Inhibition of phosphatidylinositol 3-kinase activity blocks cellular differentiation mediated by glial cell line-derived neurotrophic factor in dopaminergic neurons

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for midbrain dopaminergic neurons. To begin to understand the intracellular signaling pathways used by GDNF, we investigated the role of phosphatidylinositol 3-kinase activity in GDNF-stimulated cellular function and differentiation of dopaminergic neurons. We found that treatment of dopaminergic neuron cultures with 10 ng/ml GDNF induced maximal levels of Ret phosphorylation and produced a profound increase in phosphatidylinositol 3-kinase activity, as measured by western blot analysis and lipid kinase assays. Treatment with 1 microM 2-(4-morpholinyl)-8-phenylchromone (LY294002) or 100 nM wortmannin, two distinct and potent inhibitors of phosphatidylinositol 3-kinase activity, completely inhibited GDNF-induced phosphatidylinositol 3-kinase activation, but did not affect Ret phosphorylation. Furthermore, we examined specific biological functions of dopaminergic neurons: dopamine uptake activity and morphological differentiation of tyrosine hydroxylase-immunoreactive neurons. GDNF significantly increased dopamine uptake activity and promoted robust morphological differentiation. Treatment with LY294002 completely abolished the GDNF-induced increases of dopamine uptake and morphological differentiation of tyrosine hydroxylase-immunoreactive neurons. Our findings show that GDNF-induced differentiation of dopaminergic neurons requires phosphatidylinositol 3-kinase activation.     

Adenoviral gene transfer of ciliary neurotrophic factor and brain-derived neurotrophic factor leads to long-term survival of axotomized motor neurons

OBJECTIVE: This study was undertaken to investigate the influence of oral supplementation with creatine monohydrate on muscular performance during repeated sets of high-intensity resistance exercise. SUBJECTS/DESIGN: Fourteen active men were randomly assigned in a double-blind fashion to either a creatine group (n = 7) or a placebo group (n = 7). Both groups performed a bench press exercise protocol (5 sets to failure using each subject's predetermined 10-repetition maximum) and a jump squat exercise protocol (5 sets of 10 repetitions using 30% of each subject's 1-repetition maximum squat) on three different occasions (T1, T2, and T3) separated by 6 days. INTERVENTION: Before T1, both groups received no supplementation. From T1 to T2, both groups ingested placebo capsules. From T2 to T3, the creatine group ingested 25 g creatine monohydrate per day, and the placebo group ingested an equivalent amount of placebo. MAIN OUTCOME MEASURES: Total repetitions for each set of bench presses and peak power output for each set of jump squats were determined. Other measures included assessment of diet, body mass, skinfold thickness, and preexercise and 5-minute postexercise lactate concentrations. RESULTS: Lifting performance was not altered for either exercise protocol after ingestion of the placebos. Creatine supplementation resulted in a significant improvement in peak power output during all 5 sets of jump squats and a significant improvement in repetitions during all 5 sets of bench presses. After creatine supplementation, postexercise lactate concentrations were significantly higher after the bench press but not the jump squat. A significant increase in body mass of 1.4 kg (range = 0.0 to 2.7 kg) was observed after creatine ingestion. CONCLUSION: One week of creatine supplementation (25 g/day) enhances muscular performance during repeated sets of bench press and jump squat exercise.     

The regulatory mechanism of phosphatidylinositol 3-kinase by insulin in 3T3 L1 fibroblasts: phosphorylation-independent activation of phosphatidylinositol 3-kinase

Progress in understanding the basis of resistance to rifampicin (RifR) has allowed molecular tests for the detection of drug-resistant tuberculosis to be developed. One hundred thirteen strains of Mycobacterium tuberculosis isolated from patients with multidrug resistant tuberculosis (MDR-TB) were investigated for genotypic analysis of RifR by polymerase chain reaction-heteroduplex formation (PCR-HDF) and characterization of mutations by automated DNA sequencing of the rpoB gene. A subset of isolates (22) representative of different mutations as confirmed by sequence analysis were also evaluated by the Line Probe Assay (LiPA). In 106 of the RifR strains, 24 mutations within an 81-bp region of the rpoB gene affecting 13 amino acids were observed. Most isolates (7/8) harboring Leu533 --> Pro codon mutation required minimum inhibitory concentrations (MICs) of < or = 8 microg/ml. There was geographic variation in the frequency of occurrence of particular rpoB mutations, with the Ser531 --> Leu/Trp codon mutation found in 59/113 of isolates. Although there are certain limitations in the use of both the rapid PCR-HDF diagnostic assay and the LiPA for the detection of rifampicin susceptibility of M. tuberculosis, these provide important and convenient tools for identifying and managing patients with MDR-TB.     

