Neurons Derived from PC12 Cells Have the Potential to Develop Synapses with Primary Neurons from Rat Cortex

Acta Neurobiol Exp 2006, 66: 105-112

Neurons derived from PC12 cells have the potential to develop synapses with primary neurons from rat cortex

Tao Zhou1,2, Bainan Xu2, Haiping Que1, Qiuxia Lin1, Shuanghong Lv1, and Shaojun Liu1

1Department of Neurobiology, Institute of Basic Medical Sciences, The Academy of Military Medical Sciences, Beijing, 100850, P. R. China; 2Department of Neurosurgery, General Hospital of PLA, Beijing, 100853, P. R. China

Abstract. Neuron transplantation is considered to be a promising therapeutic method to replace functions lost due to central nervous system (CNS) damage. However, little is known about the extent to which implanted neuron-like cells can develop into mature neurons and acquire essential properties, and especially formation of synapses with host neurons. In this investigation we seeded PC12 cells labeled with GFP into primary cultured neurons isolated from rat cerebral cortex to build a co-culture system, and then induced the PC12 cells to differentiate into neuron-like cells with NGF. Seven days later, we observed the relationship between the PC12-derived neurons and primary neurons using FM1-43 imaging and immunoelectron microscopy, and found that GFP-labeled neurons could form typical synapses with host primary neurons. These observations showed that immigrant neurons differentiated from PC12 cells could develop into mature neurons and could form intercellular contacts with host neurons. Both the immigrant and host neurons could construct neuronal networks in vitro.

The correspondence should be addressed to S. Liu, Key words: neural transplantation, co-culture, PC12 cells, synapse, laser Email: [email protected] scanning confocal microscopy, electron microscopy 106 T. Zhou et al.

INTRODUCTION METHODS

Cell transplantation is an experimental approach PC12 cell culture and transfection to repairing brain injury after a stroke and also represents a possible strategy to restore brain functions Rat pheochromocytoma PC12 cells were maintained in neurodegenerative disorders such as Parkinson's in plastic flasks in a DMEM (Dulbeccos Modified and Huntington's diseases (Gross 2000, Sanberg Eagle Medium) supplemented with 10% horse serum and Brundin 1999, Saporta et al. 1999). Various cell and 5% fetal bovine serum (Gibco, NY) in a humidified types are under investigation in experimental stud- atmosphere with 5% CO2 at 37°C. The pEGFP-N1 vec- ies, of which the most promising cell sources for tor (Clontech) was transfected into the PC12 cells using neuronal transplantation are immature neurons or lipofectAMINE 2000 reagent (Invitrogen), and stable their progenitors, including fetal tissue, cell lines transfectants bearing the EGFP expression unit were and stem cells (Date et al. 2000, Flax et al. 1998; isolated under the pressure of G418 (Sigma, 500 Lindvall et al. 1990, Lundberg et al. 1997, Ono et mg/ml). EGFP positive cells were visually confirmed al. 1997, Park et al. 1999). Neuronal plasticity is under a confocal microscope in separate cultures (Yoon now widely accepted as a fundamental mechanism, et al. 2002). involving not only the rearrangement of neurons and their interconnections, but also the formation of Isolation of neurons from the cortices of newborn rats new neural cells in animals and humans during their entire lifespan (Finley et al. 1996, Gross 2000). It is Brains were taken from newborn Wistar rats. Under possible that the transplanted neurons integrate into an anatomical lens, the cerebral cortex was separated neuronal networks composed of host neurons and and sheared into scraps after removal of membranes. repair the lost functions caused by neurodegenera- The scraps were digested with 0.25% trypase at 37°C tion or CNS injury. While much interest has been for 60 min, then washed twice with culturing medium. focused on the long-term survival and differentia- After agitating the tissue was dispersed into single tion of transplanted neurons, less attention has been cells and subsequently cultured in DMEM supple- paid to the extent to which implanted neurons can mented with 10% horse serum and N3 in a 35 mm dish give rise to mature neurons and develop essential in an incubator containing 7.5% CO2 and 92.5% air at properties, especially an ability to form synapses 37°C. Four days after the initial seeding, 0.1 mM Ara- connected with host neurons. c was added to the medium and the cells were cultured The PC12 cell line, a rat pheochromocytoma cell for another day to kill the dividing cells. Then Ara-c line, has been extensively used as a tool for study- was removed by changing the medium completely. ing the function of neurotrophic factors and neu- Verification of the culture composition under a phase ronal differentiation because these cells can be contrast microscope revealed predominant presence of induced to differentiate into neuron-like cells by neurons, each with a clear nucleus and nucleolus, and NGF (Greene and Tischler 1976) or other factors long processes, forming a dense network. (Arsenijevic and Weiss 1998, Sucher et al. 1993, Sugita et al. 1999, Tyson et al. 2003). The trans- Co-culture and differentiation of PC12 cells plantation of PC12 cells into the striatum represents one potential means for the treatment of Parkinson's Prepared PC12 cells were rinsed twice and harvested. disease (Hefti et al. 1985). Studies have revealed Then the PC12 cells transfected with the pEGFP-N1 that PC12 cells survive and continue to release vector were seeded into primary neurons derived from dopamine up to 12 months after transplantation the rat cortices. After seeding, 25 ng/ml NGF was added (Ono et al. 1997), but it is not clear whether the into a medium containing DMEM supplemented with implanted PC12 cells can develop contacts with 1% horse serum. To be induced to differentiate into neu- host neurons. In this study, we established an in rons, the cells were incubated at 37°C in an atmosphere vitro co-culture system to investigate if the neurons of 7.5% CO2 and 92.5% air. Four days later 0.2 mM Ara- derived from PC12 cells could develop synaptic c was added to the medium to kill mitotic neurons. The connections with host primary cultures. co-culture was maintained for at least another 7 days. PC12 derived primary neuron synapses 107

