[Chromatographic determination of radiochemical purity - replacement of ITLC SG]. / Chromatographische Bestimmung der radiochemischen Reinheit von Radiopharmaka - Alternativen zu ITLC SG.

UNLABELLED: Thin layer chromatography is well established for quality control of radiopharmaceuticals. A convenient and widely used stationary phase are ITLC SG strips. However, the Pall Corporation stopped manufacturing of the silica gel impregnated glass fibre strips (ITLC SG). Material, Methode: As a replacement we tested silicic acid impregnated glass fibre strips from Varian (ITLC SA) and sufficient mobile phases. RESULTS: The chromatography with these strips takes two to three times longer than with ITLC SG, but it is in an acceptable range. Only three mobile phases are necessary to test most of the common in-house made radiopharmaceuticals. CONCLUSION: The proposed method is suitable for routinely measuring the radiochemical purity of radiophamaceuticals.

Fluorescence resonance energy transfer analysis of alpha 2a-adrenergic receptor activation reveals distinct agonist-specific conformational changes.

Several lines of evidence suggest that G-protein-coupled receptors can adopt different active conformations, but their direct demonstration in intact cells is still missing. Using a fluorescence resonance energy transfer (FRET)-based approach we studied conformational changes in alpha(2A)-adrenergic receptors in intact cells. The receptors were C-terminally labeled with cyan fluorescent protein and with fluorescein arsenical hairpin binder at different sites in the third intracellular loop: N-terminally close to transmembrane domain V (I3-N), in the middle of the loop (I3-M), or C-terminally close to transmembrane domain VI (I3-C). All constructs retained normal ligand binding and signaling properties. Changes in FRET between the labels were determined in intact cells in response to different agonists. The full agonist norepinephrine evoked similar FRET changes for all three constructs. The strong partial agonists clonidine and dopamine induced partial FRET changes for all constructs. However, the weak partial agonists octopamine and norphenephrine only induced detectable changes in the construct I3-C but no change in I3-M and I3-N. Dopamine-induced FRET-signals were approximately 1.5-fold slower than those for norepinephrine in I3-C and I3-M but >3-fold slower in I3-N. Our data indicate that the different ligands induced conformational changes in the receptor that were sensed differently in different positions of the third intracellular loop. This agrees with X-ray receptor structures indicating larger agonist-induced movements at the cytoplasmic ends of transmembrane domain VI than V and suggests that partial agonism is linked to distinct conformational changes within a G-protein-coupled receptor.

Conformational changes in G-protein-coupled receptors-the quest for functionally selective conformations is open.

The G-protein-coupled receptors (GPCRs) represent one the largest families of drug targets. Upon agonist binding a receptor undergoes conformational rearrangements that lead to a novel protein conformation which in turn can interact with effector proteins. During the last decade significant progress has been made to prove that different conformational changes occur. Today it is mostly accepted that individual ligands can induce different receptor conformations. However, the nature or molecular identity of the different conformations is still ill-known. Knowledge of the potential functionally selective conformations will help to develop drugs that select specific conformations of a given GPCR which couple to specific signalling pathways and may, ultimately, lead to reduced side effects. In this review we will summarize recent progress in biophysical approaches that have led to the current understanding of conformational changes that occur during GPCR activation.

Delayed priming promotes CNS regeneration post-rhizotomy in Neurocan and Brevican-deficient mice.

