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First published online July 23, 2007
doi: 10.1242/10.1242/jcs.003590
Research Article |
1 Institute of Biotechnology, University of Helsinki, Helsinki FIN-00014, Finland
2 Division of Signal Transduction, Harvard Medical School, Beth Israel Deaconess Medical Center, 77 Avenue Louis Pasteur, 10th Floor, Boston, MA 02115, USA
* Authors for correspondence (e-mail: Mikhail.Paveliev{at}helsinki.fi; Mart.Saarma{at}helsinki.fi)
Accepted 24 May 2007
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Key words: Boolean networks, Src family kinases, Axonal growth, Neurotrophic factors
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). A single cell has hundreds of different types of receptor proteins on its surface, making complex combinatorial relationships possible between signaling inputs and signaling network behavior. It remains largely unclear how different combinations of receptor signaling inputs are differentially recognized by the signaling network in such a way as to trigger execution of particular cellular events, such as gene transcription or cytoskeletal rearrangement. Signaling pathways downstream of different receptors are known to crosstalk at many levels and to converge at a limited number of signaling targets. A complex network of signaling pathways mediates the regulatory effects of neurotrophic factors (NTFs) and extracellular matrix (ECM) molecules on a wide spectrum of processes in neurons, including synaptic transmission, axonal growth and posttraumatic regeneration. Many signaling elements of those pathways have been characterized and activatory or inhibitory effects of particular elements on cell function have been described. At the same time, the integral functioning of the signaling network is just beginning to be addressed. It remains largely unclear how distinct pathways are orchestrated in the integrated network to produce highly specific cell responses to receptor stimulation.
A critical obstacle to progress in our understanding of cell signaling networks is the lack of a standard unambiguous language allowing quantitative description and analysis of the network behavior (Lazebnik, 2002). The formalism of Boolean algebra was previously used to analyze a genetic network in the specific case of the bacteriophage lambda lysis-lysogeny decision (McAdams and Shapiro, 1995) and it was theoretically predicted that protein kinase signaling networks can be described in Boolean terms (Bhalla, 2003; Bray, 1995; Huang and Ingber, 2000). Moreover, signaling molecules with particular gating behaviors have been generated by means of protein domain recombination (Dueber et al., 2003; Dueber et al., 2004). In the present work, we considered a Boolean network formalism as a framework for experimental design and the interpretation of experimental data on a signaling network behavior.
ECM molecules and NTFs are two classes of extracellular ligands that have crucial effects of axonal growth both in vivo and in vitro (Cai et al., 1999; Chen and Strickland, 2003; Ramer et al., 2000). Treatment with NTFs – especially nerve growth factor (NGF) and glial-cell-line-derived neurotrophic factor (GDNF) – is the only known way to induce axonal regeneration into the root entry zone of the spinal cord after dorsal root avulsion (Ramer et al., 2000; Schwab, 2000). GDNF also promotes axonal regeneration in propriospinal neurons after spinal cord injury (Iannotti et al., 2003). Neurturin (NRTN), which is a close homolog of GDNF, restores hindlimb cutaneous innervation in a mouse model of diabetes (Christianson et al., 2003) and activates axonal growth in dorsal root ganglion (DRG) neurons in vitro (Paveliev et al., 2004). NGF activates axonal growth by signaling through TrkA receptor tyrosine kinase (Kaplan and Miller, 2000). GDNF and NRTN bind specifically to GPI-linked receptors: GDNF Family Receptor 
1 (GFR1) and GFR2, respectively, and the ligand-receptor complex activates the transmembrane receptor tyrosine kinase Ret (Airaksinen and Saarma, 2002). Laminin is an ECM molecule that regulates axonal growth by activating integrins (Tomaselli et al., 1993) and has a crucial impact on axonal regeneration in sciatic nerve (Chen and Strickland, 2003). Peripheral axons were shown to regenerate on laminin matrix in acellular grafts of sciatic nerve (Krekoski et al., 2001). Several intracellular signaling pathways are known to mediate the positive effects of NTFs and laminin on axonal growth (Laforest et al., 2005; Liu et al., 2002; Liu and Snider, 2001; Markus et al., 2002; Paglini et al., 1998; Zhou and Snider, 2006), but the integral behavior of the signaling network converting receptor activation into axonal growth remains largely unknown (Tucker et al., 2005). Importantly, co-signaling by soluble NTFs and substrate-bound ECM molecules integrates two significantly different types of intercellular communication: long-range messages from remotely diffusing messengers and those that are highly localized to matrix.
