days using the batch mode of SAP. This process generates the total number of syllables a bird sings during the indicated time. 
    14. Recording female-directed songs
      Record female-directed songs manually between 8:00-11:00 AM (A female-directed song is defined as a song that a male sings toward a female as observed by an experimenter). A male is induced to sing female-directed songs by presenting one or two females in a nearby cage. If needed, females can be changed every 10 min.
    15. Constant fundamental frequency analysis
      1. Analyze the same set of syllables that contain a segment with a constant fundamental frequency (harmonic stacks), and that are produced in the contexts of both undirected singing and female-directed singing.
      2. Measure the constant fundamental frequency using the SAP. Typically, include 20-40 syllable renditions from 20 song files in each context in the analysis.
      3. Exemplar sonograms of pupils injected with the control or miR-9 virus are shown in Figure 3.


        Figure 3. Representative sonograms of miR-9 pupils showing examples of A) Missing syllables, B) Changes in syllable sequence and C) Syllable stuttering. A. Image showing that the song motifs of both the tutor and the control pupil have 7 syllables, whereas the miR-9 pupil’s motif has 5 syllables and syllables c and e are missing. B. Image showing the scrambled syllable sequence of a miR-9 pupil. C. Image showing two motifs of a miR-9 pupil; syllable b is repeated three times in the second motif.

    16. Statistical analysis
      For song behavioral experiments, we typically use 6-8 animals per treatment group, and include 10-20 motif renditions per animal and multiple syllables per motif in the analysis as indicated above. We use various statistical analysis methods such as t-test, paired t-test, ANOVA and/or Mann-Whitney test to evaluate the data and use P < 0.05 as the cutoff for significance.

Notes

  1. Molecular cloning
    For standard molecular biology work, such as genomic DNA isolation, PCR product purification, plasmid DNA purification, restriction enzyme digestion and ligation, we follow the manufacturers' instructions, especially when using QIAGEN kits.
  2. Handling lentivirus
    Lentiviruses should not be frozen and thawed multiple cycles and/or stored for long periods of time, which could cause a drastic drop in viral titer. We routinely prepare fresh virus for each injection experiment. Thus, the injection time (depending on the age of the animals) and viral preparation need to be coordinated. Typically, viral preparation starting from growing cells takes about 10 days. Once made, the lentivirus can be kept at 4 °C for 3-4 days without a significant drop in titer.
  3. Testing injection coordinates
    The injection coordinates can be tested by injecting a fluorescent dye into a targeted area. After injection, animals are killed, and brains are sectioned into 100 μm-thick sections. The sections are imaged with bright and fluorescent lights using a scanning scope (2x lens). Images are merged in Photoshop to check whether injection hits Area X. 
  4. Validating injection sites
    We recommend examining the injection sites after the last behavioral experiment. This can be done by sectioning the brains and imaging brain sections with both regular light and fluorescent light. The two sets of images are merged to examine whether injection sites are within Area X (Figure 2C). If the mCherry fluorescent signal is outside of Area X, the animal should be excluded from behavioral analysis or should be used as a control. The average area exhibiting strong mCherry signal typically accounts for ~20% of total Area X volume. 
  5. Tutors
    We typically use a heterogeneous group of adult male tutors to ensure that the observed impairment in song learning is not dependent on a specific tutor song. However, it is ideal that a subset of pupils injected with the experimental or the control virus are tutored by the same tutor. This will help ensure that any difference in song learning is not due to some tutor songs are more difficult to learn than others. Because tutor songs can change gradually, pupil songs should be compared to recently recorded tutor songs (within six months of tutoring).
  6. Song analysis
    Because zebra finch songs exhibit daily structural oscillation (Deregnaucourt et al., 2005), we recommend consistently analyzing songs produced during a specified time period. We also recommend that two investigators validate the key experimental results with at least one blind to the treatment groups.

Recipes

  1. Solution A
    Add components in the following order:
    Lenti-vector plasmid DNA
    67 μg
    psPAX2
    50 μg
    VSVG
    34 μg
    2 M Calcium Solution
    347.2 μl
    Sterile H2O
    to 2,800 μl
  2. Solution B
    2,800 μl 2x HBS

Acknowledgments

This work was funded by the National Science Foundation grant 1258015 (XCL) and the National Institute of Health grant R01MH105519 (XCL). The funders had no roles in study design, data collection and interpretation, or the decision to submit the work for publication. We thank Drs. M. Sheng and D. Edbauer for generously providing the lentiviral vector and Dr. H. Xia for the lentiviral packaging plasmids. We thank many members of the birdsong community for their constructive inputs throughout the course of this work.

