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Journal of Neuroscience Methods 263 (2016) 123–133
Contents lists available at ScienceDirect
Journal of Neuroscience Methods
jo ur nal ho me p age: www.elsev ier .com/ locate / jneumeth
asic Neuroscience
ong-term primary culture of neurons taken from chick embryorain: A model to study neural cell biology, synaptogenesisnd its dynamic properties
wanish Kumar, Birendra Nath Mallick ∗
chool of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India
i g h l i g h t s
Simple method of glia less primaryculture of neurons from chick embryobrain.Embryos were hatched in the lab-oratory from post-fertilized 9 dayincubated eggs.Cytomorphology and immuno-stained cultured neurons werestudied for at least 35 days.Early, late growth events and synap-togenesis were followed under livecell imaging.Axonal and SV dynamics weretracked in transfected live neuronsand evaluated.
g r a p h i c a l a b s t r a c t
r t i c l e i n f o
rticle history:eceived 11 September 2015eceived in revised form 27 January 2016ccepted 4 February 2016vailable online 12 February 2016
eywords:rowth cone
a b s t r a c t
Background: Studying neuronal growth, development and synaptogenesis are among the hot researchtopics. However, it is faced with various challenges and technical limitations that include but not limitedto donor’s species and health, threat to life, age of embryo, glial contamination, real-time tracking, andfollow-up.New method: We have successfully standardized a method for long-term primary culture of neurons col-lected from post-fertilized 9 day incubated chicken embryo brain overcoming the limitations mentionedabove. Fertilized eggs were incubated in the laboratory and neurons from the embryonic brain were col-
eurite growth lected and low-density culture, apparently without glial contamination, was studied at least for 35 daysin vitro (DIV).
euronal differentiation and growthrimary culture of neuronspinesynaptogenesisynaptic vesicles
Results: Neurons were characterized by double immunostaining using stringent neuronal and glial mark-ers. Neuronal differentiation, cytomorphology, neurite and axon formation, development and maturation,spine formation and synaptogenesis were tracked in real-time in a stage and time dependent manner.The neurons were transfected with Synaptophysin-RFP to label synaptic vesicles, which were followedin real-time under live-cell imaging.
Abbreviations: Ab, antibody; DAPI, 4′ ,6-diamidino-2-phenylindole; DIC, differential interference contrast; DIV, days in vitro; DMEM, Dulbecco’s modified Eagle’s medium;FAP, glial fibriliary acidic protein; HH, Hamburger and Hamilton; MAP-2, microtubule associated protein-2; PBS, phosphate buffer saline; PSD-95, post synaptic densityrotein-95; RFP, red fluorescence protein; SV, synaptic vesicle; EtOH, ethanol.∗ Corresponding author. Tel.: +91 11 2670 4522; fax: +91 11 2674 2558.
E-mail address: [email protected] (B.N. Mallick).
ttp://dx.doi.org/10.1016/j.jneumeth.2016.02.008165-0270/© 2016 Elsevier B.V. All rights reserved.
124 A. Kumar, B.N. Mallick / Journal of Neuroscience Methods 263 (2016) 123–133
Comparison with existing methods: Every step was carried out under controlled laboratory conditions. Eggsare easily available, easy to handle, neurons from desired day of incubation could be conveniently studiedfor long period in apparently glia-free condition. In addition to common factors affecting primary culture,selection of culture media and cover glass coating are other key factors affecting neuronal cultures.Conclusions: We describe an inexpensive, simpler pure primary neuronal culture method for studyingneuronal cell-biology, synaptogenesis, vesicular dynamics and it has potential to grow 3D-multilayeredbrain in vitro.
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. Introduction
The human brain is a complex modular organ consisting ofillions of neurons and about 1000-fold more synaptic connec-ions among them. During development, early spherical neuralrogenitor cells give rise to many processes, the neurites; one ofhese early neurites subsequently transforms into an axon whilethers develop into dendrites. The growing axons that come inontact with other neurons form terminal presynaptic swellings.hese presynaptic swellings possess specific neurotransmitter asell as cognate receptors; they also influence the post-synapticeurons to express desired receptors. The number of presynapticwellings, their morphometric characteristics, receptor decorationnd other properties may be increased, modified or lost. Thesendividual or collective changes influence somatic and autonomicehaviors including cognition as well as consciousness, sensitivity,ew and/or existing learning and memory, or recovery processes
ollowing an injury or a disease (Cajal, 1894; Kandel and Spencer,968; Mayford et al., 2012). Apparently some of these connec-ions last throughout life while others get modulated, replacedr lost or newly formed. Our understanding of how neurons formuch complex, dynamic, spatio-temporally oriented cytomorpho-ogically, neurochemically and anatomically-functional circuitry istill in its infancy.