Here we demonstrate that the ECM molecule laminin is differentially recognized by neurons in the presence versus the absence of NTFs. The essence of this difference is laminin-dependent activation of two different signaling pathways, each leading to axonal growth. NTFs activate the pathway involving Src-family kinases (SFKs), whereas in the absence of NTFs, axonal growth is independent of the SFK activity. This holds true for axonal initiation and elongation. By contrast, axonal branching is dependent on SFK activity both in the presence and in the absence of NGF. We then propose and experimentally verify a Boolean model describing differential contribution of the NTF- and laminin-triggered pathways to the converging signaling network.
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) (Fig. 2A,G,H). However, the observed outgrowth at 40 hours in the absence of NTFs was not affected by SU6656 (Fig. 2B,J,K). For the primary inhibitory analysis, the percentage of axon-bearing neurons was chosen as a reporter parameter because it allows fast and accurate quantification of multiple experimental conditions in parallel. The percentage of axon-bearing neurons reflects the axonal initiation. Axonal length and branching are analyzed in later sections of this work. To confirm the specificity of the SFK inhibition we took advantage of a dominant negative pp60src mutant (SrcDN) cloned into an adenoviral transducing construct. To allow SrcDN protein expression before the onset of axonal outgrowth, we used lower concentrations of laminin for precoating (20 ng/cm2 instead of 200 ng/cm2 in all other experiments) and quantified axonal outgrowth after longer times in culture. Again, the increase of axonal outgrowth caused by NTFs was blocked by SrcDN but not by a control GFP-expressing adenoviral vector (Fig. 2C). The outgrowth in the absence of NTFs was not affected by SrcDN (Fig. 2D). These results with SrcDN and SU6656 suggest that the NTF-dependent outgrowth is triggered by a signaling pathway that requires SFK activation whereas the NTF-independent outgrowth on laminin is triggered by a different SFK-independent signaling pathway. To verify that the two pathways are really different and independent of each other, we looked for a pharmacological tool that would inhibit the NTF-independent outgrowth without affecting the NTF-activated outgrowth. We found that roscovitine – an inhibitor of cyclin-dependent kinase 5 (Cdk5) – blocks the NTF-independent outgrowth at 40 hours in culture (Fig. 2F,L) but does not affect the outgrowth caused by NGF, GDNF or NRTN at 12 hours (Fig. 2E,I). In our experiments, the same concentration of roscovitine did not inhibit Erk1/2 activation as assessed by Thr202/Tyr204 phosphorylation (see supplementary material Fig. S2). Neither SU6656 nor roscovitine caused neuronal death under the conditions used for the axonal growth assay (supplementary material Fig. S3). Signaling mechanisms of axonal growth may undergo transcription-dependent changes when neurons are maintained for long periods in culture (Smith and Skene, 1997). As the SrcDN-transduced neurons were examined for axonal growth at 90 hours in culture, we also verified that the NTF-independent growth is blocked by roscovitine at 90 hours (supplementary material Fig. S4). Laminin induced the activation of Cdk5 in dissociated DRG cultures (supplementary material Fig. S5). Taken together these results demonstrate that there are two different and independent pathways that trigger axonal growth in the presence versus absence of NTFs (NGF, GDNF and NRTN): the NTF-dependent pathway involving SFKs activation and the NTF-independent pathway requiring Cdk5 activity. The results of inhibitory analysis (Fig. 2) allow us to describe these two pathways together as a simple Boolean network (Fig. 3A). The network includes two inputs (laminin and NTF), Boolean operations (AND, OR) and the output (axonal growth). The generic equation for this network and the electronic scheme analog are given in Fig. 3B and C, respectively.
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We then compared axonal length and branching for the NTF-dependent axonal growth at 12 hours versus the NTF-independent growth at 40 hours. The two types of axonal growth exhibited no difference in the total axonal length and the mean number of branches per neuron (Fig. 4A,B). We also took the approach proposed by Smith and Skene to compare the length and branching of the longest axon of each neuron (Smith and Skene, 1997). There was no difference between the NTF-dependent axonal growth at 12 hours and the NTF-independent growth at 40 hours (Fig. 4C).