A wealth of literature has provided evidence that reactive tissue at the site of CNS injury is rich in chondroitin sulfate proteoglycans which may contribute to the non-permissive nature of the CNS. We have recently demonstrated using a murine model of human brachial plexus injury that the chondroitin sulfate proteoglycans Neurocan and Brevican are differentially expressed by two subsets of astrocytes in the spinal cord dorsal root entry zone (DREZ) following dorsal root lesion (Beggah et al., Neuroscience 133: 749-762, 2005). However, direct evidence for a growth-inhibitory role of these proteoglycans in vivo is still lacking. We therefore performed dorsal root lesion (rhizotomy) in mice deficient in both Neurocan and Brevican. Rhizotomy in these animals resulted in no significant increase in the number of sensory fibres regenerating through the DREZ compared to genetically matched controls. Likewise, a conditioning peripheral nerve lesion prior to rhizotomy, which increases the intrinsic growth capacity of sensory neurons, enhanced growth to the same extent in transgenic and control mice, indicating that absence of these proteoglycans alone is not sufficient to further promote entry into the spinal cord. In contrast, when priming of the median nerve was performed at a clinically relevant time, i.e. 7 weeks post-rhizotomy, the growth of a subpopulation of sensory axons across the DREZ was facilitated in Neurocan/Brevican-deficient, but not in control animals. This demonstrates for the first time that (i) Neurocan and/or Brevican contribute to the non-permissive environment of the DREZ several weeks after lesion and that (ii) delayed stimulation of the growth program of sensory neurons can facilitate regeneration across the DREZ provided its growth-inhibitory properties are attenuated. Post-injury enhancement of the intrinsic growth capacity of sensory neurons combined with removal of inhibitory chondroitin sulfate proteoglycans may therefore help to restore sensory function and thus attenuate the chronic pain resulting from human brachial plexus injury.

Lesion-induced differential expression and cell association of Neurocan, Brevican, Versican V1 and V2 in the mouse dorsal root entry zone.

Lack of regeneration in the CNS has been attributed to many causes, including the presence of inhibitory molecules such as chondroitin sulfate proteoglycans (CSPGs). However, little is known about the contribution of CSPGs to regeneration failure in vivo, in particular at the dorsal root entry zone (DREZ), a unique CNS region that blocks regeneration of sensory fibers following dorsal root injury without glial scar formation. The goal of the present study was to evaluate the presence, regulation, and cellular identity of the proteoglycans Brevican, Neurocan, Versican V1 and Versican V2 in the DREZ using CSPG-specific antibodies and nucleic acid probes. Brevican and Versican V2 synthesized before the lesion were still present at high levels in the extracellular matrix of the DREZ several weeks after injury. In addition, Brevican was transiently expressed by reactive oligodendrocytes, and by a subset of astrocytes thereafter. Versican V2 mRNA appeared in NG2-positive cells with the morphology of oligodendrocyte precursor cells. Neurocan and Versican V1 levels were low before injury, and appeared in nestin-positive astrocytes and in NG2-positive cells, respectively, following lesion. Versican V1, but not V2, was also transiently increased in the peripheral dorsal root post-lesion. This is the first thorough description of the expression and cell association of individual proteoglycans following dorsal root lesion. It demonstrates that the proteoglycans Brevican, Neurocan, Versican V1, and Versican V2 are abundant in the DREZ at the time regenerating sensory fibers reach the PNS/CNS border and may therefore participate in growth-inhibition in this region.

Sustained delivery of GDNF: towards a treatment for Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease characterized by the progressive loss of nigral dopaminergic neurons. Although symptomatic therapies to substitute for the missing neurotransmitter dopamine are efficient at the early stages of the disease, the goal is to find alternative therapies which could protect dopaminergic neurons from the degenerative process. We have used two distinct gene therapy approaches to deliver the neuroprotective molecule glial cell line-derived neurotrophic factor (GDNF) in animal models of the disease: (i) an encapsulated genetically engineered cell line releasing GDNF (ex vivo gene therapy); and (ii) a lentiviral vector encoding the GDNF gene (in vivo gene therapy). Both approaches allowed protection of nigral dopaminergic neurons against lesion-induced cell death in rodent as well as monkey models of PD. Behavioral symptoms were also ameliorated in these animals. In addition, co-transplantation of embryonic dopaminergic neuronal grafts and a GDNF-releasing capsule allowed improvement of graft survival and differentiation, thereby accelerating behavioral recovery. These results should lead to clinical application in the near future.

Nerve growth factor- and neurotrophin-3-releasing guidance channels promote regeneration of the transected rat dorsal root.

Dorsal roots have a limited regeneration capacity after transection. To improve nerve regeneration, the growth-promoting effects of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) were evaluated. The proteins were continuously released by synthetic nerve guidance channels bridging a 4-mm gap in the transected dorsal root. Four weeks after lesion, the regenerated nerve cables were analyzed for the presence of myelinated and unmyelinated axons. While BDNF showed a limited effect on axonal regeneration (863 +/- 39 axons/regenerated nerve, n = 6), NGF (1843 +/- 482) and NT-3 (1495 +/- 449) powerfully promoted regeneration of myelinated axons compared to channels releasing the control protein bovine serum albumin (293 +/- 39). In addition, NGF, but not BDNF nor NT-3, had a potent effect on the regeneration of unmyelinated axons (NGF, 55 +/- 1.4; BDNF, 4 +/- 0.3; NT-3, 4.7 +/- 0.3 axons/100 microm(2); n = 6). The present study suggests that synthetic nerve guidance channels slowly and continuously releasing the neurotrophins NGF and NT-3 can overcome the limited regeneration of transected dorsal root.