Studying the neuronal growth and developmental events in vivon the brain particularly in the animals higher in evolution includ-ng humans is desirable; however, in vivo imaging of neurons hasarious technical and other limitations and concerns. Therefore,t is imperative to select appropriate model(s) to investigate theore and fundamental questions in biology in general and neurals well as brain biology in particular. Experimental studies haveeen conducted in developing brain as such and/or in primary cul-ures of neurons derived from the developing brains of variouspecies, e.g. rats, mice, fish, etc. (Darbinyan et al., 2013). Collectingmbryos is not very easy, particularly from higher mammals includ-ng humans; also it raises various ethical concerns. Surgical removalf embryos from higher animals may pose danger to donor’s life andven if one embryo is needed, the remaining embryos in the litteray be wasted or their lives could be at risk. Such complicationsay be significantly avoided using fertilized chicken eggs.Genetic manipulation in higher mammals may also lead to
mbryonic lethality due to complexity as well as complicationsuring course of the procedure in mother’s womb (Andermatt andtoeckli, 2014; Baeriswyl and Stoeckli, 2006). However, externalanipulation on chicken embryo (for example) could be relatively
asily and precisely targeted with respect to time and space (El-hali et al., 2010; Fyfe and MacMillan, 1983). It is important torecisely know the time of fertilization because it has been reportedhat some genes are on/off or some factors are expressed/repressednly during a specific post-fertilization temporal window duringevelopment (Ebendal and Persson, 1988; Godfrey and Shooter,986; Hevor et al., 1988). However, it is extremely difficult to
now the precise time of fertilization and therefore, it is inherentlyifficult to temporally correlate and synchronize the post-ertilization developmental events. As a compromise closest we can© 2016 Elsevier B.V. All rights reserved.
practically reach is to standardize the incubation period of the fer-tilized eggs to obtain embryo of desired cell-numbered stage forfurther study. Accordingly, in this report we present a protocol forprimary culture of neurons isolated from chick embryo brains at 9day incubated post-fertilized eggs. We have successfully standard-ized the method, such that it provides reproducible and consistentresults.
2. Materials and methods
2.1. Equipment
CO2 incubator (Model No. 300498-4247 Thermo Forma, USA),Egg-incubator (Model No. WZ16, Dayal Poultry Applicant, India,and Model No. OLSC-234-15, Ocean Life Science Corp., India),Haemocytometer (Neubauer improved, Marienfeld, Germany),Laminar flow (Atlantis Cleanair, India), Live cell imaging micro-scope (Axio-Observer.Z1, Carl-Zeiss, Germany fitted with CameraAxioCam MRm, CO2 Module S, Temperature Module S, Heating UnitXLS and Incubator XL-S1 fitted with temperature sensor P†100),Sceptor (Handheld automated cell counter, Cat. No. PHCC00000,Millipore, Germany), Spinning disk microscope (Nikon Eclipse Ti,Japan) fitted with spinning disk (CSU-X1, YOKOGAWA) and anEMCCD camera (Andor-iXon3 Model No. DU897, Andor Technol-ogy), and Upright microscope (Eclipse TS 100, Nikon, Japan).
2.2. Source of the eggs and their incubation
Kuroiler hatching eggs from white leghorn chicken (Gallus gal-lus domesticus) were procured from Keggfarms Pvt. Ltd., Gurgaon,Haryana, India. The eggs were incubated in the laboratory using anegg incubator Model No. OLSC-234-15, Ocean Life Science Corp.,India.
2.3. Reagents and consumables
0.25% trypsin–EDTA (Cat. No. 59428C Sigma–Aldrich, USA), 1,4-diazabicyclo[2.2.2]octane (DABCO) (Cat. No. D2522, Sigma–Aldrich,USA), 12 well tissue culture dish and 35 mm dish (Eppendorf,Germany), disposable syringes (Batch No. 315204JG1, HindustanSyringe and Medical Devices Limited, India), 100 ml plastic Petridish (Lot No. 03910501, Corning, USA), Amphotericin B (Cat.No. 15290-018, Invitrogen, USA), B-27 supplement-50× (Cat. No.17504044, Gibco, USA), Confocal cover glass bottom dish 35 mm(D35-20-1-N, 35 dish, 20 mm Microwell #1 Glass sterile, In VitroScientific, USA), Cytosine �-d-arabinofuranoside (Cat. No. C1768,Sigma–Aldrich, USA), 4′,6-diamidino-2-phenylindole (DAPI) (Cat.No. D9542, Sigma–Aldrich, USA), Dulbecco’s modified eagle’smedium (DMEM) (Cat. No. D6429, Sigma–Aldrich, USA), Ethanol(EtOH) (Reagent grade, Merck, Germany), Ethylene diamine tetra-
Chemicals, Mumbai, India), Fetal bovine serum (Cat. No. 16000044,Gibco, USA), Hank’s balanced salt solution 1×, Laminin (5 �g/ml Cat.No. L2020 Sigma–Aldrich, USA), Methanol (Cat. No. SK25620739,
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A. Kumar, B.N. Mallick / Journal of N
erck Specialties Private Limited, Mumbai, India), Microscopicound cover glass 22 mm (#1 Glass, Blue Star, Mumbai, India),icroslides (Blue Star, Mumbai, India), Mdi-sterile syringe fil-
er (0.2 �m pore size, Cat. No. SYKG0601MNXX204, Advancedicrodevices Private Limited, Ambala, India), Mowiol®4-88 (Cat.o. 81381, Sigma–Aldrich, USA), Nerve growth factor-7S (NGF)
Cat. No. N0513 Sigma–Aldrich, USA), Neurobasal medium (Cat.o. 21103049, Gibco, USA), Parafilm (Cat. No. PM-996, Parafilm,SA), Normal pipette (Eppendorf 1 ml, 200 �l, 20 �l), Pastureipette, poly-d-lysine (0.1 mg/ml, Cat. No. P6407 Sigma–Aldrich,SA), Phosphate buffer saline 1× (PBS), Glycerol (Qualigens Finehemicals, India), poly-l-lysine (0.01% solution, Cat. No. P4707,igma–Aldrich, USA), poly-l-ornithine (0.1 mg/ml, Cat. No. P4957,igma–Aldrich, USA), Penicillin, Streptomycin (Cat. No. 15140122,nvitrogen, USA), Stripette disposable serological pipette cottonlugged-5 ml (Cat. No. 4487, Corning, USA).