Embryonic mesoderm cells spread in response to platelet-derived growth factor and signaling by phosphatidylinositol 3-kinase

Abnormal mesoderm movement, leading to defects in axial organization, is observed in mouse and Xenopus laevis embryos deprived of platelet-derived growth factor (PDGF) AA signaling. However, neither the cellular response to PDGF nor the signaling pathways involved are understood. Herein we describe an in vitro assay to examine the direct effect of PDGF AA on aggregates of Xenopus embryonic mesoderm cells. We find that PDGF AA stimulates aggregates to spread on fibronectin. This behavior is similar to that of migrating mesoderm cells in vivo that spread and form lamellipodia and filipodia on contact with fibronectin-rich extracellular matrix. We go on to show two lines of evidence that implicate phosphatidylinositol 3-kinase (PI3K) as an important component of PDGF-induced mesoderm cell spreading. (i) The fungal metabolite wortmannin, which inhibits signaling by PI3K, blocks mesoderm spreading in response to PDGF AA. (ii) Activation of a series of receptors with specific tyrosine-to-phenylalanine mutations revealed PDGF-induced spreading of mesoderm cells depends on PI3K but not on other signaling molecules that interact with PDGF receptors including phospholipase C gamma, Ras GTPase-activating protein, and phosphotyrosine phosphatase SHPTP2. These results indicate that a PDGF signal, medicated by PI3K, can facilitate embryonic mesoderm cell spreading on fibronectin. We propose that PDGF, produced by the ectoderm, influences the adhesive properties of the adjacent mesoderm cells during gastrulation.     

Enhancement of neurotransmitter release induced by brain-derived neurotrophic factor in cultured hippocampal neurons

Brain-derived neurotrophic factor (BDNF), like other neurotrophins, has long-term effects on neuronal survival and differentiation; furthermore, recent work has shown that BDNF also can induce rapid changes in synaptic efficacy. We have investigated the mechanism(s) of these synaptic effects on cultured embryonic hippocampal neurons. In the presence of the GABAA receptor antagonist, picrotoxin, the application of BDNF (100 ng/ml) for 1-5 min increased the amplitude of evoked synaptic currents by 48 +/- 9% in 10 of 15 pairs of neurons and increased the frequency of EPSC bursts to 205 +/- 20% of the control levels. There was no detectable effect of BDNF on various measures of electrical excitability, including the resting membrane potential, input resistance, action potential threshold, and action potential amplitude. In addition, BDNF did not change the postsynaptic currents induced by the exogenous application of glutamate. BDNF did increase the frequency of miniature EPSCs (mEPSCs) (268.0 +/- 46.8% of control frequency), however, without affecting the mEPSC amplitude. The effect of BDNF on mEPSC frequency was blocked by the tyrosine kinase inhibitor K252a and also by the removal of extracellular calcium ([Ca2+]o). Fura-2 recordings showed that BDNF elicited an increase in intracellular calcium concentration ([Ca2+]c). This effect was dependent on [Ca2+]o; it was blocked by K252a and by thapsigargin, but not by caffeine. The results demonstrate that BDNF enhances glutamatergic synaptic transmission at a presynaptic locus and that this effect is accompanied by a rise in [Ca2+]c that requires the release of Ca2+ from IP3-gated stores.     

Transport of brain-derived neurotrophic factor across the blood-brain barrier

NMR spectroscopy of F98 glioma cell extracts showed that chronic hypertonic conditions largely increased the intracellular content of small, osmotically active molecules. Moreover, hypertonic stress decreased the incorporation of 13C-labeled amino acids into the cellular proteins albeit their cytosolic concentrations were increased, which reflects an inhibition of protein synthesis under these conditions. Reincubation with isotonic medium restored almost completely the control values for the cytosolic metabolites but not for amino acid incorporation into the protein. An increased amount of 13C label was found in the phospholipids, which indicates stimulation of membrane synthesis processes due to the recovery-induced cell swelling. On the other hand, chronic hypotonic conditions largely decreased the steady state concentration and synthesis of small, cytosolic molecules, whereas the effect on the incorporation of 13C-labeled amino acids into the cellular proteins was variable. Reincubation with isotonic medium partially restored the depressed cytosolic metabolite content and also the incorporation of labeled amino acids into cellular protein, but induced an inhibition of phospholipid synthesis. The results verify that 'readaptation' of glial cell metabolism during recovery from chronic osmotic stress is impaired or at least seriously retarded.     