FM1-43 fluorescent imaging 20 min. They were infiltrated in Epon 812 (containing DMP) for 12 hours at room temperature and then FM1-43(N-(3-triethylammoniumpropyl)-4-(4- embedded in resin. After the embedding medium was (dibutylamino)styryl) pyridinium dibromide, Molecular changed, the embedded cells were put in a thermostat to Probes) is a dye used to study cell membrane traffick- accelerate solidification. This process took 12 hours at ing. Three properties make FM dyes useful for studying 35°C, 12 hours at 45°C, and 24 hours at 60°C succes- exocytosis, endocytosis, and endosomal trafficking: (i) sively. Immunogold labeling was performed using 10 the dyes partition reversibly in membranes; (ii) the dyes nm diameter colloidal gold particles, which were cou- do not 'flip flop' and so are never free in the cytoplasm; pled to anti-rat IgG. The nickel grids were first treated (iii) the dyes are far more fluorescent in membranes than with 8% sodium hyperiodate for 20 min at room tem- in water. The excitatory wavelength of FM1-43 is 510 perature, followed by three washes in 0.05 M TBS, pH nm, and that of emitted light is 455 nm. Measurements 7.2, each for 5 min. The sections were then immersed in were performed at 37°C in modified Krebs-Ringer antibody solution which contained 5% bovine serum bicarbonate solution comprising 130 mM NaCl, 3.6 mM albumin and GFP antiserum (Clontech, dilution:

KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 10 mM Hepes, 1:200). The nickel grids were washed three times with

