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Marina E. Wolf, PhD

Marina E. Wolf, PhD
Professor and Chair

Dr. Marina Wolf is Chair of Neuroscience at the Chicago Medical School at Rosalind Franklin University of Medicine and Science. She has been a pioneer in studying the role of neuronal plasticity in drug addiction. Her laboratory uses animal models to understand why recovering addicts remain vulnerable to drug craving and relapse even after long periods of abstinence. Cell culture models are used for mechanistic experiments to complement the in vivo studies. Dr. Wolf received her PhD in Pharmacology from Yale in 1986 and trained as a postdoctoral fellow at the Center for Cell Biology, Sinai Hospital of Detroit. She was Assistant Professor of Psychiatry at Wayne State University before moving to the Chicago Medical School in 1992. Her laboratory has been continuously supported by NIDA since 1992 and she is presently PI on two R01 awards. She has previously been the recipient of a Merit Award from NIDA (R37) as well as a Senior Scientist Research and Mentorship Award (K05). She has served as a member of the NIDA Advisory Council, the NIH Council of Councils, the NIDA Board of Scientific Counselors, and the Council of the American College of Neuropsychopharmacology (ACNP). She has served on many NIH study sections, most recently as Chair of MNPS. She presently serves on the Scientific Council of the Brain & Behavior Research Foundation and is President-Elect of the ACNP.

Research Projects | Publications | Protocols

Research Projects

Glutamate is a key transmitter for neuronal plasticity and learning. My lab and others have shown that behavioral changes in animal models of addiction require glutamate-dependent forms of plasticity, and that learning and addiction involve common brain signaling pathways and cellular changes. Thus, addiction may be viewed as a form of maladaptive but extremely strong learning. An important question is how drugs like cocaine and amphetamine, which initially target dopamine (DA) systems in the brain, ultimately produce adaptations in glutamate neurotransmission. A better understanding of plasticity mechanisms engaged by drugs of abuse may lead to the development of pharmacological approaches to reduce drug craving and relapse.

Alterations in glutamate receptors are critical for controlling the strength of glutamate synapses in well-studied forms of plasticity such as LTP. My lab focuses on how alterations in glutamate receptor expression, distribution and function contribute to addiction-related plasticity. Our primary approach is to use biochemical and electrophysiological methods to investigate the functional significance of glutamate-dependent plasticity for in vivo models of addiction. We also use primary neuronal cultures to address mechanistic questions that are difficult or impossible to study in intact animals. Our work is funded by two R01 grants from the National Institute on Drug Abuse.

Our in vivo studies currently focus on the role of GluA2-lacking, calcium-permeable AMPA receptors (CP-AMPARs) in the incubation model of cocaine addiction, in which cue-induced cocaine craving progressively intensifies (“incubates”) during withdrawal from extended access cocaine self-administration (for review, see Wolf, 2016). In drug-naïve rats or rats with limited cocaine exposure, CP-AMPARs are normally expressed at very low levels by NAc neurons (Boudreau et al., 2007; Reimers et al., 2010). However, CP-AMPARs are added to NAc synapses after withdrawal from extended access cocaine self-administration and mediate the expression of “incubated” cue-induced cocaine craving (Conrad et al., 2008). Current goals include understanding mechanisms that enable CP-AMPARs to accumulate in NAc synapses during “incubation” (Ferrario et al., 2011; Li et al., 2013; Loweth et al., 2014) and strategies for normalizing synaptic transmission by stimulating mGluR1 and thereby removing CP-AMPARs from synapses (McCutcheon et al., 2011; Loweth et al., 2013, 2014). Additional areas of interest include: 1) characterization of mechanisms regulating protein translation in the NAc under normal conditions and after the incubation of craving, and how aberrant regulation of translation maintains cocaine-induced changes in excitatory synaptic transmission and drug craving (Scheyer et al., 2014; Stefanik et al., under review), 2) the role of the ubiquitin-proteasome system (UPS) in cocaine-induced changes in excitatory synaptic transmission in the NAc (Werner et al., 2015), 3) the role of NMDAR plasticity in the incubation of cocaine craving (Christian et al., in preparation), and 4) changes in AMPAR and group I metabotropic glutamate receptor transmission in the NAc accompanying the incubation of methamphetamine craving (Scheyer et al., 2016). Major techniques involved are drug self-administration, protein analysis (e.g., westerns, biotinylation and immunoprecipitation), PCR, assays of protein translation in NAc tissue (metabolic labeling and puromycin labeling), spine analysis using Lucifer yellow, and whole-cell patch-clamp recordings in acute slices.