Isolation of multipotent neural precursors residing in the cortex of the adult human brain.

Multipotent precursors able to generate neurons, astrocytes, and oligodendrocytes have previously been isolated from human brain embryos and recently from neurogenic regions of the adult human brains. The isolation of multipotent neural precursors from adult human should open new perspectives to study adult neurogenesis and for brain repair. The present study describes the in vitro isolation from adult human brains of a progenitor responsive to both epidermal and basic fibroblast growth factors that forms spheres as it proliferates. Single spheres derived from various regions of the brain generate in vitro neurons, astrocytes, and oligodendrocytes. The clonal origin of the spheres was revealed by genomic viral insertion using lentiviral vector. Interestingly, this vector appears to be a potent tool for gene transfer into human neural progeny. Ninety-six percent of the spheres investigated were multipotent. Multipotent precursors were isolated from all brain regions studied, including the temporal and the frontal cortex, the amygdala, the hippocampus, and the ventricular zone. This study is the first evidence that primitive precursors such as multipotent precursors exist in the adult human cortex and can reside far from the ventricles. Neurogenesis derived from adult human progenitors differ to murine neurogenesis by the requirement of laminin for oligodendrocyte generation and by the action of basic-fibroblast growth factor and platelet derived growth factor that prevented the formation of oligodendrocytes and neurons. Moreover, the differentiation of human adult precursors seems to differ from fetal ones: adult precursors do not necessitate the removal of mitogen for differentiation. These results indicate that the study of adult multipotent precursors is a new platform to study adult human neurogenesis, potentially generate neural cells for transplantation, and design protocols for in vivo stimulation.

Evaluation of an intrathecal immune response in amyotrophic lateral sclerosis patients implanted with encapsulated genetically engineered xenogeneic cells.

A phase I/II clinical trial has been performed in 12 amyotrophic lateral sclerosis (ALS) patients to evaluate the safety and tolerability of intrathecal implants of encapsulated genetically engineered baby hamster kidney (BHK) cells releasing human ciliary neurotrophic factor (CNTF). These patients have been assessed for a possible intrathecal or systemic immune response against the implanted xenogeneic cells. Hundreds of pg CNTF/ml could be detected for several weeks in the cerebrospinal fluid (CSF) of 9 out of 12 patients, in 2 patients up to 20 weeks after capsule implantation. Slightly elevated leukocyte counts were observed in 6 patients. Clear evidence for a delayed humoral immune response was found in the CSF of only 3 patients out of 12 (patients #4, #6, and #10). Characterization of the antigen(s) recognized by the antibodies present in these CSF samples allowed to identify bovine fetuin as the main antigenic component. The defined medium used for maintaining the capsules in vitro before implantation contains bovine fetuin. Fetuin may therefore still be adsorbed to the surface of the cells and/or the polymer membrane, or be present in the medium surrounding the encapsulated cells at the time of implantation. Because of the insufficient availability of CSF samples, as well as the relatively poor sensitivity of the assays used, a weak humoral immune response against components of the implanted cells themselves cannot be excluded. However, the present study demonstrates that encapsulated xenogeneic cells implanted intrathecally can survive for up to 20 weeks in the absence of immunosuppression and that neither CNTF nor the presence of antibodies against bovine fetuin elicit any adverse side effects in the implanted patients.

Complete and long-term rescue of lesioned adult motoneurons by lentiviral-mediated expression of glial cell line-derived neurotrophic factor in the facial nucleus.