.4. Medium composition
We used Neurobasal medium supplemented with 2% B-27, mM glutamine and 20 ng/ml NGF. DMEM (Cat. No. D6429) wasrocured from Sigma–Aldrich, USA and was supplemented with0% FBS to inhibit the proteolytic activity of trypsin.
.5. Antibodies and red fluorescence protein (RFP) construct
Anti-Synapsin I Rabbit antibody (Ab) (Cat. No. 574778, Cal-iochem, Germany), Cell Light Synaptophysin-RFP (Cat. No.10610, BacMam 2.0 Molecular Probes, USA), Donkey anti-Rabbit-ITC 2◦ Ab (Cat. No. 2090, Santacruz Biotechnology, USA), Mouseonoclonal anti-microtubule associated protein-2 (MAP-2) Alexa
luor-488 conjugated 1◦ Ab (Cat. No. MAB3418X, Millipore,ermany), Mouse monoclonal anti-NeuN 1◦ Ab (Cat. No. MAB77X, Millipore, Germany), Mouse monoclonal anti-Post Synap-ic Density-95 1◦ Ab (PSD-95) (Cat. No. 124011 Synaptic Systems,ermany), Mouse monoclonal anti-Synaptophysin 1◦ Ab (Cat. No.AB5258, Clone SY38, Millipore, Germany), Rabbit anti-Glial fib-
illary acidic protein (GFAP) 1◦ Ab (Cat. No. AB5804, Chemicon,ermany), Texas Red goat anti-mouse 2◦ Ab (Cat. No. T6390, Molec-lar Probes, USA), Texas Red goat anti-rabbit 2◦ Ab (Cat. No. T6391,olecular Probes, USA) were used.
.6. Cover glass sterilization and surface treatment
Neural cell adhesion on the surface of the cover glass isery important for growth and differentiation (Letourneau, 1975).eurons do not normally adhere easily on the glass or plasticydrophobic surfaces, which have slight negative charge and act as
non-permissive substrate. The cover glass surfaces were appro-riately modified by treating with attachment favoring biomaterial
ayer for neuronal attachment and growth. This treatment modifiedhe hydrophobic glass surface to hydrophilic nature which in turn
imicked the biophysico-chemical niche characteristics favoringeuronal adherence and growth.
Cell adhesion is an essential step for proper growth and dif-erentiation of neurons. Achieving success of neuronal adherencend their growth is quite challenging; it needs utmost attentionnd care. It depends on multiple factors; the surface chemical(s)eing most crucial. We have tested poly-l-lysine, poly-d-lysine,
aminin and poly-l-ornithine. We found that poly-d-lysine wasost effective for long term culture. Poly-lysine is a poly-cation
ue to overall net positive charge on lysine residue that leads to
lectrostatic interaction with neuronal membrane surface lead-ng to cell adhesion (McKeehan and Ham, 1976; Yavin and Yavin,974). It could be that due to its d-conformation, poly-d-lysineannot be degraded by enzymatic activity of proteases secreted byience Methods 263 (2016) 123–133 125
the cultured neurons and thus acts as a better adhesive substratethan poly-l-lysine which is actively degraded by secreted proteases(Banker and Goslin, 1991; Sabri et al., 2012). Also, poly-l-lysine,laminin and poly-l-ornithine coated cover glass could not supportlong-term neuronal growth. We followed the following stepwiseprocedure for cover glass sterilization and modification of coverglass surface using adhesives.
• We have used 22 mm circular cover glass in our study. At leastfour days prior to use they were thoroughly washed in runningwater to remove dust and other water soluble or loosely attachedparticles from the glass surface. This step was followed by placingthe cover glasses in 2 N HCl for one day.
• The next day these cover glasses were rinsed thoroughly withmilli-Q water for 4–5 h with hourly exchange of water followedby dipping in 70% EtOH for 15–20 min. They were then picked upindividually with forceps and dried by waving briefly over a flame(for a few seconds only). This step should be done with utmostcare because cover glasses may crack if they are held for longerduration directly over the flame.