Competing interests

The authors declare no competing financial interests.

Ethics

Experiments involving animals are approved by the Institutional Animal Care and use committee (IACUC) protocol (#3187) of the LSU School of Medicine.

References

  1. Deregnaucourt, S., Mitra, P. P., Feher, O., Pytte, C. and Tchernichovski, O. (2005). How sleep affects the developmental learning of bird song. Nature 433(7027): 710-716. 
  2. Edbauer, D., Neilson, J. R., Foster, K. A., Wang, C. F., Seeburg, D. P., Batterton, M. N., Tada, T., Dolan, B. M., Sharp, P. A. and Sheng, M. (2010). Regulation of synaptic structure and function by FMRP-associated microRNAs miR-125b and miR-132. Neuron 65(3): 373-384.
  3. Haesler, S., Rochefort, C., Georgi, B., Licznerski, P., Osten, P., and Scharff, C. (2007). Incomplete and inaccurate vocal imitation after knockdown of FoxP2 in songbird basal ganglia nucleus Area X. PLoS Biol 5: e321.
  4. Heston, J. B. and White, S. A. (2015). Behavior-linked FoxP2 regulation enables zebra finch vocal learning. J Neurosci 35(7): 2885-2894.
  5. Lai, C. S., Fisher, S. E., Hurst, J. A., Vargha-Khadem, F. and Monaco, A. P. (2001). A forkhead-domain gene is mutated in a severe speech and language disorder. Nature 413(6855): 519-523.
  6. Murugan, M., Harward, S., Scharff, C. and Mooney, R. (2013). Diminished FoxP2 levels affect dopaminergic modulation of corticostriatal signaling important to song variability. Neuron 80(6): 1464-1476.
  7. Shi, Z., Piccus, Z., Zhang, X., Yang, H., Jarrell, H., Ding, Y., Teng, Z., Tchernichovski, O. and Li, X. (2018). miR-9 regulates basal ganglia-dependent developmental vocal learning and adult vocal performance in songbirds. Elife 7: e29087.
  8. Tchernichovski, O., Nottebohm, F., Ho, C. E., Pesaran, B. and Mitra, P. P. (2000). A procedure for an automated measurement of song similarity. Anim Behav 59(6): 1167-1176.

简介

在这里,我们提供了一个详细的逐步协议,用于使用慢病毒操纵幼年斑胸草雀X区的miRNA表达,并分析对歌曲学习和歌曲表现的影响。 该方案有四个部分:1)使慢病毒构建体过表达miRNA miR-9; 2)包装慢病毒载体; 3)将慢病毒立体定位注入少年斑胸草雀X区; 4)青少年和成年斑马雀的歌曲学习和歌曲表演分析。 这些方法补充了近期工作中使用的方法,这些方法显示,在区域X中用慢病毒或腺相关病毒改变 FoxP2 基因表达导致歌曲行为的损害。

【背景】具有良好特征的歌曲行为和基础神经回路的斑胸草雀提供了独特的动物模型来研究声音通信和相关感觉 - 运动学习的神经机制。近年来,一些实验室开始使用病毒载体来操纵斑胸草雀脑中的基因表达并研究其功能后果。这些努力通过对 FoxP2 基因的研究得到了最好的说明,该基因编码叉头盒p2转录因子。 FoxP2蛋白控制着数百个在神经系统发育中起重要作用的下游基因的表达。人类 FoxP2 基因的突变导致言语和语言障碍(Lai et al。,2001)。在鸣禽中,斑马雀X区域的 FoxP2 基因的敲除或过表达,对于声乐学习至关重要的基底神经节核,严重损害了歌曲的行为(Haesler et al。, 2007; Murugan et al。,2013; Heston and White,2015)。这些研究显着扩展了斑胸草雀模型的用途,以研究神经回路发育,声音交流行为以及言语和语言相关神经发育障碍中的基因功能。我们最近报道,幼年斑胸草X区X区miRNA-miR-9的过表达损害了歌曲的学习和表现(Shi et al。,2018)。希望其他人可能从这项研究中受益,在这里我们提供慢病毒克隆和生产的逐步方案,将病毒立体定位注射到青少年的X区,并分析miR-9过表达对歌曲学习和表现的影响软件Sound Analysis Pro(Tchernichovski et al。,2000)。通过微小的修改,这些方法可以定制,以研究声乐学习和鸣禽表演中的其他miRNA或基因。