To date, delivery of neurotrophic factors has only allowed to transiently protect axotomized facial motoneurons against cell death. In the present report, long-term protection of these neurons was evaluated by continuously expressing the neurotrophic factor glial cell line-derived neurotrophic factor (GDNF) within the facial nucleus using a lentiviral vector system. The viral vector was injected unilaterally into the facial nucleus of 4-month-old Balb/C mice. In contrast to axotomy in other adult rodents, facial nerve lesion in these animals leads to a progressive and sustained loss and/or atrophy of >50% of the motoneurons. This model thus represents an attractive model to evaluate potential protective effects of neurotrophic factors for adult-onset motoneuron diseases, such as amyotrophic lateral sclerosis. One month after unilateral lentiviral vector injection, the facial nerve was sectioned, and the animals were killed 3 months later. Viral delivery of the GDNF gene led to long-term expression and extensive diffusion of GDNF within the brainstem. In addition, axotomized motoneurons were completely protected against cell death, because 95% of the motoneurons were present as demonstrated by both Nissl staining and choline acetyltransferase immunoreactivity. Furthermore, GDNF prevented lesion-induced neuronal atrophy and maintained proximal motoneuron axons, despite the absence of target cell reinnervation. This is the first evidence that viral-mediated delivery of GDNF close to the motoneuron cell bodies of the facial nucleus of adult mice can lead to complete and long-term protection against lesion-induced cell death.

Lentiviral vectors as a gene delivery system in the mouse midbrain: cellular and behavioral improvements in a 6-OHDA model of Parkinson's disease using GDNF.

Local delivery of therapeutic molecules represents one of the limiting factors for the treatment of neurodegenerative disorders. In vivo gene transfer using viral vectors constitutes a powerful strategy to overcome this limitation. The aim of the present study was to validate the lentiviral vector as a gene delivery system in the mouse midbrain in the perspective of screening biotherapeutic molecules in mouse models of Parkinson's disease. A preliminary study with a LacZ-encoding vector injected above the substantia nigra of C57BL/6j mice indicated that lentiviral vectors can infect approximately 40,000 cells and diffuse over long distances. Based on these results, glial cell line-derived neurotrophic factor (GDNF) was assessed as a neuroprotective molecule in a 6-hydroxydopamine model of Parkinson's disease. Lentiviral vectors carrying the cDNA for GDNF or mutated GDNF were unilaterally injected above the substantia nigra of C57BL/6j mice. Two weeks later, the animals were lesioned ipsilaterally with 6-hydroxydopamine into the striatum. Apomorphine-induced rotation was significantly decreased in the GDNF-injected group compared to control animals. Moreover, GDNF efficiently protected 69.5% of the tyrosine hydroxylase-positive cells in the substantia nigra against 6-hydroxydopamine-induced toxicity compared to 33.1% with control mutated GDNF. These data indicate that lentiviral vectors constitute a powerful gene delivery system for the screening of therapeutic molecules in mouse models of Parkinson's disease.

Increased motoneuron survival and improved neuromuscular function in transgenic ALS mice after intraspinal injection of an adeno-associated virus encoding Bcl-2.

Mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) underlie some familial cases of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder characterized by loss of cortical, brainstem and spinal motoneurons. Transgenic mice over- expressing a mutated form of human SOD1 containing a Gly-->Ala substitution at position 93 (SOD1(G93A)) develop a severe, progressive motoneuron disease. We investigated the potential of recombinant adeno-associated virus (rAAV) to transfer neuroprotective molecules in this animal ALS model. Initial experiments showed that injection of an rAAV vector encoding green fluorescent protein unilaterally into the lumbar spinal cord of wild-type mice leads to expression of the reporter gene in 34.7 +/- 5.2% of the motoneurons surrounding the injection site. Intraspinal injection of an rAAV encoding the anti-apoptotic protein bcl-2 in SOD1 (G93A) mice resulted in sustained bcl-2 expression in motoneurons and significantly increased the number of surviving motoneurons at the end-stage of disease. Moreover, the compound muscle action potential amplitude elicited by nerve stimulation and recorded by electromyographic measurements was higher in the rAAV-bcl-2-treated group than in controls. Local bcl-2 expression in spinal motoneurons delayed the appearance of signs of motor deficiency but was not sufficient to prolong the survival of SOD1 (G93A) mice. To our know-ledge, this study describes the first successful transduction and protection of spinal motoneurons by direct gene transfer in a model of progressive motoneuron disease. Our results support the use of AAVs for the delivery of protective genes to spinal cord moto-neurons as a possible way to enhance motoneuron survival and repair.