Epidermal growth factor-dependent association of phosphatidylinositol 3-kinase with the erbB3 gene product

The ErbB3 protein is a member of the ErbB subfamily of receptor protein tyrosine kinases. In the present study, the mechanism by which the ErbB3 protein is phosphorylated and the signal-transducing functions of this phosphorylated protein were investigated. When phosphorylated by the epidermal growth factor receptor in vitro, the ErbB3 protein strongly associated with the regulatory p85 subunit and the catalytic activity of phosphatidylinositol (PI) 3-kinase. The association of PI 3-kinase with ErbB3 in human breast cancer cells was found to be correlated with the constitutive phosphorylation of ErbB3 on tyrosine residues. In MDA-MB-468 breast cancer cells in which the ErbB3 protein is not constitutively phosphorylated, stimulation with epidermal growth factor led to the phosphorylation of ErbB3 on tyrosine residues and the formation of a functional signal transduction complex involving the ErbB3 protein and PI 3-kinase. These results suggest that the ErbB3 protein can be phosphorylated on tyrosine residues by a cross-phosphorylation mechanism and that the phosphorylated ErbB3 protein can couple other growth factor receptor protein tyrosine kinases to the PI 3-kinase pathway in a manner similar to the insulin receptor substrate 1 protein.     

Inhibition of phosphatidylinositol 3-kinase activity by adenovirus-mediated gene transfer and its effect on insulin action

Phosphatidylinositol 3-kinase (PI 3-K) is implicated in cellular events including glucose transport, glycogen synthesis, and protein synthesis. It is activated in insulin-stimulated cells by binding of the Src homology 2 (SH2) domains in its 85-kDa regulatory subunit to insulin receptor substrate-1 (IRS-1), and, others. We have previously shown that IRS-1-associated PI 3-kinase activity is not essential for insulin-stimulated glucose transport in 3T3-L1 adipocytes, and that alternate pathways exist in these cells. We now show that adenovirus-mediated overexpression of the p85N-SH2 domain in these cells behaves in a dominant-negative manner, interfering with complex formation between endogenous PI 3-K and its SH2 binding targets. This not only inhibited insulin-stimulated IRS-1-associated PI 3-kinase activity, but also completely blocked anti-phosphotyrosine-associated PI 3-kinase activity, which would include the non-IRS-1-associated activity. This resulted in inhibition of insulin-stimulated glucose transport, glycogen synthase activity and DNA synthesis. Further, Ser/Thr phosphorylation of downstream molecules Akt and p70 S6 kinase was inhibited. However, co-expression of a membrane-targeted p110(C) with the p85N-SH2 protein rescued glucose transport, supporting our argument that the p85N-SH2 protein specifically blocks insulin-mediated PI 3-kinase activity, and, that the signaling pathways downstream of PI 3-kinase are intact. Unexpectedly, GTP-bound Ras was elevated in the basal state. Since p85 is known to interact with GTPase-activating protein in 3T3-L1 adipocytes, the overexpressed p85N-SH2 peptide could titrate out cellular GTPase-activating protein by direct association, such that it is unavailable to hydrolyze GTP-bound Ras. However, insulin-induced mitogen-activated protein kinase phosphorylation was inhibited. Thus, PI 3-kinase may be required for this action at a step independent of and downstream of Ras. We conclude that, in 3T3-L1 adipocytes, non-IRS-1-associated PI 3-kinase activity is crucial for insulin's metabolic signaling, and that overexpressed p85N-SH2 protein inhibits a variety of insulin's ultimate biological effects.     

Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt

We have found that insulin-like growth factor I (IGF-I) can protect fibroblasts from apoptosis induced by UV-B light. Antiapoptotic signalling by the IGF-I receptor depended on receptor kinase activity, as cells overexpressing kinase-defective receptor mutants could not be protected by IGF-I. Overexpression of a kinase-defective receptor which contained a mutation in the ATP binding loop functioned as a dominant negative and sensitized cells to apoptosis. The antiapoptotic capacity of the IGF-I receptor was not shared by other growth factors tested, including epidermal growth factor (EGF) and thrombin, although the cells expressed functional receptors for all the agonists. However, EGF was antiapoptotic for cells overexpressing the EGF receptor, and expression of activated pp60v-src also was protective. There was no correlation between protection from apoptosis and activation of mitogen-activated protein kinase, p38/HOG1, or p70S6 kinase. On the other hand, protection by any of the tyrosine kinases against UV-induced apoptosis was blocked by wortmannin, implying a role for phosphatidylinositol 3-kinase (PI3 kinase). To test this, we transiently expressed constitutively active or kinase-dead PI3 kinase and found that overexpression of activated phosphatidylinositol 3-kinase (PI3 kinase) was sufficient to provide protection against apoptosis. Because Akt/PKB is believed to be a downstream effector for PI3 kinase, we also examined the role of this serine/threonine protein kinase in antiapoptotic signalling. We found that membrane-targeted Akt was sufficient to protect against apoptosis but that kinase-dead Akt was not. We conclude that the endogenous IGF-I receptor has a specific antiapoptotic signalling capacity, that overexpression of other tyrosine kinases can allow them also to be antiapoptotic, and that activation of PI3 kinase and Akt is sufficient for antiapoptotic signalling.     

Short-term response of postnatal rat vestibular neurons following brain-derived neurotrophic factor or neurotrophin-3 application

OBJECTIVES: To identify risk factors in 60 cases of mediastinitis amongst 2512 patients (2.3%) subjected to isolated coronary bypass surgery from March 1988 through December 1995, treated by a closed irrigation/drainage system. PATIENTS AND METHODS: The mean age of the 60 patients was 56.9 +/- 6.8 years (45-81 years) and 55 (91.6%) were male. Early mediastinal reexploration was performed in all cases immediately after the diagnosis of mediastinitis, with debridement of necrosed tissues, followed by implantation of a closed-circuit irrigation system of the mediastinum constituted by irrigation catheter and drain, closure of the sternum and skin, and specific systemic antibiotic therapy. The mean interval between the original surgery and reexploration was 9.4 days (range 6-14 days). No patient required more extensive procedures, namely omental or muscular flaps. Twenty potential risk factors in patients with mediastinitis, including diabetes mellitus, obesity, coexistence of peripheral vascular disease, decreased LV function, use of inotropes, mediastinal blood drainage and utilization of double IMA, were compared with the group without mediastinitis. RESULTS: Mean cardiopulmonary bypass time was 74.1 +/- 8.1 min, anesthetic time 3.5 +/- 0.8 h and postoperative mechanical ventilation 18 +/- 3 h. A total of 23 patients (38.3%) received one IMA and 35 (58.3%) two IMAs. In the postoperative period, 7 of the 60 patients (11.6%) had required inotropes because of low output. Mediastinal blood loss was 1112cc +/- 452cc and 9 patients (15%) were transfused. Cultures were positive in 40 cases (66.6%) and the most frequent infecting agent was the Staph. epidermidis in 25 cases (62.5%), followed by Candida albicans and Enterobacter and Serratia species (7.5% each); 1 patient (1.7%) died and 9 (15%) had renal failure. The irrigation/drainage was maintained for a mean of 9.1 days (5-83 days). Patients with mediastinitis had a significantly higher prevalence of diabetes (41.6% vs. 18.8%; P < 0.01), obesity (48.3% vs. 15.2%; P < 0.001), peripheral vascular disease (11.6% vs. 4.0%; P < 0.05), but a lower incidence of poor LV function (18.3% vs. 32.7%; P < 0.05). A double IMA was used more frequently in patients who had mediastinitis (58.3% vs. 23.5%; P < 0.001) CONCLUSIONS: Diabetes mellitus, obesity, co-existence of peripheral vascular disease and use of double IMA are risk factors for mediastinitis after coronary artery surgery. The efficacy of the closed method of treatment with a mediastinal irrigation/drainage system was increased with early diagnosis and reintervention.     

