Noradrenaline acting on astrocytic β2-adrenoceptors induces neurite outgrowth in primary cortical neurons

Neuropharmacology 77 (2014) 234e248

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Noradrenaline acting on astrocytic b2-adrenoceptors induces neurite outgrowth in primary cortical neurons Jennifer S. Day, Eimear O’Neill, Caroline Cawley, Nicholas Kruseman Aretz, Dana Kilroy, Sinead M. Gibney, Andrew Harkin*, Thomas J. Connor Neuroimmunology Research Group, Department of Physiology, School of Medicine & Trinity College Institute of Neuroscience, Trinity College, Dublin 2, Ireland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 April 2013 Received in revised form 17 September 2013 Accepted 30 September 2013

The neurotransmitter noradrenaline (NA) has anti-inflammatory properties and promotes expression of neurotrophic factors in the central nervous system (CNS) via activation of glial adrenoceptors. Here we examined the ability of conditioned media (CM) from NA-treated glial cells to impact upon neuronal complexity. Primary rat cortical neurons were treated either directly with NA (1e10 mM), or treated with CM from NA-stimulated primary mixed glial cells. Neuronal complexity was assessed using Sholl analysis. Exposure of neurons to CM from NA-stimulated glial cells increased all indices of neuronal complexity, whereas direct exposure of neurons to NA did not. CM from NA-stimulated astrocytes, but not microglia, also increased neuronal complexity indicating a key role for astrocytes. The b-adrenergic subtype was implicated in this response as the increase was blocked by the b-adrenoceptor antagonist propanolol, but not by the a-adrenoceptor antagonist phentolamine. CM from glial cells treated with the b2-adrenoceptor agonists salmeterol and clenbuterol, but not the b1-adrenoceptor agonist xamoterol, mimicked the ability of NA to increase neuronal complexity. NA induced expression of a range of growth factors (BDNF, NGF-b, GDNF, FGF-2 and IL-6) in glial cells. In addition to this, the phosphatidylinositol 3kinase (PI3K), mitogen activated protein kinase (MAPK) and JAK-STAT signalling pathways are implicated in NA CM-induced neuritic growth as inhibition of these pathways attenuated NA CM-induced neuritic growth. In conclusion, this study indicates a novel role for NA acting at glial b2-adrenoceptors to induce neuritic growth through the expression of soluble factors that elicit a neurotrophic action and increase neuronal complexity. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Noradrenaline Neurite growth Cortical neurons Glial cells b-adrenoceptor Trophic factors

1. Introduction Understanding the mechanism of neuronal growth is vital for unravelling the development of the nervous system and also to develop strategies to encourage neuronal regrowth following brain trauma, infection or neurodegenerative disease. Although there is limited potential of the brain to produce new neurons during a lifetime (for review, see (Gage, 2002)), the neurons of the mature central nervous system (CNS) largely fail to regenerate following damage. The neurodegeneration that occurs during brain injury, stroke, Alzheimer’s disease, Parkinson’s disease, ageing and infection is permanent, and leads to debilitating symptoms. Among the methods currently being developed to protect and encourage regrowth of neurons, the use of neurotrophic factors for therapeutic

* Corresponding author. Tel.: þ353 8962807. E-mail address: [email protected] (A. Harkin). 0028-3908/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2013.09.027

benefit represents an avenue of research to be explored for enhancement of rehabilitation following neurodegeneration (Allen et al., 2013). Neurotrophic factors, such as the neurotrophins (nerve growth factor (NGF-b), brain-derived neurotrophic factor (BDNF), neurotrophin-3 and -4/5 (NT-3, 4/5)), glial cell-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF-2), transforming growth factor (TGF-b) and cytokines, such as interleukin-6 (IL-6) and IL-10, have widely been demonstrated to be involved in the regulation of neuronal growth and protection. These growth factors are often associated with an ability to protect neurons from toxicity (Timmer et al., 2007; Nagahara et al., 2009; Xing et al., 2010; Dobolyi et al., 2012; Fang et al., 2013; Savina et al., 2013) and can also encourage neuritic growth of neurons both under basal conditions (Chao, 2003; Lee et al., 2009; Cohen-Cory et al., 2010) and following neurodegeneration (Cao et al., 2008). Unfortunately, neurotrophic factors cannot pass the blood-brain barrier (BBB) and so invasive procedures are required to directly utilise

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them in the treatment of CNS disorders. Methods of indirectly increasing expression of neurotrophic factors could therefore prove beneficial for neuroprotective-based therapies and/or regeneration of neurons following damage. Neurotrophic factors are known to be released from glial cells in the CNS, therefore, one approach involves the activation of receptors on glial cells to stimulate neurotrophic factor release. Many downstream signalling pathways have been associated with neuronal growth; of these, the mitogen-activated protein kinase (MAPK), in particular the extracellular signal-related kinase 1/2 (ERK1/2) branch, and phosphoinositide 3-kinase (PI3K) pathways predominate (Redmond et al., 2002; Read and Gorman, 2009). Many of the growth factors implicated in neuronal growth have been shown to activate these pathways, including NGF-b (Jackson et al., 1996; Sole et al., 2004), BDNF (Cohen-Cory et al., 2010), GDNF (Takahashi, 2001), FGF-2 (Reuss and von Bohlen und Halbach, 2003) and IL-6 (Heinrich et al., 2003). IL-6 also activates the Janus kinase-Signal transducer and activator of transcription (JAK-STAT) pathway (Spooren et al., 2011) which has also been shown to be involved in the induction of neurite outgrowth (Wu and Bradshaw, 1996). Therefore, it is evident that increasing expression of neuronal growth factors can lead to increased activation of the MAPK, PI3K and JAK-STAT signalling pathways and, in turn, enhancement of neuronal growth and survival. The neurotransmitter noradrenaline (NA) is involved in a wide range of CNS functions. Although there is only limited evidence that shows that NA might have a role in neuritic growth (Clarke et al., 2010), there is evidence both in vitro (Semkova et al., 1996; Junker et al., 2002) and in vivo (Zhu et al., 1998, 1999; Zeman et al., 1999) demonstrating the ability of NA to protect neuronal cells from toxic insults. NA has also been shown to drive an antiinflammatory phenotype and to limit neuroinflammation in the CNS (Gleeson et al., 2010; McNamee et al., 2010a; O’Sullivan et al., 2010). Crucially, via an action on the b-adrenoceptors, NA induces the production of a range of neurotrophic factors from various cell types (Schwartz and Costa, 1977; Follesa and Mocchetti, 1993; Yamashita et al., 1995; Culmsee et al., 1999; Counts and Mufson, 2010; Manni et al., 2011), including glial cells (Hertz et al., 2004; Pocock and Kettenmann, 2007). Therefore, the potential of utilising brain-permeable noradrenergic agents, in particular b-adrenoceptor agonists, should be investigated as a possible means for encouraging neuronal regeneration.