Our in vitro studies focus on similar scientific questions, but take advantage of the ability to more directly examine cellular mechanisms using cultured neurons. We prepare primary cultures and co-cultures of neurons from addiction-related brain regions such as the NAc, prefrontal cortex, hippocampus, and ventral tegmental area (for review, see Wolf, Neurotoxicol Res, 2010). Principal neurons in the first three regions receive convergent DA and glutamate inputs. We have shown that DA receptors regulate AMPA receptor trafficking to extrasynaptic sites on the cell surface and thereby modulate synaptic plasticity (Chao et al., 2002a,b; Mangiavacchi & Wolf, 2004a.b; Sun et al., 2005; Gao et al., 2006; Sun et al., 2008). More recently, we have demonstrated that synaptic scaling occurs in the NAc and characterized some of its features (Sun & Wolf, 2009; Reimers et al., 2014). This and other types of homeostatic plasticity may be important during drug withdrawal. Current projects focus on the role of group I metabotropic glutamate receptors in regulating AMPAR trafficking in the NAc (Loweth et al., in preparation) and characterization of the regulation of dendritic protein translation in NAc and prefrontal cortex neurons by ionotropic and metabotropic glutamate receptors (Stefanik et al., under review). Major techniques involved are cell culture, immunocytochemistry, translation assays (primarily FUNCAT), and fluorescence microscopy.

LAB MEMBERS

Michael milovanovicMichael milovanovic

Research Associate
Room: 2.271
Phone: 847-578-3000 (X8353)
Email: mike.milovanovic@rosalindfranklin.edu

Daniel ChristianDaniel Christian, Ph.D.

Postdoctoral Research Associate
Room: 2.260
Phone: 847-578-3000 (X3529)
Email: daniel.christian@rosalindfranklin.edu

Michael StefanikMichael Stefanik, Ph.D.

Postdoctoral Research Fellow
Room: 2.260
Phone: 847-578-3000 (X3529)
Email: michael.stefanik@rosalindfranklin.edu

Amanda WunschAmanda Wunsch

Postdoctoral Research Fellow
Room: 2.260
Phone: 847-578-3000 (X3529)
Email: amanda.wunsch@rosalindfranklin.edu

Johnathan FunkeJohnathan Funke

Research Assistant
Room: 2.271
Phone: 847-578-3000 (X8353)
Email: johnathan.funke@rosalindfranklin.edu

Conor MurrayConor Murray

Grad Student
Room: 2.227b
Phone: 847-578-3000 (X8172)
Email: conor.murray@my.rfums.org

RESEARCH PROJECTS | PUBLICATIONS | Protocols

Publications

Selected Publications (from over 100 peer-reviewed papers and over 40 reviews and book chapters)

Werner CT, Murray CH, Reimers JM, Chauhan NM, Molla HM, Loweth JA, Wolf ME (2017) Trafficking of calcium-permeable and calcium-impermeable AMPA receptors in nucleus accumbens medium spiny neurons co-cultured with prefrontal cortex neurons. Neuropharmacology 116:224-232. PMC5385156

Glynn RA, Wolf ME, Rosenkranz JA, Loweth JA (2017) Repeated restraint stress exposure during early withdrawal accelerates incubation of cue-induced cocaine craving. Addiction Biology [Epub ahead of print, Nov 11 2016. doi: 10.1111/adb.12475]. PMC5426993

Christian DT*, Wang XT*, Chen E, Sehgal L, Ghassemlou M, Miao J, Estepanian D, Araghi C, Stutzmann GE, Wolf ME (2017) Dynamic alterations of nucleus accumbens dendritic spines during the incubation of cocaine craving. Neuropsychopharmacology 42(3):748-756. PMC5240181

Purgianto A, Weinfeld ME, Wolf ME (2017) Prolonged withdrawal from cocaine self-administration affects prefrontal cortex- and basolateral amygdala-nucleus accumbens core circuits but not accumbens GABAergic local interneurons. Addiction Biology 22(6):1682-1694. PMC5364065

Purgianto A, Loweth JA, Miao JJ, Milovanovic M, Wolf ME (2016) Surface expression of GABAA receptors in the rat nucleus accumbens is increased in early but not late withdrawal from extended-access cocaine self-administration. Brain Research 1642:336-343. PMC4899143.

Scheyer AF*, Loweth JA*, Christian DT*, Uejima J, Rabei R, Le T, Dolubizno H, Stefanik MT, Murray CH, Sakas C, Wolf ME (2016) AMPA receptor plasticity in accumbens core contributes to incubation of methamphetamine craving. Biological Psychiatry 80:661-670. PMC5050076.

Werner CT, Milovanovic M, Christian DT, Loweth JA, Wolf ME (2015) Response of the ubiquitin proteasome system to memory retrieval after extended-access cocaine or saline self-administration. Neuropsychopharmacology 40(13): 3006-3014. PMC4864635.