• The cover glasses were sealed in a glass Petri dish and baked ina sterilized oven at 225 ◦C for 1 h followed by UV irradiation for15–20 min in a laminar hood.
• Around 200–400 �l of poly-d-lysine was uniformly spread overthe entire exposed surface of the coverglasses. The cover glasseswere then left overnight to be used the next day. Adequate carewas taken to avoid deposition of dirt and other suspended par-ticulate matter.
• As excess poly-d-lysine is toxic to the developing neurons, itwas removed by thorough washing and rinsing the coated coverglasses twice with sterile milli-Q-water for 5 min each in a lami-nar flow hood under sterile conditions. No sterilization procedurewas carried out after coating with poly-d-lysine because UV expo-sure might degrade the matrix (Sherratt et al., 2010).
• Neurons are very sensitive to O2 exposure (Brewer and Cotman,1989; Zeiger et al., 2010) and O2 imbalance is reported to causeneuronal loss. Therefore, before plating with neurons those poly-d-lysine coated cover glasses were pre-conditioned with culturemedium and incubated for 30 min to 1 h in an incubator at 37 ◦Cand 5% CO2 supply. This step presumably optimized the tempera-ture, CO2 level and pH of the medium for the cells to grow withoutshock.
2.7. Embryo dissection and surgical procedure
The fertilized eggs were incubated for 9 days at 80–85% humid-ity, 37.5 ± 0.2 ◦C in an Egg incubator. The incubator had facilitiesfor automatic tilting of the egg-holding tray at a pre-set speed andrate so that the eggs could be rocked and partially rotated. We hadset it for tilting the tray once in a 60 min by about 50◦ angles oneither side. Adequate care was taken to keep the broader ends ofthe eggs where the air sac is located, pointed upwards. This eggpositioning is an essential condition for proper egg hatching inincubator systems (Mao et al., 2007; Van Schalkwyk et al., 2000).Candling is a process to differentiate between fertile and infer-tile eggs. After a few days of incubation candling was performedusing trans-illumination with a 60 W light bulb to confirm visibleembryo formation inside the eggs. Once the embryo is formed andgrows within an egg the air sac becomes more pronounced anddark dots (i.e. the retinal pigments in the eyes) become distinctunder candling; infertile eggs lack such identifying marks. Neuronsare known to develop earlier than glial cells. Collecting neurons
early minimizes the chance of glial contamination (Parker et al.,1997; Pettmann et al., 1979). As knowing the exact time of fertil-ization is a serious practical as well as technical limitation, in thisstudy (after standardization) we have used post-fertilized 9 days
126 A. Kumar, B.N. Mallick / Journal of Neuroscience Methods 263 (2016) 123–133
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ig. 1. Dissection of the brain from a chick embryo, 9th day of incubation. (A) The
ith distinct eyes (black retinal pigment), capillaries and head. (B) An extracted emarefully removed. (F) Brain with its distinct lobes.
ncubated eggs, which corresponded to Hamburger and HamiltonHH) stage 35 as identified on the basis of presence of a nictitat-ng membrane, visceral arches and an elliptical eyeball (Hamburgernd Hamilton, 1992). Prior to removal of the embryo from the egg-hell, the eggs were observed under bright light and the shadow ofhe attachment of the membrane to the inner side of the egg-shellas marked semi-circular with a pencil on the outer side of the egg-
hell over the air sac. The eggs were sprayed with and thoroughlyiped with 70% EtOH to remove/minimize surface contamination.
hey were then introduced in a fully operational UV-sterile lami-ar flow hood. The eggs were placed on sterile Petri-dish with theroader end facing upwards. A small hole was made in the broadernd of the egg-shell through air sac to locate the embryo in themniotic fluid using curved forceps (Fig. 1). Using a pair of sterilecissors the chorioallantoic membrane was gently cut to have a bet-er view of the embryo, which was then carefully brought out withhe help of a sterile blunt forceps and placed on a sterile Petri-dish.he head was identified, severed from the body with the help of aterile scissors and transferred to a Petri dish. Then, using anotherair of sterile scissors and forceps the developing calvarium wasemoved. Meninges were gently and very carefully peeled off. Therain was excised and transferred (Fig. 1) to Ca2+ and Mg2+ freeank’s buffer. Complete removal of the meninges is essential as
hese contain many dividing cells which, if not removed, wouldontaminate the culture with fibroblasts, i.e. non-neural cells.