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As NA represents a promising therapeutic target for enhancing neuronal regrowth following neurodegeneration, the aim of this study was to investigate whether NA, acting on glial adrenoceptors, can induce neuritic growth of primary cortical neurons through the release of neurotrophic factors. We also determined the glial cell type as well as the adrenergic receptor subtype which implement NA-induced neuritic growth. Specifically, we examined the growth factors involved in NA-induced neuritic growth and the downstream signalling pathways that are activated in this response. 2. Materials and methods 2.1. Materials Noradrenaline, propranolol and phentolamine were obtained from SigmaAldrich, Ireland. Salmeterol, clenbuterol and xamoterol were obtained from Tocris Bioscience, UK. ELISA kits for GDNF and NGF-b were obtained from Promega, UK, for BDNF and VEGF from R&D Systems, UK, for FGF-2 from Biolegend, USA and for IL-6 from BD Biosciences. Wortmannin, LY294002 and PD98059 were obtained from Tocris Bioscience, UK, and S31-201 was obtained from Merck Chemicals, UK. AntibIII-tubulin was obtained from Promega, UK. All secondary antibodies were obtained from Invitrogen, USA. Gene expression assays for GDNF, NGF-b, BDNF, NT3, NT4/5, TGFb1, FGF-2, IGF-1, CNTF, VEGF, IL-10, IL-6, b-actin and Taqman master mix were obtained from Applied Biosystems. Cell culture reagents were obtained from Invitrogen (Ireland), and all other reagents were obtained from Sigma (UK) unless otherwise stated. 2.2. Primary cell culture Primary mixed glial cultures were prepared from 2-3-day-old Wistar rats (BioResources Unit, Trinity College, Dublin 2, Ireland). Cortical tissue was dissociated in complete Dulbecco’s minimal essential media (cDMEM:F12, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco), 100 IU/ml penicillin and 100 IU/ml streptomyocin (P/S) (Gibco) for 20 min at 37  C. Tissue was then triturated and filtered through a sterile mesh filter (40 mm). Cell suspension was centrifuged at 2000  RPM for 3 min at 20  C, and the pellet was re-suspended in cDMEM. Cells were seeded at 5  106 cells/ml in T75 flasks. For conditioned media (CM) studies, the mixed glial cells were re-plated into 6-well plates by trypsinisation after 10 days in vitro. Primary enriched microglia were prepared by treating mixed glial cells with GM-CSF (10 ng/ml) and M-CSF (10 ng/ml) (R&D Systems) to encourage microglial proliferation. After 10e14 days, microglia were removed by orbital shaking for 2 h at 110  RPM. The resultant suspension was centrifuged at 2000  RPM for 5 min at 20  C. Cells were plated at 1  106 cells/ml on poly-L-lysine pre-coated glass coverslips in 24-well plates. Primary enriched astrocytes were prepared by removal of microglia from mixed glial cells as described above. The remaining astrocyte monolayer was trypsinised and plated onto uncoated 6-well plates. Primary neuronal cultures were prepared from 1-day-old Wistar rats. Cortical tissue was dissociated in complete neurobasal media (cNBM, 1% (v/v) pencillin/ streptomycin, 1% (v/v) Glutamax and 0.1% (v/v) Fungizone, 1% (v/v) B27) and dissociated in trypsin-EDTA for 2 min at 37  C. cDMEM was added and tissue was triturated twice, followed by centrifugation at 2000  RPM for 4 min at 20  C. The resultant supernatant was discarded and the pellet re-suspended in cDMEM. This

Fig. 1. Neuron stained with bIII tubulin and Hoescht (A) with overlaying Sholl analysis concentric circles (B)Figure shows a neuron which has been fluorescently stained with neuronal structural protein bIII tubulin (white) with the cell body stained with Hoescht (blue). In this example, the neuron is considered to have six primary neurites (indicated with yellow stars), with three bifurcations between circles 1 and 2 (red arrows) and two more bifurcations between 2 and 3. The longest neurite terminates between circles 5 and 6 (green arrow). Scale bar ¼ 30 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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J.S. Day et al. / Neuropharmacology 77 (2014) 234e248 cell suspension was triturated, passed through a sterile mesh filter (40 mm) and centrifuged at 2000  RPM for 3 min at 20  C. The supernatant was discarded and the pellet re-suspended in cNBM. For all Sholl analysis experiments, neurons were plated at 5  105 cells/ml on poly-D-lysine-coated coverslips in 24-well plates. Cells were cultured in a humidified atmosphere containing 5% CO2:95% air at 37  C and the medium was changed every 3 days. 2.3. Cell culture treatments All drugs were dissolved in NBM for CM studies and DMEM for others. Control cultures were treated with the addition of vehicle included in the drug treatments, e.g. dimethyl sulfoxide (DMSO, Sigma-Aldrich). Neurons were treated at 3 days in vitro. Glial cells were treated at least 2 days post separation. Pre-treatments were administered 30 min prior to post-treatment. Cell cultures were incubated with the various drug treatments for either 6 h to assess gene expression, or 24 h for CM experiments and for protein production. For CM experiments, after 24 h treatment on glial cells, CM was removed and filtered through a 0.2 mm syringe filter to remove cells and cellular debris. A full CM transfer was then performed on the neurons. B-27 supplement (1% (v/v)) was added to every well after CM transfer. Neurons for Sholl experiments were treated for 24 h before fixation (day 4 in vitro), neurons for Western Immunoblotting were treated for 5 min. Cell numbers of glia (mixed glia, astrocytes or microglia) for CM experiments were kept at approximately the same numbers across experiments (approx 1  106 cells/well). Where this was not possible (e.g. for enriched microglial cultures), CM volume was adjusted appropriately. 2.4. Sholl analysis Neurite morphology was measured using Sholl analysis (Sholl, 1953). The Sholl analysis procedure was adapted from Gutierrez and Davies (2007). For the analysis, bIII tubulin immunofluorescent stained coverslips were viewed at 200 on an epifluorescent microscope. bIII tubulin is a global neuritic marker that does not distinguish between axons and dendrites. For a coverslip to be utilised in Sholl analysis, the neurons must display a healthy network phenotype, following this, individual neurons growing on the edge of the neuronal network, which were not in contact with any other neurons, were imaged. Five individual neurons from each coverslip were imaged and averaged for analysis and up to eight coverslips were analysed per experimental group. Neuronal images were imported into Microsoft PowerPoint where a calibrated image of concentric circles at 10 mm distances (up to 165 mm) was superimposed onto the cell body of the nucleus of the neuron (Fig. 1). Primary neurites, neuritic branches and neurite termination points were all counted within each circle. Primary neurites were classified as those directly stemming from the cell body within circle number 1, while a branch was counted if a neurite clearly divides in two for at least 5 mm. The longest neurite was utilised as the neuritic length. The Sholl profile represents the total number of neuritic branches for any segment plotted against the distance from the cell soma. The total number of neurites for any segment was calculated as follows: Xi ¼ Xi1 þ Bi  Ti Where Xi ¼ the number of neurites for the “ith” segment tBi ¼ the number of branching events occurring in the “ith” segment tTi ¼ the number of branching terminations occurring in the “ith” segment