关键字:斑马雀, X区, miR-9, 慢病毒, 声乐学习, 声乐表演

材料和试剂

  1. 移液器吸头和Eppendorf管
  2. 10 cm细胞培养板(Corning,目录号:430167)
  3. 24孔细胞培养板(Corning,Costar ®,目录号:3524)
  4. 0.45μm过滤器(默克,目录号:SCHVU01RE)
  5. 30毫升Polyallomer锥形离心管(Beckman Coulter,目录号:358126)
  6. 胰岛素注射器(Smiths Medical,目录号:4429-1)
  7. 25 G注射器针头
  8. Betadine Surgical Scrub(普渡产品)
  9. 斑胸草组织(例如,大脑)
  10. XL10 Gold Ultracompetent cells(安捷伦科技,目录号:200314)
  11. Oneshot Stbl3胜任 E. Coli (Thermo Fisher Scientific,Invitrogen TM ,目录号:C737303)
  12. 293LTV Cells(Cell Biolabs,目录号:LTV-100)
  13. 一种慢病毒载体,含有驱动mCherry荧光标记表达的人泛素启动子(Edbauer et al。,2010)
  14. 慢病毒包装质粒psPAX2和VSVG(Addgene,目录号:12260和35616)
  15. 用于miR-9前体扩增的PCR引物(Integrated DNA Technologies)
    正向引物:5'-GATGCTAGC TGTGTGTGTGGTTCCCGGTGGCAGCT-3'
    反向引物:5'-CATGGCGCGCC GGACCCGCAGCCCTTACCTGGAGCCC-3'
    注意:正向引物含有NheI位点,反向引物含有AscI位点(带下划线)。
  16. PfuUltraII Fusion HS DNA聚合酶(Agilent Technologies,目录号:600670)
  17. 限制酶AscI和NheI(New England BioLabs,目录号:R0558S,R0131S)
  18. T4 DNA连接酶(New England BioLabs,目录号:M0202)
  19. LB肉汤(Thermo Fisher Scientific,目录号:12780052)
  20. 琼脂(赛默飞世尔科技,目录号:22700025)
  21. 氨苄西林(Sigma-Aldrich,目录号:A0166-5G)
  22. 基因组DNA分离试剂盒(QIAGEN,目录号:69504)
  23. 凝胶提取试剂盒(QIAGEN,目录号:28704)
  24. PCR纯化试剂盒(QIAGEN,目录号:28004)
  25. EndoFree Plasmid Maxi Kit(QIAGEN,目录号:12362)
  26. 琼脂糖(Thermo Fisher Scientific,Invitrogen TM ,目录号:16500-500)
  27. 50x TAE缓冲液(QIAGEN,目录号:129237)
  28. IMDM Glutamax细胞培养基(Thermo Fisher Scientific,目录号:31980097)
  29. 胎牛血清(FBS)(赛默飞世尔科技,目录号:10437028)
  30. 2 M钙溶液
  31. 青霉素 - 链霉素5,000 U / ml(Thermo Fisher Scientific,目录号:15070063)
  32. 除非另有说明,细胞培养基IMDM补充有10%FBS和50U / ml青霉素 - 链霉素
  33. CalPhos哺乳动物转染试剂盒(Takara Bio,目录号:631312)
  34. 磷酸盐缓冲盐水(PBS,PH 7.4,Thermo Fisher Scientific,目录号:10010-023)
  35. 氯胺酮(Henry Schein Ketathesia)
  36. Xylazine(Henry Schein Vet)
  37. Metacam(Boehringer Ingelheim Vetmedica)
  38. 荧光染料(Thermo Fisher Scientific,目录号:C34775)
  39. 乙醇,200标准(Koptec)
  40. Vetbond(3M)
  41. 解决方案A(见食谱)
  42. 解决方案B(见食谱)