The brain dissection was carried out in 1× Hank’s buffer pH.3 (containing 5.36 mM KCl, 0.41 mM KH2PO4, 136.9 mM NaCl,aH2PO4·2H2O, 10 mM HEPES and 0.1% glucose/dextrose) havinga2+ chelators that included 0.002% EGTA and 0.01% EDTA, which
oosens the cells from each other in the intact brain (Brown andavis, 2002; Pitelka et al., 1983). Next, the brain was chopped intoner pieces. In each experiment we mixed brain tissues obtained
rom 1 to 4 embryos to get reasonable number of neurons. The finely
egg shell above the air sac is removed. The intact embryo at HH stage 35 is visiblen a sterile Petri-dish. (C) Head separated from the body. (D and E) Meninges being
chopped brain tissues were then placed in 0.25% trypsin–EGTAsolution for 10–15 min in a CO2 incubator for complete loosen-ing of the embryonic tissues. This trypsin mediated disaggregationstep was carried out at 37 ◦C to ensure maximum proteolysis ofthe enzyme; extra care was taken to prevent over exposure, whichmay lead to cell death. In our case, we incubated the brain tissuein trypsin for 10–15 min depending on the quantity of the tissue,which varied depending on the number of embryos taken. Thetrypsin activity was inhibited using DMEM medium containing 10%FBS, a known source of protease inhibitors. After 2–3 min the tis-sues were removed using modified/customized tips (broad boresize by removing pointed ends using scissors) attached to a 1 mlpipette. The partially digested tissues were placed in fresh Neu-robasal medium containing 2% B-27 supplement, 2 mM Glutamine,20 ng/ml NGF, Penicillin/Streptomycin and an antimycotic agent,Amphotericin B. The tissues were then gently triturated using afine bore pipette to obtain a single cell suspension. Air frothingduring pipetting was avoided as it has been reported to kill cellsat the air interface (Ren and Miller, 2003). This was avoided byejecting only about half of the pipette volume while preparingthe single cell suspension. The mixture was then passed through70 �m nylon mesh to remove cellular debris. The cells in the filtratewere counted using a Haemocytometer or using a handheld Scepter(Millipore). About 30,000–60,000 neurons were plated per 22 mmcircular cover glass or 35 mm confocal-bottom dish and cultured forstudying neuronal growth, development and synaptogenesis underlive cell imaging. The confocal-bottom dish was also coated withadhesive substratum (poly-d-lysine) following the same processas that of the cover glass as mentioned above. The cultures were
treated with 10 �M �-d-arabinofuranoside (glial inhibitor) aftertwo days of plating and at appropriate interval depending on gliadensity in the cultures. The �-d-arabinofuranoside acts on dividingcells and thus, after 2–3 cycles of media changes every 3–4 days, all
A. Kumar, B.N. Mallick / Journal of Neuroscience Methods 263 (2016) 123–133 127
Fig. 2. (A) 7 DIV cultured neurons double immunostained using rabbit anti-GFAP 1◦ Ab labeled with anti-rabbit-FITC 2◦ Ab (green) and mouse anti-synaptophysin labeled with2◦ anti-mouse Texas Red (red). GFAP is not visible, while synaptophysin is observed in the field suggesting the presence of neurons (red) devoid of glia. (B) C6-glioblastomaw ti-rabb(
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as used as positive control for GFAP using rabbit anti-GFAP 1◦ Ab labeled with anblue). Scale bars 50 �m.
he glia was lost in 8–9 days in vitro (DIV) and the cultures were leftith neurons only (Fig. 2). Glutamine addition into the medium was
estricted to the period after 4th DIV, which worked better, prob-bly because glutamine was converted into glutamate that causesxcitotoxicity in the long run (Driscoll et al., 1993). Evaporation ofedia in an incubator should be taken into consideration during
eplenishment. For example, if we removed 1 ml of the mediumrom the confocal bottom dish, we added around 1.1–1.2 ml of theresh medium.
.8. Immunocytochemistry
Neurons were grown on cover glasses and fixed with 4%araformaldehyde dissolved in 1× PBS for 15 min, followed byhree consecutive PBS washing of 10 min each. The cells wereermeabilized using 0.25% Triton-X for 5 min followed by wash-
ng with PBS for 5 min. However, for PSD-95 immunostaining theeurons were fixed in ice-cold methanol for 10 min and they didot need permeabilization. Primary antibodies at desired titer (asbserved in pilot studies), e.g. neuronal marker NeuN (1:200),AP-2 (1:200), pre-synaptic marker Synapsin (1:100) and Synap-
ophysin (1:100), post-synaptic marker PSD-95 (1:50), and glial
arker GFAP (1:200) were incubated overnight at 4 ◦C or for 3–4 ht room temperature. Fluorescent labeled isotype (species) spe-ific secondary antibodies against primary Ab were given at desiredilutions for 2 h at room temperature; followed by two PBS wash of
it-FITC 2◦ Ab (green); GFAP is visible in C6. Nuclei were counterstained with DAPI
5 min each. Primary Ab control, secondary Ab control and label con-trol were performed. In primary Ab control we checked the bindingof primary Ab to specific antigen. We have also performed con-trol experiments to check cross reactivity of multiple labeling bychanging the sequence of addition of secondary Ab, by adding bothprimary Ab and one secondary Ab or one primary Ab and both sec-ondary Abs and in other case no primary Ab and both secondaryAbs. For label controls, we incubated with different flurophoreattached secondary Abs to ascertain the specificity of flurophoreadded during the procedure. The nuclei were counterstained by1 �g/ml working solution of DAPI (in PBS) for 10 min followed bytwo PBS wash of 5 min each. The cells on the cover glasses weremounted with mounting medium consisting of mowiol® 4–88 inglycerol containing 2.5% DABCO as anti-fading agent (Harlow andLane, 2006; Johnson et al., 1982).