2.5. Real-time PCR RNA was isolated using a Total RNA isolation kit (Macherney Nagel). Any genomic DNA contamination was removed with the addition of DNase to the samples according to the manufacturer’s instructions. The yield of the resulting purified RNA was determined by measurement of the absorbance at 260 nm in a spectrophotometer, and RNA samples were subsequently equalized. RNA samples were reverse transcribed into cDNA using a high capacity cDNA archive kit (Applied Biosystems) according to the manufacturer’s protocol. Real-time PCR was performed using an ABI Prism 7300 instrument (Applied Biosystems, Darmstadt, Germany) as previously described (Boyle and Connor, 2007). Taqman Gene Expression Assays (Applied Biosystems, Darmstadt, Germany) containing primers and a Taqman probe were used to quantify each gene of

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interest. Assay IDs for the genes examined were as follows: b-actin (VIC) 4352340E, BDNF (Rn00560868_m1), CNTF (Rn00755092_m1), FGF-2 (Rn00570809_m1), GDNF (RN00755092_m1), IGF-1 (Rn00710306_m1), IL-10 (Rn00563409_m1), IL-6 (Rn00561420_m1), NGF-b (Rn01533872_m1), NT3 (Rn00579280_m1), NT4/5 (RN00566076_s1), TGF-beta 1 (Rn00572010_m1) and VEGF (Rn01511601_m1). PCR was performed in PCR plates in a 20 ml reaction volume (9 ml of diluted cDNA, 1 ml of Taqman Gene expression assay and 10 ml of TaqmanÒ Universal PCR Master Mix) and PCR (40 cycles) was run using ABIs universal cycling conditions. b-actin was used as endogenous control to normalize gene expression data, and an RQ value (2DDCt, where Ct is the threshold cycle) was calculated for each sample using Applied Biosystems RQ software (Applied Biosystems, UK). RQ values are presented as fold change in gene expression relative to the control group, which was normalised to 1. 2.6. ELISA Growth factor concentrations were measured using ELISA with protein specific antibodies and standards obtained from kit manufacturers. Assays were performed according to the manufacturer’s instructions, and absorbance read at 450 nm using a microplate reader (Biotek instruments). Absorbance was then recalculated as a concentration (pg/ml) using a standard curve derived using GraphPad Prism Software Version 4.00 (GraphPad software, Inc). 2.7. Western immunoblotting Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western Immunoblotting was performed as described previously (McNamee et al., 2010b). Briefly, protein concentration was determined by the Bradford assay method (Bradford, 1976). Proteins were separated by electrophoresis on 10% SDSpolyacrylamide gels, transferred onto PVDF membrane, and blocked for 1 h at room temperature with 5% milk in Tris-buffered saline (TBS)-Tween (0.05% Tween20, pH 7.4) prior to incubation overnight at 4  C with primary antibodies for AKT, pAKT, ERK1/2, p-ERK1/2, STAT3 and p-STAT3 (1:1000; Cell Signalling). Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000; Amersham) were applied for 1 h at room temperature and detected using chemiluminescence. Protein bands were quantified using ImageJ software (NIH). Total protein levels were used as internal loading controls and provided a measure of phosphorylated protein relative to total protein. 2.8. Statistical analysis of data All values are expressed as mean  standard error of the mean (SEM). Data were analysed using a student t-test or a one-, two- or three-way analysis of variance (ANOVA) followed, where appropriate, by a NewmaneKeuls post hoc test (GB-Stat). A p < 0.05 was considered statistically significant.

3. Results 3.1. Noradrenaline CM from glial cells increases all measures of neuronal complexity of primary cortical neurons NA is known to have extra-synaptic actions on glial cells, stimulating the release of trophic factors for neurons (for example (Madrigal et al., 2009). The purpose of these studies therefore was to determine if noradrenaline, via an action on glial cells, would provide a more trophic environment for primary cortical neurons. Treatment of primary cortical neurons with NA CM (1, 5, 10 mM) from glial cells significantly increased the number of primary neurites extending from the cell soma (p < 0.05, p < 0.01; Fig. 2a), the number of neuritic branches (p < 0.05, p < 0.01; Fig. 2b), the neuritic length (p < 0.01; Fig. 2c) and led to an enhanced Sholl profile (p < 0.05, p < 0.01; Fig. 2d) compared to control CM treated neurons. Representative images for primary cortical neurons treated with NA CM can be seen in Fig. 2eeh. A direct treatment of neurons with the same doses of NA led to no significant changes in