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). We therefore asked whether the roscovitine-sensitive outgrowth at 40 hours was independent of endogenous NTFs. Axonal outgrowth at 40 hours was not inhibited by TrkB-FC chimera (Fig. 6A). This chimeric scavenging TrkB receptor body effectively blocks the neuronal response to BDNF (Rivera et al., 2002). Axonal outgrowth at 40 hours was not significantly affected by blocking antibodies to GDNF and NGF (Fig. 6A) whereas the same concentration of these antibodies effectively blocked outgrowth in GDNF- and NGF-treated cultures (Fig. 6B). Propagating glia may become a source of axonal-growth-promoting factors by 40 hours in culture. Axonal growth at 40 hours was not affected by the antimitotic nucleoside cytosine 1-
-D-arabinofuranoside (Fig. 6A), indicating that this axonal growth was not stimulated by glia. We then showed that the conditioned medium collected at 40 hours from DRG cultures did not activate axonal growth in newly plated neurons (Fig. 6C).
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We conclude that the laminin-dependent, roscovitine-sensitive signaling pathway activates axonal growth in DRG neurons in the absence of NTFs at 40 hours in culture. We then asked the question: is the NTF/SFK-dependent pathway still able to activate axonal growth independently of the roscovitine-sensitive pathway at 40 hours? The model in Fig. 3 predicts that the NTF/SFK-dependent pathway would still activate axonal growth if the other pathway is blocked by roscovitine. Indeed roscovitine blocks axonal growth at 40 hours in the absence of NTFs (Fig. 7A,B,D) but fails to affect outgrowth in sister cultures treated with NTFs (Fig. 7A,C,E). We conclude that the NTF/SFK-activated pathway acts independently of the roscovitine-sensitive pathway. The two pathways are not additive, because NTFs do not induce additional outgrowth compared with the control (Fig. 7A). After roscovitine treatment, the NTF-activated outgrowth was further potently inhibited by SU6656 (Fig. 7A). The outgrowth was not blocked completely in this case, suggesting that there may be another NTF-activated pathway that does not involve SFK activation.
In our experiments in Fig. 7, we applied NGF and NRTN together to activate outgrowth simultaneously in two large subpopulations of mature DRG neurons that express TrkA or Ret receptors, respectively. These subpopulations are shown schematically in Fig. 8B. These two subpopulations do not significantly overlap in adult mouse DRG (Lindfors et al., 2006; Molliver et al., 1997). Also in our experiments, the values of outgrowth activated by NRTN and NGF were roughly additive (Fig. 8C). This means that each NTF (NGF or NRTN) applied separately would activate the NTF/SFK-dependent pathway in a responsive subpopulation of cultured DRG neurons. The roscovitine-sensitive pathway must be common for all neurons because roscovitine completely blocks outgrowth in the absence of NTFs at 40 hours (Fig. 2E). Thus according to the model in Fig. 3 we predict that one could block the NTF-independent outgrowth with roscovitine in all neurons at 40 hours, and simultaneously activate the NTF/SFK-dependent outgrowth in a particular subpopulation (either TrkA-expressing or Ret-expressing) by adding NGF or NRTN separately. Indeed, under roscovitine treatment, NRTN and NGF applied separately induce outgrowth in responsive subpopulations (Fig. 8A). Under NRTN+roscovitine treatment, outgrowth takes place in 44% of neurons whereas under NGF+roscovitine treatment it is 14%. Thus, outgrowth in these two subpopulations together is comparable with the outgrowth in the whole untreated population: 59.4% in control, 69.5% and 66.4% under NRTN or NGF treatment, respectively (Fig. 8A). In NGF-treated cultures, the combination of roscovitine and SU6656 causes complete inhibition of outgrowth. This result fits perfectly with the model in Fig. 3. Induction of small but significant outgrowth by NRTN in the presence of both inhibitors (Fig. 8A) suggests that some other SFK-independent pathway(s) may have a minor additional role in the NRTN-activated axonal growth.