2.9. Microscopy
Neurons in the primary cultures were monitored in day andtime dependent manner using live cell imaging, Axio-observer. Z1microscope (Carl-Zeiss, Germany). Images were acquired in phaseoptics (20×, 40× and 63×), fluorescence and differential interfer-
ence contrast (DIC) in 100× objective and analyzed using Axiovisionsoftware. Fluorescence imaging was also performed using spinningdisk microscopy (Nikon) at the Central Instrumentation Facility inour School.
128 A. Kumar, B.N. Mallick / Journal of Neuroscience Methods 263 (2016) 123–133
Fig. 3. (A) 8 DIV cultured neurons double immunostained with mouse anti-MAP2 (detected with Alexa Fluor-488, green) and rabbit anti-Synapsin (detected with Texas Red,red). MAP2 and synapsin are present in neuronal cell bodies (soma) and their projections. (B) Eight DIV neurons double immunostained with mouse anti-NeuN (detectedw a neurn
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ith FITC, green) and rabbit anti-Synapsin (detected with Texas Red, red). NeuN is
eural projections. Nuclei were counter stained with DAPI (blue). Scale bars 5 �m.
.10. Synaptic vesicle dynamics
Primary cultures of chick neurons grown on cover glasses wereransfected with Synaptophysin-RFP (Cell Light® Synaptophysin-FP, Molecular Probes) as per manufacturer’s protocol. Approxi-ately 6 �l Synaptophysin-RFP was poured on each cover glass
aving around 30,000 neurons. The neurons were monitored underhe microscope after 16 h of transfection at 100× objective withumerical aperture 1.4 attached to an Axio-observer. Z1 micro-cope (Carl-Zeiss, Germany) and the transfected vesicles could beracked. We observed the transfected cells for about 5 days post-ransfection; however, we restricted capturing dynamic events upo 25–30 h to avoid possible side effect(s) due to viral transfection.
.11. Observations
Double immunostaining showed the neurons devoid of glia inhe culture (Fig. 2); while the confirmed neurons with more thanne marker can be seen in Fig. 3. We could maintain and observehe neurons at least for 4 weeks (Fig. 4). We found that the neu-ons adhered to the coated surface after 2–3 h of plating. Neuronsrst appeared with small projections followed by neurite growth
nd retraction (Movie-1). Also, growth of some neurons appearedo track or slide along other neuronal projections giving fascicularppearance (Movie-1). Further, the neurite growth and retrac-ion slowed down as culture day progressed (Movie-1). We couldonal marker which stains nuclei. Synapsin is expressed by the cell body as well by
visualize and study the movement of the growth cones havingdistinct filopodia and lamellipodia (Movie-2). Fluorescence of RFPtagged Synaptophysin of the vesicles could be seen under fluores-cence imaging. The vesicles differed in size and speed of movement(Movie-3). Synapsin and Synaptophysin distribution patterns couldbe seen around 5 DIV and as days progressed, they showed punc-tate appearance as a reflection of possible synapse formation andmaturation.
3. Results and discussion
Growth and differentiation of cells include complex processes;the development of neurons belongs to most complex categoriesof cell differentiation and interaction. The latter involves growth,protuberance of neurites, their growth and retraction. These eventsare highly dynamic and involve remodeling of cellular morphologyat a fast rate sometimes in the order of seconds. Initially after plat-ing, cells show a lamellar appearance followed by emergence ofneurites developing growth cone at their tips. These early neuritesare highly dynamic, showing successions of growth and retraction,while sometimes they even get eliminated. In later stages, manyneurites are of comparable length. Out of these developing neu-
rites, after 2 DIV culture one of the neurites is polarized and growsmore than 100–140 �m in length possibly to grow into an axonwhile other projections show distinct tapering (Fig. 7) (Dotti et al.,1988; Kosik and Finch, 1987; Szebenyi et al., 1998).
A. Kumar, B.N. Mallick / Journal of Neuroscience Methods 263 (2016) 123–133 129
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ig. 4. Neurons isolated from chick embryo brain long term cultured after plating inell imaging microscopy after 2, 4, 8, 15, 23 and 28 days of plating. Morphological c
Neurites possess a specialized somatosensory structure at theirips: the growth cone with its distinct filopodia and lamellipo-ia (Movie-2). Presynaptic marker, e.g. synapsin staining appearedround 4–5 DIV culture; however, its intensity was low After 9–10IV punctate presynaptic vesicles appeared (Fig. 5). Around 10 DIVe followed a case of an individual neuron showing different types
f events, e.g. one projection was retracted while other projectionso nearby cell(s) remained intact for more than 7 h suggesting theossibility of developing into a matured synapse (Movie-1); we hadollowed several such cases. In this movie a fasciculation can be seenhat includes neurites belonging to different neurons and arrivingrom different directions. Post synaptic marker PSD-95 was seenfter 20 DIV (Fig. 5). Synaptic vesicles (SVs) were seen after trans-ection with Synaptophysin-RFP and became demonstrable starting0–24 h after transfection (Fig. 5). These vesicles were of variousizes and shapes; they could be tracked and their dynamic prop-rties could be estimated (Fig. 6). Vast dendritic arbors were alsoeveloped (Fig. 7). We could follow the primary cultures of the neu-ons at regular intervals until past 6 weeks after plating. Thereafter,he neurons started detaching from the cover glasses, possibly dueo apoptosis setting in.