Fig. 2. Noradrenaline CM increases all measures of neuronal complexity of primary cortical neurons. Primary cortical neurons were treated for 24 h with NA CM from glial cells or directly with NA. Sholl analysis was then performed on the neurons. All doses of NA CM significantly increased (a) number of primary neurites, (b) number of neuritic branches and (c) neuritic length. NA CM increased (d) the Sholl profile at 5e35 mm from the cell soma. Direct NA treatment had no effect on (i) number of primary neurites, (j) number of neuritic branches or (k) neuritic length and decreased (l) the Sholl profile at 15e45 mm from the cell soma. Data expressed as mean þ SEM, n ¼ 7. (aec, iek): *p < 0.05, **p < 0.01 vs. control CM/control (One-way ANOVA followed by post-hoc NewmaneKeuls). (d): **p < 0.01 NA CM 1 mM vs. control CM, #p < 0.05, ##p < 0.01 NA CM 5 mM vs. control CM, þþp < 0.01 NA CM 10 mM vs. control CM (Two-way repeated measures ANOVA followed by post-hoc NewmaneKeuls). (l): *p < 0.05, **p < 0.01 NA 1 mM vs. control, #p < 0.05, ##p < 0.01 NA 10 mM vs. control (Two-way repeated measures ANOVA followed by post-hoc NewmaneKeuls). All neurons were imaged by 200 magnification (eeh). Primary cortical neurons treated with NA CM from glial cells were stained with bIII-tubulin (white) and the cell body marker DAPI (blue). Pictures correspond to representative images for (a) control neuron treated with control CM and neurons treated with NA CM at 1 mM (b), 5 mM (c) and 10 mM (d). Scale bars in each diagram are equal to 50 mm.

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Fig. 3. Antagonism of the b-adrenoceptor blocks all NA CM-induced increases in neuronal complexity while antagonism of the a-adrenoceptor attenuates NA CM-induced increases in neuritic length. Primary cortical neurons were treated for 24 h with CM from glial cells pre-treated for 30 min with propranolol (Prop, 10 mM) or phentolamine (Phent, 10 mM) followed by NA (10 mM) stimulation for 24 h. Sholl analysis was then performed. Prop significantly reduced the NA CM-induced increases in (a) number of primary neurites, (b) number of neuritic branches and (c) neuritic length while Phent significantly reduced the NA CM-induced increases in (c) neuritic length. (aec): Data expressed as mean  SEM, n ¼ 8. **p < 0.01 vs. control CM, #p < 0.05, ##p < 0.01 vs. NA CM alone. (Two-way ANOVA followed by post-hoc NewmaneKeuls). (d,e): NA CM treated neurons had significantly more branches than control CM treated neurons at 5, 15, 25, 35 and 45 mm from the cell soma. Prop with NA CM treated neurons had significantly less branches than NA CM treated neurons alone at 5, 15, 25, 35 and 45 mm from the cell soma. Phent alone treated neurons had significantly more branches than control CM treated neurons at 25 mm from the cell soma. There were no significant differences between NA CM alone and Phent with NA CM treated neurons. Data expressed as means  SEM, n ¼ 8. **p < 0.01 NA CM vs. control CM, #p < 0.05, ##p < 0.01 NA CM þ Prop vs. NA CM, þp < 0.05 Phent vs. control CM (Three-way repeated measures ANOVA followed by post-hoc NewmaneKeuls).

the number of primary neurites (Fig. 2i), the number of neuritic branches (Fig. 2j) or the neuritic length (Fig. 2k). Direct treatment with NA (1, 10 mM) decreased the Sholl profile (p < 0.05, p < 0.01; Fig. 2l) compared to control neurons. 3.2. NA CM-induced increases in neuronal morphology are primarily attributed to the b2-adrenoceptor NA can bind to either a- or b-adrenoceptor subtypes, both of which are present on glial cells (for review, see Kimelberg, 1995). Literature suggests that the neuroprotective properties of adrenergic stimulation are mediated mainly via the b-adrenoceptor subtype (Semkova et al., 1996). Therefore, it was important to determine which glial adrenoceptor subtype was mediating the noradrenaline-induced effects on neuronal morphology in an attempt to understand the underlying mechanisms. Propranolol (b-

adrenoceptor antagonist) and phentolamine (a-adrenoceptor antagonist) were utilised in this study to block the ability of NA to bind to the b and a adrenoceptors respectively. NA CM increased the number of primary neurites, the number of neuritic branches, the neuritic length and the Sholl profile of the neurons as before (Fig. 3aee). 10 mM concentrations of propranolol (Semkova et al., 1996; Junker et al., 2002; Madrigal et al., 2009) and phentolamine (Wang and Robertson, 1997; Miyazaki et al., 1998; Klotz et al., 2003) have previously been found to abolish protective effects of NA and noradrenergic agents. Pre-treatment for 30 min with the b2-adrenoceptor antagonist, propranolol (10 mM), but not the a-adrenoceptor antagonist, phentolamine (10 mM), attenuated the NA CM induced increases in the number of primary neurites (p < 0.05; Fig. 3a), the number of neuritic branches (p < 0.01; Fig. 3b) and the neuritic length (p < 0.01; Fig. 3c). Phentolamine partially attenuated the NA CM induced increase in neuritic length

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(p < 0.05; Fig. 3c). Propranolol attenuated the NA CM induced enhancement of the Sholl profile (p < 0.05, p < 0.01; Fig. 3d) while phentolamine pre-treatment did not (Fig. 3e). Treatment of neurons with neither propranolol nor phentolamine CM alone had any effect on gross neuronal morphology. To assess the ability of activation of the b-adrenoceptor subtypes in enhancing neuronal complexity, the highly selective b2adrenoceptor agonists salmeterol (1 mM) and clenbuterol (1 mM), and the highly selective b1-adrenoceptor agonist, xamoterol (1 mM) were utilised. A 1 mM dose of each of these agonists has previously been found to elicit neuroprotective action (Semkova et al., 1996; Junker et al., 2002; McNamee et al., 2010a). Treatment of primary cortical neurons with salmeterol CM and clenbuterol CM increased the number of primary neurites extending from the cell soma (p < 0.05, p < 0.01; Fig. 4a), the number of neuritic branches (p < 0.05, p < 0.01; Fig. 4b), the neuritic length (p < 0.01; Fig. 4c) and led to an enhanced Sholl profile (p < 0.05, p < 0.01; Fig. 4d) compared to control CM treated neurons. Xamoterol CM however did not increase any of the neuritic growth parameters compared to control CM treated neurons (Fig. 4aed). 3.3. NA stimulation of astrocytic b-adrenoceptors are responsible for the NA CM-induced increases in neuronal complexity