Self-inactivating lentiviral vectors with enhanced transgene expression as potential gene transfer system in Parkinson's disease.

Glial cell line-derived neurotrophic factor (GDNF) is able to protect dopaminergic neurons against various insults and constitutes therefore a promising candidate for the treatment of Parkinson's disease. Lentiviral vectors that infect quiescent neuronal cells may allow the localized delivery of GDNF, thus avoiding potential side effects related to the activation of other brain structures. To test this hypothesis in a setting ensuring both maximal biosafety and optimal transgene expression, a self-inactivating (SIN) lentiviral vector was modified by insertion of the posttranscriptional regulatory element of the woodchuck hepatitis virus, and particles were produced with a multiply attenuated packaging system. After a single injection of 2 microl of a lacZ-expressing vector (SIN-W-LacZ) in the substantia nigra of adult rats, an average of 40.1 +/- 6.0% of the tyrosine hydroxylase (TH)-positive neurons were transduced as compared with 5.0 +/- 2.1% with the first-generation lentiviral vector. Moreover, the SIN-W vector expressing GDNF under the control of the mouse phosphoglycerate kinase 1 (PGK) promoter was able to protect nigral dopaminergic neurons after medial forebrain bundle axotomy. Expression of hGDNF in the nanogram range was detected in extracts of mesencephalon of animals injected with an SIN-W-PGK-GDNF vector, whereas it was undetectable in animals injected with a control vector. Lentiviral vectors with enhanced expression and safety features further establish the potential use of these vectors for the local delivery of bioactive molecules into defined structures of the central nervous system.

Attenuation of 6-OHDA-induced neurotoxicity in glutathione peroxidase transgenic mice.

Normal cellular metabolism produces oxidants which are neutralized within cells by antioxidant enzymes and other antioxidants. An imbalance between oxidants and antioxidants has been postulated to lead to the degeneration of specific populations of neurons in neurodegenerative diseases, e.g. Parkinson's disease. The present study investigates whether overexpression of glutathione peroxidase, the enzyme which metabolizes hydrogen peroxide to water, can prevent or slow down neuronal injury in an animal model of Parkinson's disease. Transgenic mice overexpressing the human glutathione peroxidase gene under the control of the mouse hydroxymethylglutaryl-coenzyme A promoter and genetically matched control mice were injected intracerebroventricularly with the dopaminergic neurotoxin 6-hydroxydopamine. Seven days after injection, the number of tyrosine hydroxylase-positive nigral dopaminergic neurons was decreased by 52.4% and 20.5% in 6-hydroxydopamine-injected control and glutathione peroxidase transgenic mice, respectively. Similarly, 3 days after injection of the neurotoxin, striatal dopamine was decreased by 71.2% and 56.5%, respectively. Overexpression of glutathione peroxidase therefore partially protects dopaminergic neurons against 6-hydroxydopamine-induced toxicity.

Intrathecal implants of bovine chromaffin cells alleviate mechanical allodynia in a rat model of neuropathic pain.

Intrathecal implants of adrenal chromaffin cells are known to release analgesic substances such as catecholamines and opioid peptides. In the present study, bovine chromaffin cells were encapsulated in a permselective polymer membrane which protects the cells from the host immune system and allows grafting of xenogeneic cells without immunosuppression. The effects of such implants were evaluated on the pain behavior resulting from a chronic constrictive injury (CCI) of the rat sciatic nerve. Sprague-Dawley rats with a unilateral lesion were implanted in the lumbar subarachnoid space and tested for mechanical/thermal allodynia and hyperalgesia. A significant reduction in pain was observed after mechanical non-nociceptive stimulation in animals implanted with chromaffin cells. Furthermore, these animals showed decreased signs of spontaneous pain. However, response to thermal non-noxious stimuli or to painful mechanical stimuli was not significantly decreased. Abundant clusters of viable chromaffin cells intensely labeled with the anti-tyrosine hydroxylase antibodies were observed in the retrieved implants. These results establish the analgesic efficacy of intrathecal encapsulated chromaffin cells in a chronic pain model of nerve injury. Immunoprotected allo- or xenogeneic chromaffin cells acting as 'mini pumps' continuously delivering neuroactive substances could be a useful therapy for patients suffering from neuropathic pain.