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). Investigation of connectivity and the operation logics of these networks is one of the key goals of modern cell biology (Bhalla, 2004; Dueber et al., 2004; Li et al., 2006; Sachs et al., 2002; Sachs et al., 2005) that also has an emerging potential for drug discovery (Rajasethupathy et al., 2005). The aim of the present study was to reveal connectivity of the signaling network mediating the effects of NTFs and laminin on axonal growth and to search for formal rules of signal propagation that would be consistent with experimental data. We demonstrate that the intracellular signaling network differentially recognizes two signaling inputs: laminin alone and laminin+NTF by triggering two different signaling pathways.
NGF (as well as GDNF) promotes functional regeneration of DRG axons into the spinal cord in the experimental model of dorsal root avulsion (Ramer et al., 2000). During development, DRG neurons fail to establish their peripheral projections to the mammalian limb if NGF signaling is impaired (Patel et al., 2000; Tucker et al., 2001). NGF also induces axonal outgrowth in sympathetic and trigeminal neurons (Arumäe et al., 1993; Campenot, 1982; Davies et al., 1981). This effect is known to involve both the Ras-Erk and PI3K-Akt signaling pathways (Markus et al., 2002). The role of SFKs in the NGF-dependent axonal growth remains essentially unstudied (but see Zhou et al., 2006). Here we use a SrcDN adenoviral construct and the SFK inhibitor SU6656 to demonstrate that activation of SFKs is required for NGF-dependent axonal growth in mature DRG neurons. Understanding NGF-SFK signaling during axonal growth in adult neurons may be important to reveal the mechanism of nerve regeneration, because both the protein levels and the activity of Src increase following peripheral nerve injury (Ignelzi, Jr et al., 1992; Le Beau et al., 1991). It was demonstrated previously that SFK activity is required for neuronal survival (Encinas et al., 2001) and axonal growth (Paveliev et al., 2004) induced by GDNF and NRTN.
Our results indicate that SFKs may be the convergence point for the signaling cascades triggered by laminin and NTFs. Interestingly, Tucker and co-authors have recently come to the same conclusion using a different experimental approach (Tucker et al., 2005). Our present results further suggest that convergence of laminin- and NTF-triggered signals on SFKs is a crucial step in the NTF-triggered axonal growth. Suppression of this signaling component with SrcDN blocks the increase in axonal outgrowth caused by NRTN+NGF (Fig. 2C). Furthermore SU6656 blocks NGF-triggered outgrowth both at 12 and at 40 hours in culture (Fig. 2A, Fig. 8A). This SFK-dependent signaling pathway may be remodeled so that synergy between NGF and laminin is no longer required for axonal growth if a pre-conditioning lesion is introduced (Zhou et al., 2006). Activation of SFKs is also crucial for the NRTN-dependent axonal growth at 12 hours in culture. The ability of NRTN to activate modest but significant outgrowth at 40 hours in the absence of SFK activity is a subject for further study. In contrast to the NTF-dependent axonal growth, neither SrcDN nor SU6656 was able to prevent axonal growth on laminin in the absence of NTFs (Fig. 2B,D). We conclude that the SFK enzymatic activity is not required for this type of outgrowth. By contrast, axonal branching was regulated by activation of SFKs both in the presence and in the absence of NGF (Fig. 5B,E)
In our experiments, all mature DRG neurons required laminin for axonal growth (see supplementary material Fig. S1). Laminin is known to bind 11 and 31 integrin receptors on DRG neurons providing the signaling input for activation of axonal growth (Tomaselli et al., 1993). Spatiotemporal regulation of laminin-induced signaling by integrins during posttraumatic regeneration in the nervous system is still poorly understood (Silver and Miller, 2004). Developmental loss of regenerative potential is thought to be the crucial obstacle for posttraumatic regeneration of central projections by DRG neurons (Cai et al., 2001). Laminin is expressed in the glial scar in posttraumatic adult CNS but its regeneration-promoting potential is masked by chondroitin sulfate proteoglycans (McKeon et al., 1991). The laminin receptor integrin 1, which was expressed in the adenoviral vector, was shown to improve dramatically the ability of mature DRG neurons to regenerate axons in vitro both on laminin-precoated substrate and in the presence of aggrecan – the condition that mimicks glial scar (Condic, 2001). It was also shown that disruption of the laminin 