设备

  1. 移液器
  2. 凝胶电泳仪(Bio-Rad)
  3. 水浴(37°C和42°C,精密)
  4. 带振动器的培养箱(32°C或37°C,用于培养细菌)
  5. 组织培养罩
  6. 组织培养箱温度为37°C
  7. 超速离心机和SW28转子(Beckman Coulter,Optima,型号:LE-80K)
  8. 台式离心机(Eppendorf,型号:5804 R)
  9. 台式离心机(Eppendorf,型号:5414 R)
  10. ND-1000分光光度计(Thermo Fisher Scientific,型号:NanoDrop TM 1000,目录号:ND-1000)
  11. 热循环仪(Bio-Rad)
  12. 立体定位头架(MyNeurolab)
  13. 油压微操纵器(NARISHIGE,型号:MO-10)
  14. 玻璃拔针器(NARISHIGE,型号:PC-10)
  15. 玻璃毛细管(WIRETROL1-5μl)(Drummond Scientific,Wiretrol ®,目录号:5-000-1001)
  16. 轨道灯(Motic,型号:MLC-150C)
  17. Thermal pat(Kent Scientific,型号:DCT-15)&nbsp;
  18. 用荧光灯扫描显微镜
  19. 手术工具:剪刀和镊子(精细科学工具)
  20. 麦克风(Audio-Technica,目录号:AT803b)
  21. 放大器(M-Audio,型号:2626)
  22. 窗口电脑
  23. 声音证明室(按照Sound Analysis Pro用户手册构建)
  24. LED灯(超亮LED,目录号:RLBN-NW30SMD)

软件

  1. 声音分析程序(SAP)版本1.02(Tchernichovski 等人,2000), http:// soundanalysispro。 COM

程序

涉及慢病毒和动物的实验应得到机构动物护理和使用委员会以及机构生物安全委员会的批准,并遵循机构或国家法规。使用细菌或病毒时,所有玻璃器皿,移液器吸头,管子和溶液应在使用前进行高压灭菌。所有手术工具在使用前都应进行高压灭菌,外科手术应在无菌条件下进行。


  1. 将斑胸草雀miR-9基因克隆成慢病毒载体
    1. 使用QIAGEN基因组DNA分离试剂盒从任何斑胸草雀组织(例如,大脑)中分离基因组DNA。
    2. 使用PCR从基因组DNA扩增斑胸草雀miR-9基因(变性:95℃/ 10秒;退火:54℃/ 25秒;和延伸:72℃/ 25秒; 40个循环)。
    3. 通过在1.5%琼脂糖凝胶上电泳分离PCR产物。
    4. 从凝胶中切下290bp的条带,并使用QIAGEN凝胶提取试剂盒纯化DNA片段。
    5. 用限制酶NheI和AscI在37℃消化PCR产物2-3小时。
    6. 用限制酶NheI和AscI在37℃消化慢病毒载体2-3小时。
    7. 通过凝胶电泳纯化消化的慢病毒载体,然后凝胶提取。
    8. 使用T4 DNA连接酶在20μl连接缓冲液中于4℃将miR-9片段连接至慢病毒载体(摩尔比:5:1)过夜。
    9. 用DNA连接混合物转化Stbl3细胞。
    10. 将转化的Stbl3细胞平板接种到含有氨苄青霉素(100mg / ml)的LB琼脂平板上。
    11. 在32°C下培养细菌20小时。
    12. 挑取一个菌落,在32毫升LB肉汤/氨苄青霉素中于32°C生长15-20小时。
    13. 使用QIAGEN EndoFree Plasmid Maxi Kit纯化质粒DNA。
    14. 将质粒DNA重悬于10mM Tris缓冲液(pH7.5)中。
    15. 用Nanodrop定量质粒DNA。
    16. 通过用限制酶NheI和AscI测序和/或消化来验证质粒DNA(参见图1中的质粒图谱)。
    17. 类似于步骤A9-A15中所述制备包装质粒psPAX2和VSVG,除了使用XL10 Gold Ultracompetent细胞并且细菌在37°C生长。