The current protocol supports primary cultures of neuronserived from chick embryos; glial contamination is effectively sup-ressed. The neurons in cultures differentiated phenotypically,nd developed toward cells fully resembling matured neurons.n the method reported here we detected the presence of vari-us neuronal markers and we were able to study the dynamicsf SVs as well. Using immunocytochemistry, we have demon-trated the presynaptic neuronal marker synapsin, synaptophysinnd post-synaptic marker PSD-95 (Fig. 5), presence of stringenteuronal marker like NeuN, MAP-2 and synapsin (Fig. 3). Usingirus-mediated transfection we could study the SV-dynamics. Weoted different shapes of spines on neuronal branches near the
ell bodies (soma) including mushroom shaped spines having anpproximate diameter of 2.5 �m (Fig. 8). We observed how a sin-le neuron made contacts with target neurons located even atonger distance, i.e. bypassing neurons on the way (Fig. 9A and B);focal bottom dish. Cultured neurons were observed under phase optics 20× in lives occur during growth and differentiation. Scale bars 50 �m.
projections from different neurons were traced to the somata ofother neuron(s) (Fig. 9C and D). As compared to presently availablemethods, by this protocol we could maintain healthy cultures forsignificantly longer time apparently free from glial contamination,observe the neuronal growth and development at different timeintervals, synaptogenesis, interstitial axonal branching and tracethem live, we could also study growth cone and vesicular dynam-ics after transfection; the details of which have been mentionedalong with respective methods and results.
Different types of media containing various supplements arecommercially available; they are either glial defined or neuronaldefined. Various combinations of mixed culture and sandwichculture have been used to study neuronal growth (Chen et al.,2011; Jones et al., 2012; Todd et al., 2013); each has its ownmerits and demerits. We have developed a primary neuronal cul-ture using neuronal defined medium that needs no mixing orsandwich with glia and hence is apparently free from glial contam-ination. Notwithstanding, although our preparation could grow inthe absence of glia, it is possible that the presence of a few gliacells or their secretions at the start of the culture could have beensufficient to grow neurons alone, which needs confirmation. Celldensity also plays a significant role in the neuronal growth andsurvival. In this method, we have standardized a low-density neu-ronal culture that is ideal for live cell imaging studies. As neuronswere sparsely distributed, it was easier to monitor, track and imageacquisition of individual neurons for long hours for off-line analy-sis. We have used chick neurons collected from post-fertilized eggsincubated for 9 days corresponding to stage HH 35 (Hamburgerand Hamilton, 1992) as around this time a majority of the neu-ronal population has already been formed, however, there is littleor no glia. Isolation of neurons at this stage has its own advantageas at this stage we can isolate neurons from glial cells which leadto uniform and homogenous population(s) of neurons in the cul-
tures with developing cells. Further, even a few glia was present,they could be easily inhibited by �-d-arabinofuranoside treatmentin a few successive medium changes. It was also relatively easyand convenient to dissociate embryonic tissue and their rate of
130 A. Kumar, B.N. Mallick / Journal of Neuroscience Methods 263 (2016) 123–133
Fig. 5. 30 DIV primary neuron culture seen in DIC and stained with rabbit anti-Synapsin (presynaptic marker; detected with FITC, green), mouse anti-PSD-95 (postsynapticmarker; detected with Texas Red, red). Nuclei were counterstained with DAPI (blue). The merged image shows yellow punctate appearances, which are the point of contacts(synapses). The yellow color is caused by overlap of FITC and Texas Red signals. Scale bars 10 �m.
Fig. 6. 33 DIV neurons transfected with RFP tagged Synaptophysin. (A) DIC image of a transfected neuron. (B) RFP-labeled synaptic vesicles are present in the cell body (soma)as well as in neuronal projections. (C) Overlay of RFP and DIC images verifying localization of synaptic vesicles in the cell body (soma) and neuronal branches (neurites). (D)Gray scale image of the vesicular fluorescence shown in (B). Scale bars 10 �m.