Ma Y-Y, Lee BR, Wang X, Guo C, Liu L, Cui R, Lan Y, Balcita-Pedicino JJ, Wolf ME, Sesack SR, Shaham Y, Schluter OM, Huang YH, Dong Y (2014) Bi-directional modulation of incubation of cocaine craving by silent synapse-based remodeling of prefrontal cortex to accumbens projections. Neuron 83(6):1453-1467. PMID: 25199705 (PMCID in progress).

Reimers JA, Loweth JA, Wolf ME (2014) BDNF contributes to both rapid and homeostatic alterations in AMPA receptor surface expression in nucleus accumbens medium spiny neurons. Eur J Neuroscience 39(7):1159-1169 (special issue on Synaptic Basis of Disease). PMID: 24712995 (PMCID in progress).

Scheyer AF*, Wolf ME*, Tseng KY* (2014) A protein synthesis-dependent mechanism sustains calcium-permeable AMPA receptor transmission in nucleus accumbens synapses during withdrawal from cocaine self-administration. J Neurosci 34(8):3095-3100. PMC3929765.

Loweth JA, Scheyer AF, Milovanovic M, LaCrosse AL, Flores-Barrera E, Werner CT, Li X, Ford KA, Le T, Olive MF, Szumlinski KK, Tseng KY*, Wolf ME* (2014) Synaptic depression via positive allosteric modulation of mGluR1 suppresses cue-induced cocaine craving. Nat Neurosci 17(1):73-80. PMC3971923.

Dec AM, Kohlhaas KL, Nelson CL, Hoque KE, Leilabadi SN, Folk J, Wolf ME, West AR (2014) Impact of neonatal NOS-1 inhibitor exposure on neurobehavioral measures and frontal-temporolimbic integration in the rat nucleus accumbens. Int J Neuropsychopharmacology 17(2):275-287. PMID: 24025168 (PMCID in progress)

Selvakumar B, Campbell PW, Milovanovic M, Park DJ, West AR, Snyder SH, Wolf ME (2014) AMPA receptor upregulation in the nucleus accumbens shell of cocaine-sensitized rats depends upon S-nitrosylation of stargazin. Neuropharmacology 77:28-38. PMC3934570

Plaza-Zabala A, Li X, Milovanovic M, Loweth JA, Maldonado R, Berrendero F, Wolf ME (2013) An investigation of interactions between hypocretin/orexin signaling and glutamate receptor surface expression in the rat nucleus accumbens under basal conditions and after cocaine exposure. Neurosci Lett 557 Pt B:101-106. PMC3869201

Lee BR, Ma Y-Y, Huang YH, Wang X, Otaka M, Ishikawa M, Neumann PA, Graziane NM, Brown TE, Suska A, Guo C, Lobo MK, Sesack SR, Wolf ME, Nestler EJ, Shaham Y, Schlüter OM, Dong Y (2013) Maturation of silent synapses in amygdala-accumbens projection contributes to incubation of cocaine craving. Nat Neurosci 16(11):1644-1651. PMC3815713

Wang X, Cahill ME, Werner CT, Christoffel DJ, Golden SA, Xie Z, Loweth JA, Marinelli M, Russo SJ, Penzes P, Wolf ME (2013) Kalirin-7 mediates cocaine-induced AMPA receptor and spine plasticity, enabling incentive sensitization. J Neurosci33:11012-11022. PMC3718375

Purgianto A*, Scheyer AF*, Loweth JA, Ford KA, Tseng KY, Wolf ME (2013) Different adaptations in AMPA receptor transmission in the nucleus accumbens after short versus long access cocaine self-administration regimens. Neuropsychopharmacol 38:1789–1797. PMC3717554

Li X, DeJoseph MR, Urban JH, Bahi A, Dreyer J-L, Loweth AJ, Ferrario CR, Ford KA, Wolf ME (2013) Different roles of BDNF in nucleus accumbens core versus shell during the incubation of cue-induced cocaine craving and its long-term maintenance. J Neurosci 33: 1130-1142. PMC3711541

Ferrario CR, Goussakov I, Stutzmann GE, Wolf ME (2012) Withdrawal from cocaine self-administration alters NMDA receptor-mediated Ca2+ entry in nucleus accumbens dendritic spines. PLoS One 7(8): e40898. PMC3411692

Boudreau AC, Milovanovic M, Conrad KL, Nelson CL, Ferrario CR, Wolf ME (2012) A protein crosslinking assay for measuring cell surface expression of glutamate receptor subunits in t he rodent brain after in vivo treatments. Curr Protoc Neurosci 59:5.30.1-5.30.19. PMC3356776

McCutcheon JE, Loweth JA, Ford KA, Marinelli M, Wolf ME*, Tseng KY* (2011) Group I mGluR activation reverses cocaine-induced accumulation of calcium-permeable AMPA receptors in nucleus accumbens synapses via a protein kinase C-dependent mechanism. J Neurosci 31:14536 –14541. PMC3220940