      图1.Lenti-miR-9载体的质粒图谱

  2. 生产慢病毒
    1. 在IMDM培养基中种子293LTV细胞3×10 6个 / 10cm平板(通常6个平板),在转染前一天补充抗生素(除非另有说明)和10%FBS。
    2. 在转染前2小时,第二天用IMDM(无FBS)替换75%的培养基(细胞约70%汇合)。
    3. 在单独的试管中制备溶液A和溶液B(参见下面的配方)。
    4. 将溶液B逐滴加入溶液A中,同时轻轻摇动转染混合物(A + B)。
    5. 让转染混合物在室温下静置15分钟。
    6. 轻轻地将转染混合物滴加到细胞培养板(每10cm板1.4ml转染溶液)中。
    7. 将转染的细胞在37℃孵育8-10小时。
    8. 取出并丢弃含磷酸钙的培养基,并用含有2%FBS的8ml IMDM替换。
    9. 转染后48小时收集含有病毒的细胞培养基(在4℃下储存直至步骤B11)并用含有2%FBS的8ml IMDM替换培养基。
    10. 转染后72小时收集含病毒的细胞培养基(之后可丢弃细胞)。
    11. 将收集的细胞培养基合并,并在4℃下以720×g×(2,000rpm,Eppendorf离心机,5804R)/ 10分钟旋转。
    12. 保存上清液,用无菌0.45μm过滤器过滤。
    13. 将上清液在82,700(r av ) x g (25,000rpm,超速离心)在4℃下旋转2小时。
    14. 弃去上清液,用PBS短暂冲洗沉淀。
    15. 将沉淀重悬于50-60μlPBS中,4°C过夜。
    16. 在将它们扔掉之前,将所有废弃介质和塑料制品漂白。

    滴定病毒
    1. 在24孔板中将293LTV细胞接种到IMDM w / 10%FBS中的每孔2×10 4个细胞。
    2. 二十四小时后,将培养基换成IMDM w / 2%FBS。
    3. 用IMDM培养基进行连续病毒稀释:10 -1 ,10 -2 ,10 -3 ,10 -4 ,10 -5 ,和10 -6 。
    4. 向每个孔中的细胞中加入1μl每种病毒稀释液,每个病毒稀释液加入三个孔。
    5. 72小时后,从10 -5 或10 -6 稀释度开始计算每孔荧光细胞的数量,并从一式三份孔中平均细胞计数。
    6. 滴度是每孔荧光细胞的数量乘以稀释倍数。
      例如,如果细胞计数为3 /孔,10 -6 稀释,则滴度为3×10 6 IU。 通常,我们获得约2-3×10 6 sup IU /μl(IU =感染单位)的滴度。

  3. 将慢病毒注入幼年区X
    1. 在第10天通过移除他们的父亲准备注射用的雄性幼鱼,并将它们与母亲一起放在声音减弱室中直到第30天。病毒注射在25±1日龄时进行。
    2. 通过肌肉注射每克体重24μg/氯胺酮-12μg/甲苯噻嗪,对动物进行称重和麻醉。
    3. 将动物安装在立体定位头部固定器平台上,尾部向上10度,并拧紧嘴杆和耳杆。
    4. 用碘消毒头皮,将羽毛从头顶拔出。
    5. 沿着中线打开头皮,使用一把剪刀约1-1.2厘米。
    6. 使用拔针器拉动玻璃注射针。通过转动刻度盘可以调节加热温度,使针尖的内径为25-30μm(可以预先进行)。
    7. 在4℃下以9,300 x g (10,000 rpm,Eppendorf离心机,5414 R)注射5分钟之前简单地旋转病毒溶液。
    8. 用1μl矿物油,1-2μl病毒溶液和0.5μl矿物油填充注射针头。
    9. 将注射针安装到立体定位机械手上。
    10. 使用立体定位操纵器将注射针移动到前囟点并记录前/后和内侧/外侧坐标(这是注射坐标的参考点)。
    11. 将注射针移动到区域X上方(A / P中间点和M / L注射坐标)并做一个标记。
    12. 使用25 G注射器针在标记部位的头骨上打开1-1.5 mm 2 的小窗口。&nbsp;
    13. 用25 G针在硬脑膜上打开一个开口,以方便玻璃针的进入。
    14. 在下面的坐标处将每个区域X注入6或8个位置(图2B):前/后,2.8和3.2 mm;内侧/外侧,1.3和1.5毫米;背/腹,4.2和4.4毫米(从头骨表面)。对于行为实验,病毒是双侧注射的。
    15. 使用液压装置在2分钟内在每个部位注射120nl病毒溶液。
    16. 让注射针在注射前保持2分钟,注射后5分钟,然后取出,以促进病毒溶液的扩散。
    17. 将颅骨放回开口并闭合皮肤(一侧略微超过另一侧)并使用Vetbond密封头皮。
    18. 将动物置于温度为30°C的温热处理,直至其醒来(约需30分钟)。
    19. 将动物放回家笼。
    20. 用70%乙醇对手术区域进行消毒,将注射针漂白并将其扔进尖锐的废物容器中,清洗并高压灭菌手术工具。
    21. 记录以下信息:注射日期,动物ID,注射剂,注射剂的坐标和体积。