3.4. NA stimulation of glial cells results in the release of GDNF, IL-6 and FGF-2 As the previous sections have described, NA treatment of primary glial cells via an action on the astrocytic b2-adrenoceptor, induces morphological changes in primary cortical neurons, potentially via the release of soluble factors. It was therefore important to examine potential mediators which might be resulting in these changes. NA (10 mM) treatment led to the increase in mRNA expression after 6 h of GDNF, NGF-b, BDNF, FGF-2 and IL-6 within the glial cells (p < 0.01; Fig. 6a). Treatment of glial cells with the short-acting b2-adrenoceptor agonist salbutamol (10 mM) resulted in a similar mRNA expression profile (data not shown). Furthermore, NA treatment of glial cells for 24 h led to an increase in the release of GDNF, IL-6 and FGF-2 protein (p < 0.01; Fig. 6b). To confirm noradrenergic activation

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Mori et al., 2002), it was important to assess the roles of each cell type in enhancing neuronal complexity via the b2-adrenoceptor. Treatment of primary cortical neurons with NA CM from enriched astrocytes, led to increases in the number of primary neurites (p < 0.01; Fig. 5a), the number of neuritic branches (p < 0.01; Fig. 5b), the neuritic length (p < 0.01; Fig. 5c) and enhanced the Sholl profile (p < 0.05, p < 0.01; Fig. 5c). Treatment of primary cortical neurons with CM from enriched microglia however led to a decrease in the number of primary neurites (p < 0.01; Fig. 5e), and no changes for the neuritic branches (Fig. 5f), the neuritic length (Fig. 5g) or the Sholl profile (Fig. 5h).

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Primary mixed glial preparations are primarily composed of astrocytes (80e90%) and microglia (10e20%). As both cell types have been shown to express the b2-adrenoceptor (Kimelberg, 1995;

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Distance from Cell Soma ( m) Fig. 4. Salmeterol CM and clenbuterol CM but not xamoterol CM increase all measures of neuronal complexity of primary cortical neurons. Primary cortical neurons were treated for 24 h with the CM from glial cells treated with Salm (1 mM), Clen (1 mM) or Xam (1 mM) for 24 h. Sholl analysis was then performed on the neurons. Both Salm CM and Clen CM treated neurons significantly increased (a) number of primary neurites (b) number of neuritic branches and (c) neuritic length. Xam CM treatment of neurons had no effect on any parameter. (aec) Data expressed as mean  SEM, n ¼ 5e7. *p < 0.05, **p < 0.01 vs. control CM (One-way ANOVA followed by post-hoc NewmaneKeuls). (d): Clen CM had significantly more branches at 5, 15, 25 and 35 mm from the cell soma than control CM treated neurons while Salm CM treated neurons had significantly more branches at 5, 15, 25, 35 and 45 mm from the cell soma. Data expressed as means  SEM, n ¼ 5e7, **p < 0.01 Salm CM vs. control CM, ##p < 0.01 Clen CM vs. control CM (Two-way repeated measures ANOVA followed by post-hoc NewmaneKeuls).

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Fig. 5. NA CM from enriched astrocytes but not from enriched microglia increases all measures of neuronal complexity of primary cortical neurons. Astrocytes (aed): Primary cortical neurons were treated for 24 h with NA (10 mM) CM from astrocytes. Sholl analysis was then performed on the neurons. NA CM significantly increased (a) number of primary neurites (b) number of neuritic branches (c) neuritic length and the Sholl profile (d) compared to control CM treated neurons. Data are expressed as mean þ SEM, n ¼ 8, *p < 0.05 vs. control CM and **p < 0.01 vs. control CM ((aec) Student’s t-test, (d) Two-way repeated measures ANOVA followed by post-hoc NewmaneKeuls). Microglia (eeh): Primary cortical neurons were treated for 24 h with the NA CM (10 mM) from microglia. Sholl analysis was then performed on the neurons. NA CM significantly reduced the number of primary neurites and had no effect on the number of neuritic branches or the neuritic length. CM from NA treated enriched microglia reduced the Sholl profile. Data are expressed as mean þ SEM, n ¼ 8, **p < 0.01 vs. control CM ((eeg) Student’s t-test, (h) Two-way repeated measures ANOVA followed by post-hoc Newman Keuls).

and involvement of the b-adrenoceptor, pre-treatment of glial cells with the b-adrenoceptor antagonist propranolol (10 mM) was shown to block the increase in IL-6 mRNA expression by NA CM, whereas a-adrenoceptor antagonist phentolamine (10 mM) did not (Fig. 6c).

3.5. Inhibition of GDNF and FGF-2 signalling partially attenuates NA CM-induced increases in neuronal morphology As expression of the neurotrophic factors GDNF and FGF-2 was increased following NA treatment of glial cells, it was next

J.S. Day et al. / Neuropharmacology 77 (2014) 234e248

mRNA expression (Fold chan ge vs. control)

32 Control NA

28

**

241

attenuated NA-CM induced increases in number of primary neurites (p < 0.05; Fig. 7d) and the neuritic length (p < 0.01; Fig. 7f) but not primary neurites (Fig. 7e). 3.6. Inhibition of IL-6 receptors and neurotrophin receptors attenuates NA CM-induced increases in neuronal morphology

24 5

**

**

** 0

As expression of IL-6 and the neurotrophins NGF-b and BDNF was increased following NA treatment of glial cells, it was next assessed whether inhibition of IL-6, NGF-b and BDNF signalling using receptor antibodies could attenuate NA CM-induced increases in neuritic complexity. Pre-treatment for 30 min with antiIL6R (0.1 mg/ml) attenuated NA CM-induced increases in the number of neuritic branches (p < 0.05; Fig. 8b) and the neuritic length (p < 0.01; Fig. 8c). Y1036 is a small molecule inhibitor that binds both NGF-b (KD ¼ 3 mM) and BDNF (KD ¼ 3.5 mM) and prevents their binding to neurotrophin receptors (Eibl et al., 2010). Y1036 (40 mM) attenuated NA CM-induced increases in the number of primary neurites (p < 0.05; Fig. 8d) number of neuritic branches (p < 0.01; Fig. 8e) and the neuritic length (p < 0.01; Fig. 8f).

**

** NGF-β BDNF NT3 NT4/5 GDNF TGFβ

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3.7. The growth factors NGF-b, GDNF, FGF-2 and IL-6 increase the neuritic length of primary cortical neurons

**

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Expression of the growth factors NGF-b, GDNF, FGF-2 and IL-6 was significantly increased by NA treatment of glial cells. Therefore, the ability of these factors to induce morphological changes in primary cortical neurons was assessed. Primary cortical neurons were treated directly with NGF-b, GDNF, FGF-2 and IL-6 (1, 5, 10 ng/ ml). Neuritic length was significantly increased following treatment with NGF-b (p < 0.05; Fig. 9a), GDNF (p < 0.01; Fig. 9b), FGF-2 (p < 0.05; Fig. 9c) and IL-6 (p < 0.01; Fig. 9d).