Li X, Wolf ME (2011) Visualization of virus-infected brain regions using a GFP-illuminating flashlight enables accurate and rapid dissection for biochemical analysis. J Neurosci Meth 201:177-179. PMC3176337

Li X, Wolf ME (2011) Brain-derived neurotrophic factor rapidly increases AMPA receptor surface expression in rat nucleus accumbens. Eur J Neurosci 34:190-198. PMC3936351

McCutcheon JE, Wang X, Tseng KY, Wolf ME*, Marinelli M* (2011) Calcium-permeable AMPA receptors are present in nucleus accumbens synapses after long withdrawal from cocaine self-administration but not experimenter-administered cocaine. J Neurosci 31:5737-5743. PMC3157976

Ferrario CR, Loweth JA, Milovanovic M, Ford KA, Galiñanes GL, Heng L-J, Tseng KY, Wolf ME (2011) Alterations in AMPA receptor subunits and TARPs in the rat nucleus accumbens related to the formation of Ca2+-permeable AMPA receptors during the incubation of cocaine craving. Neuropharmacology 61:1141-1151. PMC3094740

Ferrario CR, Loweth JA, Milovanovic M, Wang X, Wolf ME (2011) Distribution of AMPA receptor subunits and TARPs in synaptic and extrasynaptic membranes of the adult rat nucleus accumbens. Neurosci Lett 490:180-184. PMC3038183

Reimers JM, Milovanovic M, Wolf ME (2011) Quantitative analysis of AMPA receptor subunit composition in addiction-related brain regions. Brain Res 1367:223-233. PMC3005033

Ferrario CR, Li X, Wolf ME (2011) Effects of acute cocaine or dopamine receptor agonists on AMPA receptor distribution in the nucleus accumbens. Synapse 65:54-63. PMC2965794

*joint first or last authorship

SELECTED RECENT REVIEW ARTICLES AND BOOK CHAPTERS

Dong Y, Taylor JR, Wolf ME, Shaham Y (2017) Circuit and synaptic plasticity mechanisms of drug relapse. J Neurosci 37(45):10867-10876. PMC in progress

Wolf ME (2016) Synaptic mechanisms underlying persistent cocaine craving. Nat Rev Neurosci 17(6):351-65. PMC5466704

Li X, Wolf ME (2016) Multiple faces of BDNF in cocaine addiction. Behav Brain Res 279C:240-254. PMC4277902

Loweth JA, Tseng KY, Wolf ME (2014) Neuroadaptations in the nucleus accumbens contributing to incubation of cocaine craving. Neuropharmacol 76 Pt B: 287-300. PMC3836860

Loweth JA, Tseng KY, Wolf ME (2013) Using metabotropic glutamate receptors to modulate cocaine’s synaptic and behavioral effects: mGluR1 finds a niche. Current Opin Neurobiol 23(4):500-506. PMC3664109

Wolf ME, Tseng KY (2012) Calcium-permeable AMPA receptors in the VTA and nucleus accumbens after cocaine exposure: when, how and why? Front Molecular Neurosci 5:72. PMC3384237

Wolf ME (2010) The Bermuda triangle of cocaine-induced neuroadaptations. TINS 33:391-398. PMC2935206

Wolf ME (2010) Regulation of AMPA receptor trafficking in the nucleus accumbens by dopamine and cocaine. Neurotoxicol Res 18:393–409. PMC3935242

Wolf ME, Ferrario CR (2010) AMPA receptor plasticity in the nucleus accumbens after repeated exposure to cocaine. Neurosci & Biobehav Rev 35(2):185-211. PMC2962767

RESEARCH PROJECTS | PUBLICATIONS | PROTOCOLS

Lab Photos

Conor Lab Group in 2016From left to right: Jessica Loweth, Mike Milovanovic, Dan Christian, Marina Wolf, Michael Stefanik, Aaron Caccamise, Conor Lab Group in 2016

Stop that dogStop that dog!

Kelly Conrad's graduation party

From left to right: Xuan Li, Carrie Ferrario, Jeremy Reimers, Xiu Sun, Xiaoting Wang, Kelly Conrad, and Chris Nelson, at Kelly Conrad's graduation party.

Proving that we work here

From left to right: Carrie Ferrario, Marina Wolf, Xuan (Anna) Li, Xiaoting Wang, Jeremy Reimers, and Mike Milovanovic proving that we work here.

Jeremy Reimers

Jeremy Reimers, former Ph.D. student in Dr. Wolf's Lab at his graduation party.

4th percentile party

Kuei Tseng, Marina Wolf, Xuan (Anna) Li, and Jessica Loweth, at the 4th percentile party.

Kuei Tseng and Marina Wolf are happy.

Kuei Tseng and Marina Wolf are happy.

Anna Li and Jessica Loweth are happy.

Anna Li and Jessica Loweth are happy.