2.0 mM NaHCO3, 1.5 mM CaCl2, and 5.5 mM glucose, TBS, followed by incubation with colloidal gold-cou- equilibrated with O2/CO2 (95:5, v/v)(KRB) (Pouli et al. pled IgG (1:10) for 50 minutes at room temperature. 1998). Synaptic terminals were loaded with FM1-43 (10 They were then washed again another three times in mM) using a depolarizing stimulus (50 mM external K+, distilled water. Finally, they were counterstained with 60 s) to induce exocytosis. All the cells were fixed by saturated uranyl acetate and lead citrate, and then 4% paraformaldehyde and 0.05% glutaraldehyde in 0.1 observed with an electron microscope (Philips 120). M phosphate buffer for 30 minutes. Confocal imaging was performed on a confocal laser RESULTS scanning system (Radiance 2100TM, Bio-Rad, USA) attached to a TE300 microscope (Nikon, Japan) using a Choice of PC12 cells labeled with GFP 100× Nikon Plan Fluro oil immersion objective. The GFP was excited using a 488-nm argon/krypton laser To label the PC12 neuronal cells, undifferentiated and detected with a 515530-nm band pass filter. The PC12 cells were transfected with pEGFP-N1 vector so FM-143 was excited using a 543-nm argon/krypton that the cells could stably express EGFP. Forty-eight laser and detected with a 590570 nm band pass filter. hours after transfection, about 5% PC12 cells could be Images were acquired using a single scan and analyzed visually confirmed as EGFP positive under a fluores- by LaserSharp2000 software (Bio-Rad, USA).The cence microscope. About 30% of the labeled cells aperture size was optimized to increase signal-to-noise expressed EGFP strongly, while the remaining cells ratio. Images were digitally enhanced to identify active expressed EGFP weakly. After two weeks culture synaptic terminals, which appeared as fluorescent under the pressure of G418, EGFP positive cells puncta (Trudeau et al. 1996). became dominant and grew into clones, remained polygonal with an undifferentiated morphology and Immuno-electron microscopy without any neural processes.

Under a phase-contrast microscope the co-cultured Isolation of primary cells neurons were marked and fixed for electron microscop- ic study. The culture cells were fixed with 4% The primary cells mainly comprised neurons and paraformaldehyde and 0.05% glutaraldehyde in a 0.1 M astrocytes. Most of the neurons had a clear nucleus and phosphate buffer for 30 min, followed by post-fixation a nucleolus. They also had a single axon and one or with 0.5% osmium tetroxide in a 0.24 M phosphate more dendritic branches emerging from various loca- buffer for 60 min. The fixed cells were dehydrated tions of the cell body, and their processes became very through a series of graded ethanol (50%, 70%, 90%, long, forming a dense network. The other cells were 100%), each for 5 min. Finally, the cells were infiltrat- flat astrocytes, which lay under the neurons and had ed in 100% ethanol with Epon 812 (without DMP) for short, thick processes. 108 T. Zhou et al.

Co-culture of primary neurons and PC12-derived FM1-43 imaging neurons Synaptic terminals were loaded with FM1-43 (10 EGFP positive PC12 cell clones were picked out mM) using a depolarizing stimulus (50 mM external before co-culture to ensure that each implanted PC12 K+, 60 s) to induce exocytosis. In this investigation, the cell could express EGFP (Fig. 1b, h, k). One day after labeled synaptic terminals could be seen on the surface 25 ng/ml NGF was added to the co-culture system, of primary perikarya as well as on the surface of neu- neurites began to grow out from the PC12 cell bodies. rons differentiated from PC12 cells. The neurites became longer and the number of neurites In the co-culture system intercellular contact forma- increased, suggesting that the PC12 cells had differen- tion between neurons derived from PC12 cells (EGFP tiated to neuron-like cells. In the differentiated neuron- labeled) and primary cortex neurons occurred within like PC12 cells, cell bodies and main processes were one week, as indicated by staining with FM1-43. A raw EGFP positive, viewed under a fluorescence micro- FM1-43 image (Fig. 1g) shows vesicles of a synapse scope. Observed with laser confocal scanning stained by FM1-43 in a cell body and its neurites, and microscopy, EGFP labeled PC12 neuronal cells in the a raw GFP image (Fig. 1h) shows that the cell was co-culture system dispersed in the network were com- EGFP-. A merged image (Fig. 1i) suggested that the posed of neurons and their processes. After 7 days of cell was a neuronal PC12 cell. The other three images co-culture, the network was stained with DiI (Fig. 1a). (Fig. 1j,k,l) show vesicles of synapses stained by FM1- Figure 1b shows an EGFP positive PC12 cell in the co- 43 in another neuronal PC12 cell body and its neurites. culture system. Fig 1c is the merged image of Fig. 1a As a comparison, Fig. 1d shows vesicles stained by and Fig. 1b, showing a PC12 cell in the network of FM1-43 in a cell body and its neurites, and Fig.1e primary neurons. shows that the cell was EGFP negative. The merged image (Fig. 1f) shows that the cell was a primary neuron.