20 10 0

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IL-6 mRNA expre ession (Fold change vs. control)

4 3

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*** ***

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Phent

Fig. 6. NA treatment of glial cells leads to the increase in mRNA expression and enhanced release of several growth factors. Glial cells were treated for 6 h with NA (10 mM) and then harvested for mRNA expression. NA increased the expression of NGFb, BDNF, GDNF, FGF-2 and IL-6 (a). NA significantly reduced mRNA expression of NT3. NT4/5, TGF-b1 and IL-10 remained unchanged. Data expressed as means þ SEM, n ¼ 5e6, **p < 0.01 vs. control (Student’s t-test). Glial cells were treated for 24 h with NA (10 mM), after which time the CM was analysed for protein release by ELISA (b). NA significantly increased the release of GDNF, FGF-2 and IL-6 from glial cells. There was no effect of NA on NGF-b, and BDNF was undetected in the samples. Data expressed as means þ SEM, n ¼ 6e7. **p < 0.01 vs. own control. ND ¼ not detected. (Student’s ttest). Glial cells were pre-treated with Prop (10 mM) or Phent (10 mM) for 30 min followed by stimulation with NA (10 mM) for 6 h and then harvested for mRNA expression (c). NA increased expression of IL-6. This expression was blocked by Prop but not by Phent. Data expressed as mean  SEM, n ¼ 8. ***p < 0.001 vs. control. (Twoway ANOVA followed by post-hoc NewmaneKeuls).

assessed whether inhibition of GDNF and FGF-2 signalling using neutralising antibodies (nABs) could attenuate NA CM-induced increases in neuritic complexity. Pre-treatment for 30 min with GDNF nAB (0.5 mg/ml) attenuated NA-CM induced increases in neuritic length (p < 0.01; Fig. 7c) but not primary neurites or neuritic branching (Fig. 7a and b). FGF-2 nAB (2.5 mg/ml)

3.8. NA CM-induced neurite outgrowth involves the PI3K, MAPK and STAT3 signalling pathways Increases in neuronal morphology have previously been associated with activation of the PI3K pathway (Ditlevsen et al., 2003), the ERK1/2 MAPK pathway (Lee et al., 2009) and the STAT3 pathway (He et al., 2005). To verify activation of these pathways in primary cortical neurons, neurons were treated for 5 min with control or NA CM and then prepared for Western Immunoblotting. NA CM significantly increased the ratio of phosphorylated AKT to total AKT (p < 0.05; Fig. 10a), phosphorylated ERK1/2 to total ERK1/ 2 (p < 0.01; Fig. 10b) and phosphorylated STAT3 to total STAT3 (p < 0.05; Fig. 10c). To assess the role of these pathways in NA CMinduced neuritic outgrowth, we examined the ability of 30 min pretreatment with the PI3K inhibitors wortmannin (100 nM) and LY294002 (10 mM), the ERK1/2 inhibitor PD98059 (10 mM) and the STAT1 inhibitor S31-201 (10 mM) to attenuate NA CM-induced increases in neuronal morphology. Both PI3K inhibitors attenuated the NA CM-induced in the number of primary neurites (p < 0.05; Fig. 10d), the number of neuritic branches (p < 0.01; Fig. 10e) and the neuritic length (p < 0.01; Fig. 10f). PD98059 attenuated the NA CM-induced in the number of primary neurites (p < 0.05; Fig. 10g), the number of neuritic branches (p < 0.01; Fig. 10h) and the neuritic length (p < 0.01; Fig. 10i). S31-201 attenuated the NA CM-induced in the number of primary neurites (p < 0.01; Fig. 10j), the number of neuritic branches (p < 0.01; Fig. 10k) and the neuritic length (p < 0.01; Fig. 10l). 4. Discussion Here we demonstrate that NA, via an action on astrocytic b2adrenoceptors, can induce neurite outgrowth in primary cortical

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Control CM NA CM

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Fig. 7. Neutralization of GDNF and FGF-2 attenuates NA CM-induced increases in neuronal complexity. Primary cortical neurons were treated for 24 h with NA CM (10 mM) which had been incubated for 30 min with normal goat IgG (control, 0.5 mg/ml) or the GDNF nAB (0.5 mg/ml) (aec), or were treated for 24 h with NA CM (10 mM) which had been incubated for 30 min with mouse IgG1k (control, 2.5 mg/ml) or the FGF-2 nAB (2.5 mg/ml) (def). Sholl analysis was then performed. NA CM significantly increased (a, d) primary neurites, (b, e) number of neuritic branches and (c, f) neuritic length. The GDNF nAB attenuated NA CM-induced increases in neuritic length (c) but not primary neurites (a) or neuritic branching (b). The FGF-2 nAB attenuated NA CM-induced increases in (d) primary neurites and (f) neuritic length but not (e) neuritic branching. Data expressed as means þ SEM, n ¼ 6e8. *p < 0.05, **p < 0.01 vs. control CM, #p < 0.05, ##p < 0.01 vs. NA CM alone (Two-way ANOVA followed by post-hoc NewmaneKeuls).

neurons. NA CM but not direct NA treatment, significantly enhanced the number of primary neurites, the number of neuritic branches, the neuritic length and the Sholl profile of primary cortical neurons. Direct NA treatment in fact resulted in a decreased Sholl profile. There is limited evidence in the literature supporting a role for NA to reduce neuritic growth. Physiological levels of NA have been proposed to act to inhibit axonal growth and innervation in the rat heart (Clarke et al., 2010). Basal levels of NA acted to continuously inhibit neuritic growth while inhibition of endogenous NA signalling using b-adrenoceptor antagonists increased the neuritic growth. NA-induced inhibition of neurite outgrowth was proposed to occur primarily through presynaptic neuronal b1adrenoceptors. In this study, NA CM from glial cells increased

neurite outgrowth. This is the first time that NA, via an action on glial cells, has been demonstrated to alter the morphology of neurons. This study implies that the NA is stimulating the glial cells to release substances into the CM which act upon the neurons and result in morphological changes. Neurons in vivo co-exist with glial cells, and thus the action of NA on glia is more comparable to ongoing NA stimulation in the brain. Subsequent work was undertaken in this study to ascertain potential mediators of NA CMinduced neuronal changes. Lack of demonstration of the effect of NA on neuronal complexity in vivo represents a limitation of this work. Neurons in vivo are retained in a network, and thus have many additional signals through electrical activity and cell-contact to the soluble mediators investigated here. Live-cell imaging would