Interdepartmental Neuroscience & Neuropharmacology Retreat

Marina, Jessica, Anna and Xiaoting at the Interdepartmental Neuroscience & Neuropharmacology Retreat

Jeremy Reimer's graduation party

Kuei Tseng and Bob Messing at Jeremy Reimer's graduation party.

Andrew Scheyer Defense Party 2015

Andrew Scheyer Defense Party 2015

Craig Werner Defense Party 2015

Craig Werner Defense Party 2015

Craig with committee 2015

Craig with committee 2015

Craig's Defense 2015

Craig's Defense 2015

Mike and Michael Winter 2014

Mike and Michael Winter 2014

RESEARCH PROJECTS | PUBLICATIONS | PROTOCOLS

Protocols

Nucleus Accumbens Cell Culture Protocol

1. PREPARATION

2 Days before dissection
1.1 Prepare glass coverslips and poly-d-lysine coated plate or flasks.
Needed: glass coverslips (Fisher brand 12mm,12-454-82)
Poly-d-lysine
1M HCl
95% EtOH
24 well plate
1) Put some clean coverslips in a small beaker with 1M HCl (~ 30ml) and sonicate for 15 min
2) Wash the coverslips with ddH2O twice
3) Wash the coverslips with EtOH 95% 2x and sonicate for 5 min
4) Rinse in fresh EtOH 95% (the coverslips can be stored at 4°C for a couple months)
5) Individually flame dry the coverslips and place in a 24-well-plate.
6) Sterilize under UV light for about 1-2 hr
7) Add 0.4 -0.5ml Poly-d-Lysine/well (24 well plate) and incubate at 37°C overnight
8) Remove Poly-d-Lysine
9) Rinse 2x with sterile H2O or PBS 1X and air-dry the plates in the hood (dark) for 2hrs, store at 4°C until use (covered with Aluminum Foil).

24 hours before dissection
1.2 Prepare Conditioned Media for NAc cells
1) Prepare 50 ml Initial Neurobasal Media
Initial Neurobasal Media is used for primary NAc cultures only on first day of culture. Fifty ml of Initial Neurobasal Media is sufficient for two plates - this amount can be obtained from 4 flasks (25cm2) of glial cells (12.5ml/flasks).
50 ml Neurobasal Media
500 ml Glutamine
250 ml Gentamicin
500 ml Fungizone
2) Filter (0.22mm filter)
3) Add 12.5 ml above media to each flask of glial cells, condition the media for 8-24 hr.
1.3 Prepare solutions
1) 100 ml PBS 1X
90 ml H2O sterile
10 ml PBS 10X
Store at 4°C
2) 50 ml CMF Buffer
45 ml Sterile H2O
5 ml PBS 10X
250 ml 20% Glucose
500 ml Fungizone
250 ml Gentamicin
Filter and store at 4°C
1.4 Autoclave the following equipment and supplies
One 16G sharp edged cannula (for making tissue punches), two 27G needles, 1 blade handle, 2 forceps, 1 spatula, 1 scissors, long and short pipettes, 500ml and 200ml tips
1.5 Wax-plate
Melt 1 wax sheet (Ted Pella, Inc. Cat # 109) on 60mm plate, sterilize the wax filled plate under UV (2h or overnight). The wax can be remelted.

The Dissection Day
1.6 Continue preparation of Conditioned Media for NAc cells (see 1.2 above).
Collect conditioned media from glial cells (to be used today; day 1 of culture) and prepare additional medium to be conditioned for use on the next day.
1) Thaw Glutamic acid and B27 under the hood
2) Make 50 ml Regular Neurobasal Media for conditioning today, use tomorrow
50 ml Neurobasal Media
500 ml Glutamine (200 mM)
250 ml Gentamicin (10mg/ml)
3) Remove Initial Neurobasal Media conditioned from glia cultures and add
500 ml Glutamic Acid
500 ml B27
4) Filter, then store at 4°C until needed.
5) Add the Regular Neurobasal Media to glial cultures to condition it for next day use.
1.7 Prepare 0.5M Kynurenic acid
Dissolve 1mg kynuerenic acid in 10.57ul 1N NaOH
1.8 Prepare Cysteine water
30 ml 0.5M CaCl2
500 ml 0.02M Cysteine
7.35 ml H2O
Filter
1.9 Prepare equipment and supplies
1) 2 ice buckets, 2 empty sterile beakers, 2 absorbent bench underpads, blade, wax plate, syringe, transfer pipette,2 beakers of PBS, paper towels
2) Put PBS at -80°C until ice crystals just start forming.
3) Get ice, set up ~15 ml CMF Buffer on ice with O2 bubbling through a 22G needle
4) Thaw 5 ml FBS and 0.5 ml DNAse
5) Set up microscope, turn on Hood, Centrifuge, water bath