Fig 1. Fluorescence images showing the relationship between primary neurons and migrated neurons derived from PC12 cells. Image (a) shows host primary neurons labeled with DiI as red. A substantial number of perikarya and their processes were noted. A green fluorescence labeled neuron with elaborated processes is the neuron differentiated from PC12. A substantial number of varicosities were noted on the processes. Image (c) presents a merged image showing the relationship between the primary neurons and the migrated neurons derived from a PC12 cell. Image (d) shows a primary neuron and its processes labeled by FM1- 43. Many red fluorescence spots form the outline of the neuron and the processes. Image (e) shows the same neuron showing no labeling for GFP. Image f shows the merged image. Another FM1-43 positive neuron is shown in image (g). The same neuron derived from a PC12 cell was labeled by stronger green fluorescence (h). On the merged image (i), two fluorescence labels can be seen clearly. Images (j)(l) show a double process labeled by two kinds of fluorescence. Many red fluorescence spots [arrow in (j)] and green labeling processes (k) were noted to be superimposed (l). Scale bars are 50 µm. PC12 derived primary neuron synapses 109

Synapse structures under electron microscopy one PC12-derived neural cell. We paid special attention to the last type of synapse since it suggested the Under an electron microscope, neurons differentiat- existence of a new, potentially functional relationship ed from PC12 cell could be distinguished from pri- between these two neurons. Since PC12 derived neu- mary ones due to the chromaffin granules in their ron-like cells were sprinkled into the network com- perikarya and processes (Fig. 2ab). Comparing the posed by primary neurons and their processes (Fig. terminal swelling of the two kinds of neurons, there 1c), connections were formed predominantly with pri- were obvious differences (Fig. 2cf): (i) microtubules mary neuronal processes and also with the synapses in axons of PC12 neuronal cells were thicker and themselves. We oriented primary neurons alone in the straighter than those in axons of primary neurons; (ii) cluster of EGFP positive PC12 neuronal cells to search axons and terminal varicosity swellings of the PC12 for synapses between primary neuronal soma and neuronal cells were darker than those of the primary processes of PC12 neuronal cells. These synaptic neurons; (iii) the terminal swellings of PC12 neuronal structures were indistinguishable from those between cells were much more regular and bigger than those of primary neurons in terms of size of active zones and primary neurons. number of vesicles. In these synapse structures, presy- In this study we observed three types of typical naptic and postsynaptic membranes, synaptic clefts synapses and synapse-like structures. They were and vesicles could be found and all the synaptic vesi- between either two primary neurons, two neurons dif- cles were round and clear. Both asymmetric and sym- ferentiated from PC12 cells, or one primary neuron and metric synapses were observed.

Fig 2. The fine structures of host primary and immigrant neurons and their relationship. Image (a) shows the close relationship between a primary perikaryon and a processes derived from a PC12 cell, characterized by its high electron density structure. A synapse was established between these cells [see inset, shown in boxed section of image (a)]. Image (b) shows that a perikaryon of a PC12 cell, characterized by its chromaffin granules, was in close contact with a varicosity (asterisks) from a primary neuron. A synapse-like structure (arrow) was noted. Many dense chromic grains were seen in the perikaryon. A varicosity was observed in close contact with this soma with chromic grain (c). Image (d) shows high magnification of the box appearing in image (c), to illustrate this close relationship. Images (e) and (f) show the fine structure and synapse between a varicose-derived PC12 cell and primary perikaryon. Many rough endoplasmic reticula, free ribo- somes, and mitochondria were noted in the cytoplasm of the perikaryon. A varicosity was observed in close contact with this perikaryon [see box in image (e)]. Image (g) shows a complex synaptic structure among the primary neuronal processes and a varicosity differentiated from PC12 cells. At least three synapses (arrows and a box) were seen to form. Typical parallel arrangement of neurotubules is seen. At high magnification [in the box in image (g)], a typical Gray I synapse (including presynaptic and postsynaptic membrane, synaptic cleft and vesicles) was exhibit- ed. The presynapse varicosity was also labeled with colloidal gold particles [(see box and inset in image (h)]. However, we did not see any colloidal gold particles in postsynaptic structures. Scale bars are 500 nm. 110 T. Zhou et al.