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Anti-IL6R

Control

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Fig. 8. Neutralization of both IL-6 and the neurotrophins attenuate NA CM-induced increases in neuronal complexity. Primary cortical neurons were treated for 24 h with NA CM (10 mM) which had been incubated for 30 min with IgG2bk (control, 0.1 mg/ml) or anti-IL6R (0.1 mg/ml) (aec) or the neurotrophin antagonist, Y1036 (def). Sholl analysis was then performed. NA CM significantly increased (d) primary neurites, (b, e) number of neuritic branches and (c, f) neuritic length. Anti-IL-6R significantly reduced the NA CM-induced increases in (b) number of neuritic branches and (c) neuritic length. Furthermore, Y1036 attenuated all NA CM-induced increases in complexity. Data expressed as mean þ SEM, n ¼ 7e8. *p < 0.05, **p < 0.01 vs. Control CM, #p < 0.05, ##p < 0.01 vs. NA CM alone. (Two-way ANOVA followed by post-hoc NewmaneKeuls).

be required to examine neuritic growth in vivo. For example, increased expression of BDNF has been found to increase axonal length and branching of neurons in vivo in a real-time imaging study (Alsina et al., 2001). NA CM-induced increases in neuritic growth were shown to be primarily attributed to the b-adrenoceptor, as increased complexity was blocked by the b-adrenoceptor antagonist propranolol but not by the a-adrenoceptor antagonist phentolamine. Furthermore, NAinduced increase in IL-6 expression was blocked only by propranolol and not by phentolamine, indicating that the neuritic growthenhancing effects of NA were b-adrenoceptor-mediated. Further experiments demonstrated that NA CM-induced increased complexity was mediated by the b2-adrenergic subtype as the selective b2-adrenoceptor agonists salmeterol and clenbuterol increased all measures of neuronal complexity while the selective b1-adrenoceptor agonist xamoterol did not. As the b2-adrenoceptor

primarily signals via the downstream messenger cAMP, it was investigated if artificially increasing cAMP levels in the glial cells could also increase neuronal morphology. The cell permeable cAMP analogue dbcAMP significantly increased all measures of neuronal complexity (data not shown). As previously discussed, this is the first time that NA has been shown to induce neuritic growth of primary neurons via an action on glial cells. However, NA has been shown to induce neuronal protection following various toxic insults. For example, primary rat astrocytes treated with NA (10 mM) resulted in a CM which attenuated NMDA-dependent glutamate release from primary cortical neurons (Madrigal et al., 2009). Furthermore, co-culture of NA-treated astrocytes with primary neurons protected the neurons from oxygen glucose deprivation and this was attributed to an increase in monocyte chemoattractant protein (MCP-1) release from the astrocytes. It also appears that the b2-adrenoceptor is vital for NA-induced neuronal protection, as the

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Fig. 9. The growth factors NGF-b, GDNF, FGF-2 and IL-6 increase the neuritic length of primary cortical neurons. Primary cortical neurons were treated for 24 h with NGF-b (a), GDNF (b), FGF-2 (c) or IL-6 (d). Sholl analysis was then performed. All growth factors investigated significantly increased the neuritic length of the neurons compared to control. Data expressed as means  SEM, n ¼ 6e8 **p < 0.01, *p < 0.05 vs. control. (One-way ANOVA followed by post-hoc NewmaneKeuls).

b2-adrenoceptor antagonists, propranolol and ICI 118551, inhibited this release of MCP-1 (Madrigal et al., 2009). Similar results were also observed by Junker et al. (2002) who showed that mixed neuronal and astrocytic hippocampal cultures were protected from glutamate-induced neuronal toxicity by clenbuterol stimulation. In addition, clenbuterol attenuated neuronal damage following a stroke model in mice (Junker et al., 2002), and has also been shown to be beneficial in a rat model of spinal cord injury (Zeman et al., 1999), ischaemic damage (Zhu et al., 1998), and is associated with an increase in the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xl following ischaemia (Zhu et al., 1999). Thus the ability of NA to induce neuritic growth via the glial b2-adrenoceptor fits with the previously discovered neuroprotective functions of this receptor. Astrocytes and not microglia were shown to be involved in NA CM-induced increases in neuritic growth. This result is in line with the literature, which implicates the ability of astrocytes to provide protection to neurons and to secrete a variety of growth factors. For example CM from untreated primary astrocytes was shown to protect primary neurons from amyloid b-induced neuronal toxicity, and although the study did not demonstrate what factors might be present in the CM, the neuroprotection relied on activation of the ERK1/2 pathway, which is induced by many trophic factors (Yamamuro et al., 2003). Similarly, hydrogen peroxide-induced neurotoxicity in primary neurons is attenuated upon the increasing presence of astrocytes in the culture (Desagher et al., 1996). Astrocytes have also been previously demonstrated to be involved in encouraging neuritic growth of neurons. For example, neurons in co-culture with astrocytes show enhanced axonal growth compared to neurons grown with fibroblasts (Dijkstra et al., 1999). It therefore appears that astrocytes need to be stimulated to increase production of trophic factors to induce neuritic growth; basal release of trophic factors is insufficient to induce