      图2.将慢病毒注射到斑胸草雀脑区X区。 A.外科手术的立体定位设置。 B.示意图,显示病毒注射到区域X中的坐标.C。示例区域显示区域X中病毒表达的mCherry信号。区域X中的D.mCherry标记的神经元。

  4. 歌曲录制和分析
    1. 将注射的幼鱼与母亲一起饲养至第30天,给每个注射幼年的成年男性导师,并在第30天至第70天将其保持在声音减弱的室内。
    2. 在没有导师的情况下,为指定年龄的每个少年学生记录两天的无向歌曲。
    3. 手动排序从上午8点到下午12点记录的所有歌曲文件,并消除表示笼子噪音的文件(此步骤可以使用SAP自动完成)。
    4. 选择20个歌曲文件大致均匀分布在整个排序的歌曲文件集中(例如,如果有200首歌曲文件,请选择第一个,第11个,第21个,第31个,等 )为每个学生。
    5. 计算每个主题的平均音节数
      手动计算20个瞳孔歌曲文件(50-80个主题演绎)和10个家庭歌曲文件(25-40个主题演绎)中的音节总数和主题总数。在瞳孔或导师歌曲具有多个版本的图案的情况下,包括所有版本的计数并排除通常出现在歌曲文件的开头或结尾的部分图案。将音节总数除以学生及其导师的主题总数。比较每个学生的每个主题的音节数与其导师的音节数。
    6. 计算缺失音节的数量
      手动计算20个瞳孔歌曲文件(50-80个主题演绎)和10个家庭歌曲文件(25-40个主题演绎)中的音节类型(A,B,C,D,等)的数量。如果音节类型仅出现在导师的歌曲中,而不出现在瞳孔的歌曲中,或者如果瞳孔的歌曲中的音节的频率小于其在教师的歌曲中的频率的10%,则将其定义为缺失的音节。
    7. 主题相似性分析
      将20个瞳孔图案与10个辅导图案进行比较,并使用SAP的默认非对称时间课程模式获得每个比较的主题相似性得分。 200个成对比较的平均%相似性以获得基序相似性得分。
    8. 最大主题相似性分析
      为每个瞳孔排列200个主题相似性测量值(20个瞳孔图案×10个导师图案)并平均10个最高值(前5%)以获得最大图案相似性得分。
    9. 音节精度分析
      使用SAP的默认非对称模式,在20个再现中测量学生歌曲主题的每个音节的准确度分数。平均瞳孔主题中所有音节的准确度分数,以获得该瞳孔的音节准确度分数。
    10. 音节特征分析
      使用SAP测量20个瞳孔图案再现和10个教师图案再现中每个音节的每个音节特征(持续时间,平均频率,音高,调频和维纳熵),并对所有演绎进行平均测量。
      计算每个声学特征和每个音节与导师(%)的差异:
      (学生的测量 - 导师的测量)/导师的测量。
      平均每个音节特征的所有音节的百分比差值。
    11. 音节特征变化
      计算每个声学特征的变异系数,用于20个音节的再现,并平均每个声学特征的所有音节类型的变异系数。
    12. 音节转换熵分析
      1. 使用SAP的自动分段功能对从上午8点到下午12点记录的所有歌曲进行分段(通常可以获得10,000-19,000个音节)。
      2. 使用SAP的群集模块将这些音节分类为类型(群集)。
      3. 通过在声谱图中匹配具有音节类型的聚类来视觉验证聚类,并手动纠正明显的错误分类情况(例如。,由于分段不一致)。
      4. 计算所有音节类型对之间的过渡频率,从而得到矩阵。例如,对于包含五个音节类型(A,B,C,D和E)的歌曲主题,计算A到A,A到B,A到C,A到D,A到E的音节转换频率; B到A,B到B,B到C,依此类推。&nbsp;
      5. 对于每个音节类型t(矩阵中的每一行),计算相对转移概率:pt =音节对之间的转换频率除以一行中所有音节对的转换频率之和。
      6. 计算每个音节类型t的转移熵:Entropyt = sum [pt x log(pt)]。
      7. 计算每个音节类型的加权转换熵:
        Entropytw = Entropyt x音节权重,以便为更频繁的音节类型赋予更高的权重。音节权重定义为给定音节类型的转换频率(矩阵中行的总和)除以所有音节类型的转换频率之和(整个矩阵的总和)。&nbsp;
      8. 最后,通过对其所有音节类型的过渡熵进行平均来计算歌曲的整体过渡熵。
    13. 量化歌唱量
      使用SAP的批处理模式,在两天内在上午8点到下午12点之间分割为每只鸟记录的所有歌曲文件。此过程生成一只鸟在指定时间内唱歌的总音节数。&nbsp;
    14. 录制女性导演的歌曲
      在8:00-11:00 AM之间手动录制女性导演的歌曲(女性导演的歌曲被定义为男性在实验者观察时向女性唱歌的歌曲)。通过在附近的笼子中呈现一个或两个女性,诱导男性唱出女性导演的歌曲。如果需要,女性可以每10分钟更换一次。
    15. 恒定基频分析
      1. 分析包含具有恒定基频(谐波叠加)的片段的同一组音节,并且这些音节是在无向歌唱和女性导演歌唱的背景下产生的。
      2. 使用SAP测量恒定的基频。通常,在分析的每个上下文中包括来自20个歌曲文件的20-40个音节再现。
      3. 注射对照或miR-9病毒的瞳孔的示例声像图如图3所示。