Immunoreactive (IR) EGFP-IR with a permissive transplantation environment. In the co-culture system composed by PC12 cells and pri- Because chromic grains were located mainly in cell mary neurons, the PC12 cells induced by NGF out- bodies and near processes of neurons differentiated grew neurites and formed synapses with primary neu- from PC12 cells, it was difficult to identify finer rons. These data suggest that differentiated neurons processes. Here, we employed immunogold-labeled derived from undifferentiated precursors could be antibodies against GFP to display these fine processes used to generate high-throughput functional neurons, and their relationship with primary neurons. We detect- which can serve as a reproducible source of cells for ed that some typical synapses or synaptic structures reconstructive therapies where synapse function is occurred between PC12-derived neuronal cell process- necessary. es (labeled with colloidal gold particles) and primary neuron terminal swellings (Fig. 2gh). In these synap- Comparison of synaptic structures tic structures, presynaptic and postsynaptic membranes, synaptic clefts and vesicles could be found and Synapses formed between PC12-derived neuronal all the synaptic vesicles were round and clear. cell bodies and processes of primary neurons, and those between PC12-derived neuronal cell processes DISCUSSION and cell bodies of primary neurons, were indistinguishable from those between primary neurons in In this study, we have demonstrated by FM1-43 terms of size of active zones and number of vesicles. imaging and electron microscopy, that neuron-like However, there were some ultrastructural differences cells derived from PC12 cells have the potential to between terminal swellings. The PC12 cell line is able develop synapses by connecting with primary neurons to form cholinergic synapses with a clonal cell line of when provided with a permissive transplantation envi- skeletal muscle origin (Schubert et al. 1977). Our study ronment. The manipulation of the co-culture system showed that the vesicles of synapses were round clear allowed a precise analysis of neuronal maturation and vesicles but not large dense-core vesicles. connection formation of transplanted neurons, and their relationships with host neural cells. Environment in co-culture system