morphological changes in neurons. In agreement with this, treatment of astrocytes with lysophosphatidic acid, a phospholipid derived from cell membranes, leads to a CM which induces neuritic growth of primary cortical neurons via activation of the MAPK pathway, while control CM had no effect (Spohr et al., 2011). In contrast, microglia have been shown to have inhibitory effects on neurite outgrowth in various studies. CM from myeloid lineage cells (microglia, monocytes and macrophages) inhibit neurite outgrowth via myosin light chain phosphorylation in primary rat cerebellar neurons (Pool et al., 2011). CM from primary rat microglia is required for progesterone-induced inhibition of neurite outgrowth in neuron-astrocyte cocultures (Bali et al., 2013). Microglia also contribute to neurite outgrowth deficits induced by unconjugated bilirubin by increasing nitric oxide (NO) production and glutamate release (Silva et al., 2012). A large number of growth factors, hormones, cytokines and synthetic molecules have been shown to modulate the morphology of neurons from both in vitro and in vivo models (Greene and Tischler, 1976; Walicke et al., 1986; Patel and McNamara, 1995; Costantini and Isacson, 2000; Chao, 2003; Ditlevsen et al., 2003; Paratcha et al., 2003; Khaibullina et al., 2004; Cui, 2006; Ducray et al., 2006). Thus attempting to investigate the role of each of these in NA CM-induced increases in neuritic morphology was beyond the scope of this study. Instead, the mRNA expression of a range of growth factors associated with neuritic outgrowth and/or induction by NA as determined by a literature search, was investigated following NA stimulation of glial cells. NA increased the expression of GDNF, NGF-b, BDNF, FGF-2 and IL-6. The NA CM was also found to contain GDNF, NGF-b, FGF-2 and IL-6 protein. Although levels of NGF-b were not increased in the NA CM compared to control CM, this factor was present. The NA CM therefore contains a combination of NGF-b, IL-6, FGF-2 and GDNF and potentially many more trophic factors which were not

J.S. Day et al. / Neuropharmacology 77 (2014) 234e248

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Fig. 10. The PI3K inhibitors wortmannin and LY294002, the MAPK inhibitor PD98059 and the STAT3 inhibitor S31-201 block NA CM-induced changes in neuronal morphology. Primary cortical neurons were treated for 5 min with CM from NA-treated or control glia before Western Immunoblotting was performed. NA CM increased phosphorylation of (a) AKT, (b) ERK1/2 and (c) STAT3. Data expressed as mean  SEM, n ¼ 5e6. *p < 0.05, **p < 0.01 vs. control CM. (Student’s t-test). Primary cortical neurons were treated for 30 min with the PI3K inhibitors Wort (100 nM) and LY (10 mM) (def), the MAPK inhibitor PD98059 (10 mM) (gei), or the STAT3 inhibitor S31-201 (10 mM) (jel) and then treated for 24 h with NA CM from glial cells (10 mM). Sholl analysis was then performed. Both Wort and LY attenuated the NA CM-induced increases in (d) number of primary neurites, (e) number of neuritic branches and (f) neuritic length. PD also attenuated the NA CM-induced increases in (g) number of primary neurites, (h) number of neuritic branches and (i) neuritic length. S31-201 treatment attenuated the NA CM-induced increases in (j) number of primary neurites, (k) number of neuritic branches and (l) neuritic length. Data expressed as means þ SEM, n ¼ 6e8. *p < 0.05, **p < 0.01 vs. control CM, #p < 0.05, ##p < 0.01 vs. NA CM alone. (Two-way ANOVA followed by post-hoc NewmaneKeuls).

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investigated here. Interactions between the above mentioned trophic factors therefore are more than likely contributing to the ability of NA CM to induce neuritic growth. Various evidence suggests that such interactions between growth factors have a synergistic effect in NA-induced increase in neuronal morphology. NGF-b, for example, is capable of activating the STAT3 signalling pathway commonly associated with IL-6 (Zhou and Too, 2011) and RET, the GDNF receptor (Tsui-Pierchala et al., 2002) and synergistic neuroprotective activity of NGF-b together with both IL-6 and GDNF has been observed in neuronal cultures (Hama et al., 1989; Madduri et al., 2009). Many trophic factors can induce their own and other trophic factor expression and release from cells. For example, BDNF can stimulate its own release from hippocampal neurons in culture (Canossa et al., 1997). FGF-2 can increase GDNF mRNA in C6 cells (Suter-Crazzolara and Unsicker, 1996) and can increase both the mRNA (Vige et al., 1991) and the secretion of NGF-b from primary astrocytes in culture (Fukumoto et al., 1991). The ability of FGF-2 to protect primary hippocampal neurons from glutamate toxicity has also been attributed to GDNF release by the neurons following FGF-2 stimulation (Lenhard et al., 2002). Additionally, GDNF has also been shown to induce the expression of FGF-2 from glial cells which was dependent on the MAPK pathway (Hauck et al., 2006). From these studies it is obvious that trophic factors often synergise and combine to create downstream effects. Often the abilities of one trophic factor rely on the presence of another. Thus in the present study, it is interesting that inhibition of GDNF, FGF-2, IL-6, NGF-b and BDNF by antibody neutralization or by receptor antagonism attenuated to some degree the NA CM-induced increases in neuritic growth. Neurons were also treated with a cocktail of neutralising antibodies against the neurotrophic factors that were increased following NA treatment (GDNF, FGF-2 and IL-6; data not shown) and a more robust effect was observed, complimentary to the effects seen following neutralization of each factor alone. It therefore is possible that neurons require the combination of factors to grow in response to the CM although additional experimentation into molecular mechanisms and signalling pathways initiated by the factors in neuronal cells would be required to demonstrate synergy. Activation of the IL-6, NGF-b, GDNF and FGF-2 receptors results in activation of the PI3K/AKT, MAPK and STAT3 signalling pathways and we have shown that inhibition of these pathways attenuates NA CM-induced increase in neuronal complexity. Over-expression of Ras, upstream of MAPK, leads to increases in dendritic length, and inhibition of the MAPK or PI3K pathways blocks the ability of Ras to induce the dendritic changes, thus suggesting that Ras is essential in dendritic growth and that both pathways are utilised for this ability (Kumar et al., 2005). Furthermore, a study by Jang et al. (2009) demonstrated that blockade of the PI3K and MAPK pathways inhibited the NGF-b-induced increases in neurite outgrowth of PC12 cells (Jang et al., 2009). Moreover, activation of the MAPK pathway has been shown to result in the serine phosphorylation of STAT3 (for review, see Bowman et al., 2000). 5-HTinduced neuritogenesis has been shown to be dependent on all three of these pathways; PI-3K, MAPK and STAT3 (Fricker et al., 2005). The growth factors which mediate the NA CM-induced neuritic growth can activate some if not all of the three pathways demonstrated to be involved in NA CM-induced neuritic growth. As there is cross-talk between the growth factors and between the signalling pathways, it may be that a threshold of signalling activation is required prior to neuritic growth. Understanding the mechanisms involved in the neuronal growth is imperative for further understanding the regenerative potential of CNS tissue. This work raises the possibility that brainpermeable noradrenergic agonists could be utilised to encourage repair after brain trauma or infection. NA CM led to a robust

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