2. DISSECTION, DIGESTION, TRITURATION AND PLATING
(7-8 rat pups for 2 plates)
1) Clean the wax plate with CMF and PBS 1x, put PBS with ice forming on the wax plate.
2) Put the pups in the ice bucket to anesthetize them (2-3min).
3) Take the pups out, disinfect with alcohol, cut the head off and gently remove the brain.
a) Sagittal dissection method
Put the brain in the wax plate on ice, cut it in two hemispheres along the midline. Using anterior commissure as marker to localize the peripheral border of the NAc, punch out the tissue containing NAc out with 16G cannula, remove lateral 1/3 and medial ¼ of the cylinder, then remove remaining tissue (NAc) to the beaker containing CMF and O2.
b) Coronal dissection method
Place the whole brain, ventral surface facing up, on a wax plate (petrie dish filled with wax) on ice. It may be necessary to fix the brain in place with a needle. Make a coronal cut just rostral to where the rostral poles of the two hemispheres come together - at the level shown in Coronal Plate 5 (Atlas of Prenatal Rat Brain Development, J Altman & SA Bayer, CRC Press, 1995). Make a second coronal cut just rostral to the circle of willis - at a level that is approximately in between those shown in Coronal Plates 7 and 8. Place the resulting coronal section, caudal surface facing down, in a glass petrie dish that has been pre-chilled. Punch out the nucleus accumbens with a sharpened 16 G cannula, then transfer it to the beaker containing CMF and O2.
4) While there are 4 rats left, make up the papain enzyme solution
Papain Enzyme Solution (Total volume 2.5 ml)
1.87 ml Cysteine Water
52 ml Papain Suspension - before adding, sonicate for 15" and vortex for 5 min)
500 ml Hanks' Balanced Salt Soultion (5X)
2.5 ml 0.5M Kynurenate
5 ml 3M HCl
Filter with 0.2 mM filter. The pH should be ~6.8. Keep at RT for 30'- 60'.
5) After dissection take tissue under the hood and transfer tissue (with long glass pipette, sterile) into 15 ml sterile tube.
6) Rinse tissue 4X with cold CMF, swirl gently to rinse. (Change pipette for each rinse). Take off CMF slowly.
7) Remove as much CMF as possible. Add 1 ml -1.5ml of the papain enzyme solution.
8) Incubate at 36-37°C for 30 min. Mix gently by tapping with hand intermittently.
9) prepare Inactivation Solution and filter 5 ml of FBS
3.5 ml Hanks BSS
1.0 ml FBS
500 ml DNase
Filter
10) Remove papain enzyme solution using glass pipette.
11) Rinse 2x with CMF, and remove CMF.
12) Add 1.5 ml of inactivation solution
13) Triturate tissue
15x with the sterile long glass pipette
6-8x with 3cc syringe with 22G syringe needle
14) Using the syringe, gently layer cell suspension on top of the sterilized 4ml FBS in the 15 ml tube).
15) Spin at 1,000 RPM for 10 min at 4°C.
16) Get the Initial Neurobasal Media which has been stored at 4 °C and warm up under the hood.
17) After cells have spun, discard as much supernatant as possible
18) Suspend cells in 1.0 ml of Initial Neurobasal Media, using pipetman with 1000ml blue tip 4-5X gently.
19) Check and count cells under microscope, do viability test
20) Dilute to the correct concentration (4x104 cells/ml) and plate on 24 well plates (1 ml/well)
3. MEDIUM CHANGE

3.1 FIRST CHANGE
(The media is changed for the first time after 24 hr in culture.)
As described above, prepare 50 ml Regular Neurobasal Media on the day of the dissection, condition with glia cultures for 24h. After conditioning, add 500ml B27, and filter. Remove all of the old media from the wells and add 1 ml of the Regular Neurobasal Media to each well.
3.2 SUBSEQUENT CHANGES
(Following the first change, half of the media is replaced every 4-5 days.)
Prepare 25 ml Regular Neurobasal Media (for 2 flasks), condition for 24h. After conditioning, add 500 ml B27, filter. Remove 500ml of old media from each well and add 500ml fresh media to each well.
4. NOTES
1) Each medium must be conditioned for 8h-24h in Glial cultures
2) Regular NB Med must be changed every 4-5 days.
3) For cortical cell cultures, dissect cortex, cut it into 2X2X2mm cubes and follow the above protocol.

Solutions for Nucleus Accumbens Cell Culture
Regular NeuroBasal Medium

Ingredient Stock Conc. Amount Final Conc.
NeuroBasal Media 10ml
Gentamicin 10mg/ml 50ul 0.05mg/ml
Glutamine 200mM 100ul 2mM
B27 200ul
  1. Condition the medium without B27 in glial cultures (80% confluent) for 8-24 hrs.
  2. Add B27, filter, store at 4 degrees C till needed.