Differentiating and constructing synapses In the co-culture system, there should be many kinds of factors that affect differentiation, maturation and/or For a long time many research groups have been synapse formation of implanted neurons, many of searching for neural cells for transplantation to restore which are produced by host cells. It will be helpful to brain functions lost due to injuries or neurodegenera- manipulate the implanted neurons function if we make tive disorders (Date et al. 2000, Flax et al. 1998, the role of these factors clear (Kirschenbaum and Lindvall et al. 1990, Lundberg et al. 1997, Ono et al. Goldman 1995, Mehler et al. 1993, Noma et al. 1999, 1997, Park et al. 1999, Ryder et al. 1990). The ideal Shetty and Turner 1998, Vicario-Abejon et al. 1995). neural cells for transplantation should have these fea- Interestingly, we noticed that when the PC12 cells tures: (i) the ability to remain undifferentiated and to alone were induced by NGF, they would differentiate self-renew; (ii) the ability to change phenotype to neu- into neurons with simple and immature morphology. ral cells; (iii) be genetically manipulatable for trans- When the co-culture system had a certain number of plantation of modified cells into the brain. The last glial cells in it, the PC12 cell-derived neurons showed feature represents a promising strategy for the expres- complex and mature appearance with many more and sion of specific neurotrophic factors and neurotrans- longer processes. This might contribute to the environ- mitter-synthesizing enzyme (Henningson Jr et al. ment supplied by primary neural cells, especially by 2003, Singh 2001, Wyman et al. 1999, Zhang et al. glia. Data have demonstrated that astrocytes increase 2003). Our investigation demonstrated that neuron- the number of functional synapses of neurons by sev- like cells derived from cell lines, such as the PC12 enfold and they are required for synaptic maintenance cell, have the potential to develop functional synapses in vitro (Ullian et al. 2001). Glial cells are important to by connecting with primary neurons when provided synaptic strength at excitatory synapses and may play PC12 derived primary neuron synapses 111 a role in synaptic plasticity and modulating responses the dopaminergic nigrostriatal neurons. Brain Res 348: to neural injury (Beattie et al. 2002, Fields and 283288. Stevens-Graham 2002). Henningson CT Jr, Stanislaus MA, Gewirtz AM (2003) Embryonic and adult stem cell therapy. J Allergy Clin CONCLUSION Immunol 111: S745753. Kirschenbaum B, Goldman SA (1995) Brain-derived neu- In the present study, the co-culture system that rotrophic factor promotes the survival of neurons arising retained glial cells reconstructed the environment of the from the adult rat forebrain subependymal zone. Proc brain, which suggests that when PC12 cells would be Natl Acad Sci U S A 92: 210214. transmitte into the brain, they could successfully differ- Lindvall O, Brundin P, Widner H, Rehncrona S, Gustavii B, entiate into neuron-like cells and have the potential to Frackowiak R, Leenders KL, Sawle G, Rothwell JC, develop functional synapses with host neurons. These Marsden CD, et al. (1990) Grafts of fetal dopamine neu- facts indicate the possible significance of PC12 cells in rons survive and improve motor function in Parkinson's clinical application to treat neural diseases. disease. Science 247: 574577. Lundberg C, Martinez-Serrano A, Cattaneo E, McKay RD, REFERENCES Bjorklund A (1997) Survival, integration, and differentiation of neural stem cell lines after transplantation to the Arsenijevic Y, Weiss S (1998) Insulin-like growth factor-I is adult rat striatum. Exp Neurol 145: 342360. a differentiation factor for postmitotic CNS stem cell- Mehler MF, Rozental R, Dougherty M, Spray DC, Kessler derived neuronal precursors: Distinct actions from those JA (1993) Cytokine regulation of neuronal differentiation of brain-derived neurotrophic factor. J Neurosci 18: of hippocampal progenitor cells. Nature 362: 6265. 21182128. Noma T, Yoon YS, Nakazawa A (1999) Overexpression of Beattie EC, Stellwagen D, Morishita W, Bresnahan JC, Ha NeuroD in PC12 cells alters morphology and enhances BK, Von Zastrow M, Beattie MS, Malenka RC (2002) expression of the adenylate kinase isozyme 1 gene. Brain Control of synaptic strength by glial TNFalpha. Science Res Mol Brain Res 67: 5363. 295: 22822285. Ono T, Date I, Imaoka T, Shingo T, Furuta T, Asari S, Date I, Shingo T, Yoshida H, Fujiwara K, Kobayashi K, Ohmoto T (1997) Evaluation of intracerebral grafting of Ohmoto T (2000) Grafting of encapsulated dopamine- dopamine-secreting PC12 cells into allogeneic and xeno- secreting cells in Parkinson's disease: Long-term primate geneic brain. Cell Transplant 6: 511513. study. Cell Transplant 9: 705709 Park KI, Liu S, Flax JD, Nissim S, Stieg PE, Snyder EY Fields RD, Stevens-Graham B (2002) New insights into (1999) Transplantation of neural progenitor and stem neuron-glia communication. Science 298: 556562. cells: Developmental insights may suggest new therapies Flax JD, Aurora S, Yang C, Simonin C, Wills AM, for spinal cord and other CNS dysfunction. J Billinghurst LL, Jendoubi M, Sidman RL, Wolfe JH, Kim Neurotrauma 16: 675687. SU, Snyder EY (1998) Engraftable human neural stem Pouli AE, Karagenc N, Wasmeier C, Hutton JC, Bright N, cells respond to developmental cues, replace neurons, and Arden S, Schofield JG, Rutter GA (1998) A phogrin- express foreign genes. Nat Biotechnol 16: 10331039. aequorin chimaera to image free Ca2+ in the vicinity of Finley MF, Kulkarni N, Huettner JE (1996) Synapse forma- secretory granules.Biochem J 330: 13991404. tion and establishment of neuronal polarity by P19 Ryder EF, Snyder EY, Cepko CL (1990) Establishment and embryonic carcinoma cells and embryonic stem cells. J characterization of multipotent neural cell lines using Neurosci 16: 10561065. retrovirus vector-mediated oncogene transfer. J Neurobiol Greene LA, Tischler AS (1976) Establishment of a nora- 21: 356375. drenergic clonal line of rat adrenal pheochromocytoma Sanberg PR, Brundin P (1999) Cell transplantation and neu- cells which respond to nerve growth factor. Proc Natl roscience. Cell Transplant 8: 36. Acad Sci U S A 73: 24242428. Saporta S, Borlongan CV, Sanberg PR (1999) Neural trans- Gross CG (2000) Neurogenesis in the adult brain: Death of plantation of human neuroteratocarcinoma (hNT) neu- a dogma. Nat Rev Neurosci 1: 6773. rons into ischemic rats. A quantitative dose-response Hefti F, Hartikka J, Schlumpf M (1985) Implantation of analysis of cell survival and behavioral recovery. PC12 cells into the corpus striatum of rats with lesions of Neuroscience 91: 519525. 112 T. Zhou et al.