A. Kumar, B.N. Mallick / Journal of Neuroscience Methods 263 (2016) 123–133 131
F d tapei
sppsmocOsoWn
alais2fbiTwa2ft
ttoeicy
malian counterparts (Andermatt and Stoeckli, 2014; Baeriswyland Stoeckli, 2006). Compared with mammalian embryos, chickembryos have much shorter gestation period while this period
ig. 7. 15 DIV neurons showing distinct polarity which is characteristic of axon anndicated by white arrow. Scale bar 50 �m.
urvival was higher than that of neurons collected from an adult orost-natal day brains. Notwithstanding, one may encounter a fewractical difficulties (see Section 2) which we have addressed andolved. Difficulties included exposure of the cultures to optimumixture of O2 and CO2. Issues like these were solved by taking care
f appropriate cell adhesive substratum, adequate and optimumover glass washing, coating and care during medium exchange.ther issues were the need of proper trophic support and pre-
enting growth cues in their proper temporal sequence. These kindf issues needed special care and attention throughout the study.e are yet to study the ultrastructural details which we are plan-
ing to do after adequate standardization.Our results show that brain from chicken embryo can be used
s a good model for studying cellular and molecular neurobio-ogy as well as synaptogenesis. The chicken has been used as
model to study developmental events from early days of civ-lization. Chicken as a model organism has been used to studyeveral biological processes including organ development (Stern,004, 2005). This includes Aristotle’s embryological work in hisamous treatise “Historia Animalium”, William Harvey’s study oflood circulation, discovery of cholera vaccine by Louis Pasteur, and
solation and characterization of NGF by Levi-Montalcini (1952).he chick embryo develops into a vertebrate, homeotherm chickenhose brain development has been well characterized (Andermatt
nd Stoeckli, 2014; Baeriswyl and Stoeckli, 2006; Burt, 2007; Stern,005). In contrast to mammalian embryos, chicken embryos, i.e.ertilized chicken eggs are readily and easily available any timehroughout the year.
Although it is difficult to know the precise moment of fertiliza-ion, the actual start of incubation may be safely considered as therigger that starts post-fertilization events. Thus, both the demandsf synchronization of developmental stages between individual
mbryos and the comparability of series of experiments are sat-sfied. Chicken eggs are large, mechanically robust, extremelyost-effective, easy to transport and handle, and available allear long. Because they are self-sufficient, chick embryos posered dendritic arborization in neurons can be seen. Interstitial axonal branching is
less complexity to experimental manipulation than their mam-
Fig. 8. 11 DIV primary cultures of neurons showing the presence of spines (indicatedby arrows). Scale bars 20 �m.
132 A. Kumar, B.N. Mallick / Journal of Neuroscience Methods 263 (2016) 123–133
Fig. 9. (A) Collage showing a neuronal projection reaching a cell body (soma) of another neuron indicated by white arrow. (B) The same neuron as in (A) has been tracedu n has
n et in (
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Banker G, Goslin K. Culturing nerve cells. MIT Press; 1991, 65 pp.
sing Axiovision software. In this panel it can be seen that the projection of a neuroeuronal projections contacting a target neuron. Scale bar 10 �m. (D) Magnified ins
s still sufficiently long to study or manipulate inter and intra-ay events. Another important advantage of the self-sufficiencyf chick embryos is that incubation and hatching can be per-ormed under controlled conditions. Thus, the chicken embryo
odel represents an easy and very convenient method to studyeuronal growth, development, vesicular transport, synaptogene-is and synaptic transmission under live cell imaging.
Gallus domesticus has a compact genome size (approximately0,000 genes) which to some extent is closer in number to humanshan many species. Thanks to the emergence of cutting edge tech-ologies (Cogburn et al., 2004; Das et al., 2006; Novina and Sharp,004; Pandey et al., 2004) and whole genome sequencing, it isow becoming possible to study the function of individual genessing reverse and forward genetic approaches in developing neu-ons derived from chicken embryos. Also, in a phylogenetic sensehe chicken embryo may be a good model to compare the functionf particular genes from one species with that of humans. Suchtudies are less easy with mice and rats due to the nearness of theodent genome to the human genome (Ankra-Badu and Aggrey,005; Hardison, 2000; Uchikawa, 2008).
. Conclusion
We describe here an easy and convenient protocol of primaryulture of neurons from chick embryo grown by hatching eggs inn incubator in the laboratory. The method can be convenientlysed as a model to study neuronal growth, development, neuriterowth and retraction, axonal path-finding, growth cone dynam-cs, vesicular transport and synaptogenesis under live cell imaging
icroscopy, immunostaining and transfection. We have success-ully monitored all these aspects.
onflict of interest
The authors declare that they do not have any conflict of interest.
grown more than 300 �m to reach the target neuron. (C) DIC image showing manyC). Scale bar 5 �m.
Acknowledgements
We thank Prof. Anil K. Tyagi and Prof. Debi P. Sarkar as wellas their associates from Delhi University South Campus for kindlyhelping us with fertilized eggs during our initial phase of standard-ization and setting-up of the process of primary culture until wedeveloped egg-fertilization facility in our laboratory. We acknowl-edge the help extended by Dr. Gunjan Dhawan during the initialphase of setting-up of the primary culture. We also thank Ms. TriptiPanwar for her help in image acquisition on the spinning diskmicroscope at the Central Instrumentation Facility of our school.A.K. received CSIR fellowship for carrying out Ph.D. Research fund-ing to B.N.M. from JC Bose Fellowship; DBT-BUILDER; DST-FIST;DST-PURSE; UGC, UPE-II; UGC-RNW are acknowledged.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.jneumeth.2016.02.008.
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