1 gene in Schwann cells leads to impaired regeneration of motor axons in sciatic nerve (Chen and Strickland, 2003). Here we demonstrate that the laminin-dependent and NTF-independent axonal growth is blocked by the Cdk5 inhibitor roscovitine. Cdk5 is known to regulate neuronal migration and axonal growth in neurons of central and peripheral nervous system (Dhavan and Tsai, 2001). Ledda and co-authors showed previously that Cdk5 mediates GDNF signaling through soluble GFR1 receptor whereas GPI-linked GFR1 signals through a different pathway (Ledda et al., 2002). It was also shown that the Cdk5-dependent pathway did not involve SFKs because it was not affected by the SFK inhibitor PP2. These data further support our conclusion that Cdk5-dependent activation of axonal growth represents a signaling pathway that acts as an alternative to the NTF/SFK-dependent pathway for axonal growth. Laminin is also known to activate Cdk5 in cerebellar macroneurons and in a differentiated neuroblastoma cell line, and in both cases, this signaling pathway leads to activation of axonal growth (Li et al., 2000; Paglini et al., 1998). In our experiments, the Cdk5-dependent pathway was not regulated by GDNF or soluble GFR1 signaling because GDNF-blocking antibodies did not affect the roscovitine-sensitive axonal growth (Fig. 6A).
Description of signaling pathways with conventional diagrams, where arrows represent positive and negative regulations between signaling molecules, are often useless for quantitative analysis, limiting the predictive and investigative value of this traditional approach (Lazebnik, 2002). Development of consistent and unambiguous rules for network representation is prerequisite to understanding bioregulatory networks (Ideker et al., 2001; Kohn et al., 2006; Kurata et al., 2003; Sachs et al., 2002). In the present work, we looked for a simple and intuitive interface between formal logic and `wet' biology of cell signaling. To understand how a physiological output is regulated by multiple ligand inputs (which act separately or simultaneously), a description of the signaling network is required, which allows an output prediction provided that the inputs are known. A prediction should be testable experimentally. For this purpose, the type of the input-output relationship at the signal convergence points should be defined explicitly. Here we proposed Boolean logics as a way to define the type of signal integration in a signaling network. Using a Boolean network formalism we predicted the neuron behavior under a complex condition of stimulation of two signaling pathways at 40 hours in culture and successfully designed experiments for the predicted model verification (Figs 5, 6). McAdams and Shapiro previously used an electrical engineering representation of Boolean networks to describe regulation of gene expression in bacteriophage lambda (McAdams and Shapiro, 1995). Using this method, the network in Fig. 3A can be presented as Fig. 3C.
It is now evident that NGF and GDNF family ligands promote axonal growth through a pathway that involves SFK activity. A different, SFK-independent pathway activates axonal growth in the absence of neurotrophic factors. We believe that our findings on the operation of the signaling network triggering neuritogenesis in sensory neurons contribute to a better understanding of posttraumatic recovery in the nervous system, and to development of therapeutic approaches for the treatment of nerve injury.
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). Studies were performed according to National Institutes of Health guidelines and approved by the local Ethical Committee. One-month-old NMRI or BALB/C mice were obtained from local animal house (University of Helsinki) and sacrificed by cervical dislocation under CO2 anaesthesia. Cells were plated (culture density 400-800 neurons/cm2) on glass coverslips. Coverslips were pre-coated with poly-DL-ornithine (1 mg/ml) overnight at 4°C and then with laminin-1 (Invitrogen, 100 ng/cm2) for 4 hours at 37°C. In some experiments, axonal growth was estimated after 40 hours in culture. For those experiments, a lower density (200-400 neurons/cm2) was used. Culture medium included F12 (50%) and DMEM (50%), supplemented with glutamine, penicillin, streptomycin and serum substitute, containing 0.35% bovine serum albumin, 60 ng/ml progesterone, 16 µg/ml putrescine, 400 ng/ml L-thyroxine, 38 ng/ml sodium selenite and 340 ng/ml triiodothyronine. NGF (Promega), GDNF, NRTN (PeproTech), roscovitine and SU6656 (Calbiochem), blocking antibodies to NGF (Chemicon) and to GDNF (Amgen) were applied at the time of plating neurons.