        图3. miR-9瞳孔的代表性超声图显示A)缺少音节的例子,B)音节序列的变化和C)音节口吃。 A.图像显示导师和歌曲的主题对照瞳孔有7个音节,而miR-9瞳孔的主题有5个音节,音节c和e缺失。 B.显示miR-9瞳孔的扰乱音节序列的图像。 C.图像显示miR-9瞳孔的两个图案;音节b在第二个主题中重复三次。

    16. 统计分析
      对于歌曲行为实验,我们通常每个处理组使用6-8只动物,并且如上所述在分析中包括每只动物10-20个基序表达和每个基序的多个音节。我们使用各种统计分析方法,例如t检验,配对t检验,ANOVA和/或Mann-Whitney检验来评估数据并使用P <0.05。 0.05作为显着性的截止值。

笔记

  1. 分子克隆
    对于标准分子生物学工作,如基因组DNA分离,PCR产物纯化,质粒DNA纯化,限制酶消化和连接,我们遵循制造商的说明,特别是在使用QIAGEN试剂盒时。
  2. 处理慢病毒
    慢病毒不应多次冷冻和解冻和/或长期储存,这可能导致病毒滴度急剧下降。我们经常为每次注射实验准备新鲜病毒。因此,需要协调注射时间(取决于动物的年龄)和病毒制剂。通常,从生长细胞开始的病毒制备需要约10天。一旦制成,慢病毒可以在4℃保持3-4天而滴度没有显着下降。
  3. 测试注射坐标
    可以通过将荧光染料注入目标区域来测试注射坐标。注射后,杀死动物,将脑切成100μm厚的切片。使用扫描示波器(2x镜头)对部分进行明亮和荧光灯成像。在Photoshop中合并图像以检查注射是否命中区域X.&nbsp;
  4. 验证注射部位
    我们建议在最后一次行为实验后检查注射部位。这可以通过对大脑进行切片并使用常规光和荧光照射脑切片来完成。合并两组图像以检查注射部位是否在区域X内(图2C)。如果mCherry荧光信号在X区之外,则应将动物排除在行为分析之外,或者将其用作对照。显示强mCherry信号的平均面积通常占面积X总量的约20%。&nbsp;
  5. 导师
    我们通常使用异质的成年男性导师组来确保观察到的歌曲学习障碍不依赖于特定的导师歌曲。然而,理想的是注射实验或对照病毒的学生的子集由同一导师辅导。这将有助于确保歌曲学习的任何差异不是由于某些导师歌曲比其他歌曲更难学习。因为导师歌曲可以逐渐改变,所以应该将学生歌曲与最近录制的导师歌曲(在辅导的六个月内)进行比较。
  6. 宋分析
    由于斑胸草雀歌曲表现出日常的结构振荡(Deregnaucourt et al。,2005),我们建议不断分析在特定时间段内产生的歌曲。我们还建议两名研究人员验证关键实验结果,至少有一名盲人治疗组。