Schubert D, Heinemann S, Kidokoro Y (1977) Cholinergic of autophosphorylation sites. Int J Dev Neurosci 21: metabolism and synapse formation by a rat nerve cell 6374. line. Proc Natl Acad Sci U S A 74: 25792583. Ullian EM, Sapperstein SK, Christopherson KS, Barres BA Shetty AK, Turner DA (1998) In vitro survival and differen- (2001) Control of synapse number by glia. Science 291: tiation of neurons derived from epidermal growth factor- 657661. responsive postnatal hippocampal stem cells: Inducing Vicario-Abejon C, Johe KK, Hazel TG, Collazo D, McKay effects of brain-derived neurotrophic factor. J Neurobiol RD (1995) Functions of basic fibroblast growth factor 35: 395425. and neurotrophins in the differentiation of hippocampal Singh G (2001) Sources of neuronal material for implanta- neurons. Neuron 15: 105114. tion. Neuropathology 21: 110114. Wyman T, Rohrer D, Kirigiti P, Nichols H, Pilcher K, Sucher NJ, Brose N, Deitcher DL, Awobuluyi M, Gasic GP, Nilaver G, Machida C (1999) Promoter-activated expres- Bading H, Cepko CL, Greenberg ME, Jahn R, sion of nerve growth factor for treatment of neurodegen- Heinemann SF, et al. (1993) Expression of endogenous erative diseases. Gene Ther 6: 16481660. NMDAR1 transcripts without receptor protein suggests Yoon S, Ban E, Yoo YS (2002) Direct monitoring of the post-transcriptional control in PC12 cells. J Biol Chem expression of the green fluorescent protein-extracellular 268: 2229922304. signal-regulated kinase 2 fusion protein in transfected Sugita S, Khvochtev M, Sudhof TC (1999) Neurexins are cells using capillary electrophoresis with laser-induced functional alpha-latrotoxin receptors. Neuron 22: fluorescence detection. J Chromatogr A 976: 8793. 489496. Zhang RL, Zhang L, Zhang ZG, Morris D, Jiang Q, Wang L, Trudeau LE, Emery DG, Haydon PG (1996) Direct modula- Zhang LJ, Chopp M (2003) Migration and differentiation tion of the secretory machinery underlies PKA-dependent of adult rat subventricular zone progenitor cells trans- synaptic facilitation in hippocampal neurons. Neuron 17: planted into the adult rat striatum. Neuroscience 116: 789797. 373382. Tyson DR, Larkin S, Hamai Y, Bradshaw RA (2003) PC12 cell activation by epidermal growth factor receptor: Role Received 26 August 2005, accepted 19 May 2006