Immunocytochemical procedures
Cultures were fixed for 15 minutes in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and stained with rabbit polyclonal antiserum to protein gene product 9.5 (PGP 9.5; Affiniti). This marker was shown to be neuron specific in DRG and stains axons and cell bodies of all subpopulations of DRG neurons (Calzada et al., 1994; Wilson et al., 1988). Phospho-Erk1/2Thr202/Tyr204 antibody was from Cell Signaling. Alexa Fluor 488- and Alexa Fluor 350-conjugated goat anti-rabbit secondary antibodies were from Molecular Probes. Upon staining all samples were mounted in gelvatol.
SrcDN recombinant adenovirus
The SrcDN construct was a generous gift from David Kaplan (Popsueva et al., 2003). The construct co-expresses SrcDN and GFP, therefore neuron transduction was followed by GFP fluorescence. DRG cultures were transduced with SrcDN or with GFP adenoviral construct when plated. In these experiments, axons were visualized using primary antibodies for PGP 9.5 and Alexa Fluor 350-conjugated secondary antibodies with 420-nm-long pass barrier filter. In our experiments, axonal growth on laminin-coated substrate was proportional to the laminin concentration, which is why we used low laminin concentrations in the experiments with adenoviruses to allow the construct expression before the onset of axonal growth (see Results).
Morphometry
The immunostained cultures were viewed using the 20x objective (NA 0.50) of a Zeiss Axioplan 2 fluorescent microscope (Zeiss) and 300-500 neurons per sample were counted to measure the percentage of process-bearing neurons. Neurons with processes at least twice as long as cell body diameter were defined as axon bearing. Axonal length and the number of branches were measured using ImagePro Plus 5.1 software (Media Cybernetics). Neurons were selected by scanning through a sample and the first ten randomly encountered neurons in each culture were measured. In all experiments, statistical significance was calculated for the data from at least three independent experiments using one-way ANOVA (Excel, Microsoft). Error bars represent s.e.m. *P<0.05; **P<0.01; ***P<0.001.
Image acquisition and processing
All images were collected at room temperature on gelvatol-embedded samples. Epifluorescent images were collected with a Zeiss Axioplan 2 fluorescent microscope equipped with objective lenses 20x (air, NA 0.50), 40x (oil, NA 0.75) or 60x (oil, NA 1.40), AxioCam CCD camera and AxioVision acquisition software (all from Zeiss).
In vitro kinase assay
Dissociated DRG cultures were plated on laminin, or in the absence of laminin precoating, and maintained for 40 hours. The lysis buffer contained 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM DTT, 1 mM Na3VO4 and Complete Mini protease inhibitors (Roche). Relative protein concentrations were measured by SDS-PAGE and staining with silver nitrate. Cdk5 was immunoprecipitated from cell lysates by incubation with anti-Cdk5 antibody (Santa Cruz) and then with protein-G-Sepharose beads (Amersham). Kinase assay was performed for 2 hours at room temperature in kinase buffer containing 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM MgCl2, 1 mM MnCl2, 1 mM DTT, 1 mM Na3VO4, 20 mM NaF, Complete Mini protease inhibitors with precipitated Cdk5 beads, 10 µg of histone H1 (Upstate) and 5 µCi [-32P]ATP (GE Healthcare) per sample. 5x Laemmli buffer (300 mM Tris-HCl (pH 6.8), 25% glycerol, 25% SDS, 0.015% Bromophenol Blue and 325 mM DTT) was added to the reaction and the sample was then separated on 12% SDS-polyacrylamide gel. Histones were visualized by Coomassie Blue staining of the gel (45% methanol, 10% acetic acid, 0.25% Coomassie Blue) and by autoradiography (Fuji films) of the dried gel.
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, AND; 
, OR; upper line, NOT. Boolean operator NOT means that the output is positive only if the input to the operator is negative. (C) Electronic scheme analog for the network in A. The color code in A and C is the same as in 
, SU6656 treatment (C,F) and roscovitine treatment (I,L). For each condition, data are shown for 30 neurons from three independent experiments; the data in A,B,D,E,G,H,J,K are means ± s.e.m. Where indicated, NGF was applied at 100 ng/ml, SU6656 at 2 µM, roscovitine at 50 µM.