Initial NeuroBasel Medium

Ingredient Stock Conc. Amount Final Conc.
NeuroBasal Media 10ml
Gentamicin 10mg/ml 50ul 0.05mg/ml
Glutamine 200mM 100ul 2mM
B27 100ul
Glutamic Acid 2.5mM 100ul 25uM
  1. Condition the medium without B27 in glial cultures (80% confluent) for 24 hrs.
  2. Add B27, filter, store at 4 degrees C till needed.

CMF Buffer

Ingredient Stock Conc. Amount Final Conc.
H2O (Sterile) 45ml
PBS 10x 5ml 1X
Glucose (sterile) 20% 0.25ml 1%
Fungizone 250ug/ml 0.5ml 2.50ug/ml
Gentamicin 10mg/ml 0.25ml 0.05mg/ml

Filter and store at 4 degrees C.

Cysteine Water

Ingredient Stock Conc. Amount Final Conc.
H2O (Sterile) &nbsp 7.35ml
Cysteine 0.02M(15.7mg/5ml) 0.5ml 1.25mM
CaCl2 0.5M(736mg/10ml) 30ul 1.90mM

Glia Media (10% FBS)

Ingredient Stock Conc. Amount Final Conc.
MEM 45ml
FBS 5ml 10%
Glutamine 20mM 500ul 2mM
Glucose 20% 845ul 0.34%
Gentamicin 10mg/ml 250ul 0.5mg/ml
Insulin 25mg/ml 20ul 0.01mg/ml

Papain Enzyme Solution

Ingredient Stock Conc. Amount Final Conc.
Cysteine Water 1.25mM 2ml 1mM
Papain suspension 1151unit/ml (Sonicate for 15', vortex for 5') 43ul 20unit/ml
Hank's Balanced Salt Solution 5X 0.5ml 1X
Kynurenic Acid 0.5M 2.5ul 0.5mM
HCl 3N Varies (around 5ul) varies

The pH should be adjusted with HCl to 7.3. Then filter.
It takes about 30-60 min at room temperature to activate the papain.

Hank's Balanced Salt Solution 5X
(Modified) 50ml

Ingredient Stock Conc. Amount Final Conc.
NaCl 5M 5.8ml 580mM
KCl 1M 1.35ml 27mM
NaHCO3 0.5M 13ml 130mM
NaH2PO4 1M 0.5ml 10mM
MgSO4 0.5M 0.5ml 50mM
Glucose 20% 5.625ml 2.25%
H2O 23.225ml

Filter and store at 4 degrees C.

Kynurenic acid (0.5M)
1mg KA
10.57ul 1N NaOH
Poly-d-Lysine solution (0.1 mg/ml)
5 mg Poly-d-Lysine
50ml ddH2O
store at -20°C in dark.

Stock Solution Preparation

L-Glutamic acid (2.5mM)
20mg L-Glutamic acid
54.4ml ddH2O
DNAse (stock conc. 2.0mg/ml)
10ml 1X PBS
20mg DNAse.

Make 1ml aliquots of the above stock solutions.
Make 1ml aliquots of Glutamine, B27, fungizone, and 200ul aliquots of insulin
Store at -20°C

SIGMA CATALOGUE NUMBERS:
L-Glutamic Acid G1251 MW 147.1
Cysteine C-1276 MW 157.6
Polysine-D-lysine P-7280 MW 30,000-70,000
Insulin I-5500 MW 5733
Kynurenic acid K-3375 MW 189.2
Hank's Balanced salt solution H-8389

GIBCO CATALOGUE NUMBERS:
MEM 11090-081
NeuroBasal Media 21103-049
B27 17504-044
Gentamicin 15710-064 (10mg/ml)
L-Glutamine 25030-081 (200mM)
Fungizone 15290-018 (250ug/ml)

WORTHINGTON BIOCHEMICAL CORP:
PAP Papain suspension LS003126 (52.1mg/ml, 22.1u/mg)
DNAse I LS002006 (3.017u/mg)
Ted Pella, Inc. :
wax sheets #109 (1lb. box)

Coronal Plate 5

Coronal Plate 5

Coronal Plate 7

Coronal Plate 7

Coronal Plate 8

Coronal Plate 8

Glia Cell Culture Protocol

PREPARATION

A. Prepare solutions

1) 100 ml PBS 1X
90 ml H2O sterile
10 ml PBS 10X
Store at 4°C

2) 50 ml CMF Buffer
45 ml Sterile H2O
5 ml PBS 10X
250 ml 20% Glucose
500 ml Fungizone
250 ml Gentamicin
Filter and store at 4°C

3) 100 ml 10 % FBS Glia Media
45ml MEM (Sigma)
5ml FBS (Gibco)
845ul 20% Glucose (sterile)
500ul Glutamine (200mM stock, Gibco)
250ul Gentamicin (Gibco)
20ul Insulin (sigma) final conc 0.01 mg/ml (stock concentration 25mg/ml of sterile water)
Filter

4) Papain Enzyme Solution (Total volume 2.5 ml)
1.87 ml Cysteine Water
52 ml Papain Suspension - before adding, sonicate for 15" and vortex for 5 min)
500 ml H&B (5X)
2.5 ml 0.5M Kynurenate
5 ml 3M HCl
Filter with 0.2 mM filter. The pH should be ~6.8. Keep at RT for 30'- 60'.