A requirement for phosphatidylinositol 3-kinase in pseudopod extension

Phagocytosis requires actin assembly and pseudopod extension, two cellular events that coincide spatially and temporally. The signal transduction events underlying both processes may be distinct. We tested whether phagocytic signaling resembles that of growth factor receptors, which induce actin polymerization via activation of phosphatidylinositol 3-kinase (PI 3-kinase). Fcgamma receptor-mediated phagocytosis was accompanied by a rapid increase in the accumulation of phosphatidylinositol 3,4,5-trisphosphate in vivo, and addition of wortmannin (WM) or LY294002, two inhibitors of PI 3-kinase(s), inhibited phagocytosis but not Fcgamma receptor-directed actin polymerization. However, both compounds prevented maximal pseudopod extension, suggesting that PI 3-kinase inhibition produced a limitation in membrane required for pseudopod extension. Availability of plasma membrane was not limiting for phagocytosis, because blockade of ingestion in the presence of WM was not overcome by reducing the number of particles adhering to macrophages. However, decreasing bead size, and hence the magnitude of pseudopod extension required for particle engulfment, relieved the inhibition of phagocytosis in the presence of WM or LY294002 by up to 80%. The block in phagocytosis of large particles occurred before phagosomal closure, because both compounds inhibited spreading of macrophages on substrate-bound IgG. Macrophage spreading on IgG was accompanied by exocytic insertion of membrane from an intracellular source, as measured by the dye FM1-43. These results indicate that one or more isoforms of PI 3 kinase are required for maximal pseudopod extension but not phagocytosis per se. We suggest that PI 3-kinase is required for coordinating exocytic membrane insertion and pseudopod extension.     

Production and characterization of recombinant rat brain-derived neurotrophic factor and neurotrophin-3 from insect cells

Rat brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were engineered for expression in a baculovirus-infected Spodoptera frugiperda insect cell system. The BDNF and NT-3 from the culture supernatants were purified by ion-exchange and reverse-phase chromatography to apparent homogeneity. The purification procedure yielded approximately 2 mg of pure rat BDNF or NT-3 per liter of culture supernatant. A single N-terminus only was found for either secreted molecule and was analogous to that predicted from the corresponding cDNA sequence. The recombinant neurotrophins obtained were also homogeneous with regard to molecular weight and amino acid sequence. In their native conformation, the insect cell-produced rat BDNF and NT-3 molecules were homodimers consisting of 119 amino acid polypeptide chains. Thus, although the genes transfected into the S. frugiperda cells coded for proBDNF or proNT-3, the BDNF and NT-3 recovered after purification were > 95% fully processed, mature protein. Mature recombinant rat BDNF and NT-3 were found not to be significantly glycosylated. Pure, recombinant rat BDNF and NT-3 promoted the survival of embryonic dorsal root ganglion neurons in the low picomolar range. Because recombinant rat BDNF and NT-3 can be obtained in large quantities, purified to near homogeneity, and are identical in amino acid sequence to the corresponding human proteins, they are suitable for evaluation in animal models.     

Presynaptic modulation of synaptic transmission and plasticity by brain-derived neurotrophic factor in the developing hippocampus

In addition to the regulation of neuronal survival and differentiation, neurotrophins may play a role in synapse development and plasticity. Application of brain-derived neurotrophic factor (BDNF) promotes long-term potentiation (LTP) in CA1 synapses of neonatal hippocampus, which otherwise exhibit only short-term potentiation. This is attributable, at least in part, to an attenuation of the synaptic fatigue induced by high-frequency stimulation (HFS). However, the prevention of synaptic fatigue by BDNF could be mediated by an attenuation of synaptic vesicle depletion from presynaptic terminals and/or a reduction of the desensitization of postsynaptic receptors. Here we provide evidence supporting a presynaptic effect of BDNF. The effect of BDNF on synaptic fatigue depended on the stimulation frequency, not on the stimulus duration nor on the number of stimulation pulses. BDNF was only effective when the synapses were stimulated at frequencies >50 Hz. Treatment with BDNF also potentiated paired-pulse facilitation (PPF), a parameter reflecting changes in the properties of presynaptic terminals. This effect of BDNF was restricted only to PPF elicited with interpulse intervals 相似文献   

An essential role of phosphatidylinositol 3-kinase in myogenic differentiation

The oncogene p3k, coding for a constitutively active form of phosphatidylinositol 3-kinase (PI 3-kinase; EC 2.7.1.137), strongly enhances myogenic differentiation in cultures of chicken-embryo myoblasts. It increases the size of the myotubes and induces elevated levels of the muscle-specific proteins MyoD, myosin heavy chain, creatine kinase, and desmin. Inhibition of PI 3-kinase activity with LY294002 or with dominant-negative mutants of PI 3-kinase interferes with myogenic differentiation and with the induction of muscle-specific genes. PI 3-kinase is therefore an upstream mediator for the expression of the muscle-specific genes and is both necessary and rate-limiting for the process of myogenesis.     