食谱

  1. 解决方案A
    按以下顺序添加组件:
    class =“ke-zeroborder”bordercolor =“#000000”style =“width:300px;” border =“0”cellspacing =“0”cellpadding =“2”>Lenti-载体质粒DNA
    67微克
    psPAX2
    50μg
    VSVG
    34微克
    2 M钙溶液
    347.2μl
    无菌H 2 O
    至2,800μl
    >
  2. 解决方案B
    2,800μl2xHBS

致谢

这项工作由国家科学基金会拨款1258015(XCL)和国家卫生研究院拨款R01MH105519(XCL)资助。资助者在研究设计,数据收集和解释或决定提交出版作品方面没有任何作用。我们感谢Drs。 M. Sheng和D. Edbauer慷慨地提供慢病毒载体,H。Xia博士为慢病毒包装质粒。我们感谢鸟鸣社区的许多成员在整个工作过程中给予的建设性意见。

利益争夺

作者声明没有竞争性的经济利益。

伦理

涉及动物的实验由LSU医学院的机构动物护理和使用委员会(IACUC)协议(#3187)批准。

参考

  1. Deregnaucourt,S.,Mitra,P.P.,Feher,O.,Pytte,C。和Tchernichovski,O。(2005)。 睡眠如何影响鸟鸣的发展学习。 自然 433(7027):710-716。&nbsp;
  2. Edbauer,D.,Neilson,J.R.,Foster,K.A.,Wang,C.F.,Seeburg,D.P.,Batterton,M.N.,Tada,T.,Dolan,B.M.,Sharp,P.A。和Sheng,M。(2010)。 通过FMRP相关microRNA miR-125b和miR-132调节突触结构和功能。 神经元 65(3):373-384。
  3. Haesler,S.,Rochefort,C.,Georgi,B.,Licznerski,P.,Osten,P。和Scharff,C。(2007)。 在鸣禽基底神经节核中敲除 FoxP2 后不完整且不准确的声音模仿区域X. PLoS Biol 5:e321。
  4. Heston,J.B。和White,S.A。(2015)。 与行为相关的FoxP2规则可实现斑马雀的声乐学习。 J Neurosci 35(7):2885-2894。
  5. Lai,C。S.,Fisher,S.E.,Hurst,J.A.,Vargha-Khadem,F。和Monaco,A.P。(2001)。 叉头域基因在严重的言语和语言障碍中发生突变。 自然 413(6855):519-523。
  6. Murugan,M.,Harward,S.,Scharff,C。和Mooney,R。(2013)。 减少的FoxP2水平影响对歌曲变异性重要的皮质纹状体信号的多巴胺能调节。 Neuron 80(6):1464-1476。
  7. Shi,Z.,Piccus,Z.,Zhang,X.,Yang,H.,Jarrell,H.,Ding,Y.,Teng,Z.,Tchernichovski,O。和Li,X。(2018)。 miR-9在鸣禽中调节基底神经节依赖性发育声乐学习和成人声乐表现。 Elife 7:e29087。
  8. Tchernichovski,O.,Nottebohm,F.,Ho,C.E。,Pesaran,B。和Mitra,P.P。(2000)。 自动衡量歌曲相似度的程序。 Anim Behav 59(6):1167-1176。
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Copyright Shi et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Shi, Z., Tchernichovski, O. and Li, X. (2018). Studying the Mechanisms of Developmental Vocal Learning and Adult Vocal Performance in Zebra Finches through Lentiviral Injection. Bio-protocol 8(17): e3006. DOI: 10.21769/BioProtoc.3006.
  2. Shi, Z., Piccus, Z., Zhang, X., Yang, H., Jarrell, H., Ding, Y., Teng, Z., Tchernichovski, O. and Li, X. (2018). miR-9 regulates basal ganglia-dependent developmental vocal learning and adult vocal performance in songbirds. Elife 7: e29087.
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