5) H&B Solution (5X) 50ml

Ingredient Stock Conc. Amount Final Conc.
NaCl 5M 5.8 ml 580mM
KCl 1M 1.35 ml 27mM
NaHCO3 0.5M 13 ml 130mM
NaH2PO4 1M 0.5 ml 10mM
MgSO4 0.5M 0.5 ml 50mM
Glucose 20% 5.625 ml 2.25%
H2O 23.225 ml

Filter and store at 4°C

6) Cysteine Water

Ingredient Stock Conc. Amount Final Conc.
Sterile H2O 7.35ml
Cysteine 0.02M(15.7mg/5ml) 0.5ml 1.25mM
CaCl2 0.5M(736mg/10ml) 30ul 1.90mM

B. Prepare Other Supplies

1) Coat flasks with poly-d-lysine
Coat each flask with poly-d-lysine (0.1 mg/ml; solution shown below)
Incubate at 37°C overnight
Remove Poly-d-Lysine
Rinse 2x with sterile H2O and air-dry the flasks for 2hrs, store at 4°C until use.

Poly-d-Lysine solution (0.1 mg/ml)

5 mg Poly-d-Lysine
50ml ddH2O
store at -20°C

2) Prepare Wax-plate
Re-melt and sterilize the wax-plate (Petri dish with wax) under UV light (2hrs or overnight)

DISSECTION, DIGESTION, TRITURATION AND PLATING

1) Postnatal (P2-3) rats are anesthetized by hypothermia on ice and brains are removed into ice-cold phosphate-buffered saline (PBS) and bubbled with O2 continuously. The forebrain is split sagitally at the midline. The meninges are removed and the cortex is isolated. The cortex is cut into 1mm3 cubes with a scalpel blade, transferred to ice-cold CMF solution.

2) After dissection take tissue under the hood and transfer tissue (with long glass pipette, sterile) into 15 ml sterile tube.

3) Rinse tissue 4X with cold CMF, swirl gently to rinse. (Change pipette for each rinse). Take off CMF slowly.



4) Remove as much CMF as possible. Add 1 ml -1.5ml of the papain enzyme solution.

5) Incubate at 37°C for 45 min. Shake gently by hand intermittently.

6) Filter 5 ml of FBS

7) Prepare Inactivation Solution

3.5 ml Hanks BSS

1.0 ml Filtered FBS

500 ml DNase

Filter

8) Add approximately 2 ml CMF to tube with tissue and remove

9) Rinse 2 more times with CMF, and remove CMF

10)Add 1.5 ml of Inactivation Solution

11)Triturate tissue with a 9 inch Pasteur pipette followed by a 22G needle attached to a 3cc syringe

12)Using the syringe, gently layer cell suspension on top of the filtered FBS (~4 ml of FBS in the 15 ml tube).

13)Spin at 1,000 RPM for 10 min at 4°C

14)Discard as much supernatant as possible and suspend the pellet in 2ml of 10% FBS Glia Media

15)Check and count cells under microscope, do viability test

16)Cells are plated in poly-d-lysine coated flasks at 400,000 cells/cm2 final concentration in 10% FBS Glia Media (2 pups produce about 5 flasks).

17)Incubate at 37°C for 1.5h

18)Rinse the flask with ice cold 10% FBS Glia Media (about 5ml/flask)

19)Coat the flask with fresh 10% FBS Glia Media (about 7ml/flask)

Media is replaced every 5-7 days, when the confluence of the cells is about 50% of the flask surface. After the first feeding in 10% FBS Glia Media (step 17), feed the glia culture with 4% FBS Glia Media

SIGMA CATALOGUE NUMBERS

Cysteine C-1276 MW 157.6
Polysine-D-lysine P-7280 MW 30,000-70,000
Insulin I-5500 MW 5733
Hank's Balanced salt solution H-8389
Fetal Bovin Serum (FBS) F4135

GIBCO CATALOGUE NUMBERS
MEM 11090-081
Gentamicin 15710-064 (10mg/ml; stock as purchased)
L-Glutamine 25030-081 (200mM; stock as purchased)
Fungizone 15290-018 (250ug/ml; stock as purchased)

WORTHINGTON BIOCHEMICAL CORP
PAP Papain suspension LS003126 (52.1mg/ml, 22.1u/mg)
DNAse I LS002006 (3.017u/mg)

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