Activation of PI 3-kinase by G protein betagamma subunits

We have reported that fMLP-induced activation of pertussis toxin-sensitive GTP-binding proteins in THP-1 cells potentiates the insulin-induced accumulation of PtdIns(3,4,5)P3, a product of phosphoinositide 3-kinase (T. Okada et al., Biochem. J. 317, 475-480, 1996). The synergism in PtdIns(3,4,5)P3 accumulation was observed in Chinese hamster ovary cells expressing both insulin and fMLP receptors. In rat adipocytes, which represent the physiological target cells of insulin, receptor-mediated activation of GTP-binding protein by adenosine and prostaglandin E2 potentiated the insulin-induced PtdIns(3,4,5)P3 accumulation. In cell-free systems, the activity of the p85/p110beta subtype of phosphoinositide 3-kinase was, while that of p85/p110alpha was not, stimulated by the betagamma subunits of the GTP-binding proteins. We propose here a hypothesis that the p85/p110beta subtype is under the control of both the insulin receptors and the GTP-binding protein-coupled receptors in intact cell systems.     

Expression of brain-derived neurotrophic factor protein in the adult rat central nervous system

We have generated and characterized a multi-functional polyclonal anti-brain-derived neurotrophic factor antibody. Western blot analysis, dorsal root ganglion neurite outgrowth and dorsal root ganglion neuron survival assays showed that this antibody specifically recognized brain-derived neurotrophic factor and not the other neurotrophins. Furthermore, it was capable of blocking the functional effects of brain-derived neurotrophic factor. Using this antibody, we examined the expression of brain-derived neurotrophic factor in adult rat brains by immunohistochemistry. We found distinct brain-derived neurotrophic factor immunoreactivity in several structures of the brain. These included the neocortex, piriform cortex, amygdaloid complex, hippocampal formation, claustrum, some thalamic and hypothalamic nuclei, the substantia nigra and some brainstem structures. In contrast to brain-derived neurotrophic factor messenger RNA expression, brain-derived neurotrophic factor immunoreactivity was also found in the lateral septum, bed nucleus of the stria teminalis, medial preoptic nucleus, olivery pretectal nucleus, lateral paragigantocellular nucleus and the dorsal horn of the spinal cord. In normal adult rat brains, there was little or no staining in the CA1 region or the granule cell layer of the dentate gyrus of the hippocampus. However, kainate treatments greatly increased brain-derived neurotrophic factor immunoreactivity in the pyramidal cells of the CA1 region, as well as in the dentate gyrus, CA2 and CA3 hippocampal regions. We present evidence for both the subcellular localization and anterograde transport of endogenous brain-derived neurotrophic factor in the central nervous system. The detection of brain-derived neurotrophic factor protein in several discrete regions of the adult brain, and brain-derived neurotrophic factor's dramatic up-regulation following kainate treatment, strongly supports a role of brain-derived neurotrophic factor in the maintenance of adult neurons and synapses. Since several populations of neurons lost during neurodegenerative diseases synthesize brain-derived neurotrophic factor protein, modulation of brain-derived neurotrophic factor levels may be clinically beneficial. The antibody described in this paper will be helpful in determining more precisely the functional activities of brain-derived neurotrophic factor in the adult.     

Evidence for a tropic role of brain-derived neurotrophic factor in transplanted embryonic retinae

Developing retinal axons must follow a stereotypic course directing them to the subcortical visual centres which on arrival they must recognise. Transplantation studies suggest that local substrate cues close to the surface of the brainstem and diffusible factors emanating from the target region are important. To test a role for diffusible factors, we transplanted retinae to the cerebral cortex and have shown that outgrowth can be promoted by BDNF secreting fibroblasts.