Neurturin protects dopaminergic neurons following medial forebrain bundle axotomy.

To determine whether neurturin (NTN), a recently identified homologue of glial cell line-derived neurotrophic factor (GDNF), is able to preserve tyrosine hydroxylase immunoreactivity (TH-IR) in a rat model of Parkinson's disease, polymer encapsulated cells genetically engineered to release NTN were implanted near the substantia nigra 1 week before a unilateral medial forebrain bundle axotomy. Animals were allowed to survive for 1 week post-axotomy. Upon sacrifice, animals that received a NTN capsule had a significantly higher percentage of TH-IR (lesioned side vs non-lesioned side) than animals that had received a capsule containing non-transfected parent cells. However, in contrast to GDNF, no reduction of turning was observed upon amphetamine rotation with NTN. Nevertheless, these results suggest that NTN might have a therapeutic value for the treatment of Parkinson's disease.

Implants of polymer-encapsulated genetically modified cells releasing glial cell line-derived neurotrophic factor improve survival, growth, and function of fetal dopaminergic grafts.

Neural transplantation as an experimental therapy for Parkinsonian patients has been shown to be effective in several clinical trials. Further benefit, however, may be expected if the grafting is combined with a treatment of neurotrophic factors thus improving the survival and growth of grafted embryonic dopaminergic neurons. Continuous trophic support may be needed and therefore requires the long-term delivery of neurotrophic factors to the brain. We demonstrate here that the implantation of polymer-encapsulated cells genetically engineered to continuously secrete glial cell line-derived neurotrophic factor to the adult rat striatum improves dopaminergic graft survival and function. Near complete compensation of 6-hydroxydopamine-induced rotation was already achieved within 3 weeks postgrafting in rats that received glial cell line-derived neurotrophic factor-releasing capsules in addition to dopaminergic cell grafts of cultured tissue. Rats without trophic factor supply showed only little recovery at the same time point and sham grafted rats showed no recovery. The number of tyrosine hydroxylase-immunoreactive cells per graft was increased 2.6-fold in the presence of glial cell line-derived neurotrophic factor 6 weeks postgrafting. Similarly, tyrosine hydroxylase-immunoreactive fibers around the graft were increased by 53%. Moreover, these fibers showed a preferential growth towards the trophic factor-releasing capsule. Taken together, these results provide evidence that encapsulated genetically engineered cells are an effective means of long-term trophic factor supply into the adult rat brain and that the delivery of glial cell line-derived neurotrophic factor can sustain dopaminergic graft function and survival.

The copper chelator d-penicillamine delays onset of disease and extends survival in a transgenic mouse model of familial amyotrophic lateral sclerosis.

A subpopulation of familial cases of amyotrophic lateral sclerosis has been linked to mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). There is in vitro evidence that certain SOD1 mutants, in addition to their normal dismutation function, show increased ability of the enzyme to act as a peroxidase. This reaction is sensitive to inhibition by copper chelators. To test this hypothesis in vivo, we administered the copper chelator d-penicillamine to a transgenic mouse model of familial amyotrophic lateral sclerosis overexpressing a mutated form of human SOD1. We demonstrate that oral administration of d-penicillamine is able to delay the onset of the disease and extend the survival of these mice. Histological studies also showed a decreased loss of facial motor neurons in d-penicillamine-treated transgenic mice, corroborating the slower evolution of the disease in these animals. These results suggest that copper chelators may benefit patients with familial amyotrophic lateral sclerosis linked to mutations in the SOD1 gene.

Neuronal GDNF expression in the adult rat nervous system identified by in situ hybridization.

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor which has been purified on the basis of its ability to promote the survival of dopaminergic neurons in vitro. GDNF has subsequently been cloned and its sequence shown to be distantly related to transforming growth factor-beta (TGF-beta). To identify GDNF expressing cells in the adult rat brain, in situ hybridization using a digoxygenin (DIG)-labelled riboprobe has been performed. Our results show that GDNF mRNA is mainly expressed in neurons and that its synthesis is not restricted to dopaminergic areas. It is widely expressed in the cortex, the hippocampus, the striatum, the substantia nigra, the thalamus, the cerebellum and the spinal cord. Neuronal GDNF expression varies among brain regions as determined by the intensity of the in situ signal. Double labelling of the substantia nigra using tyrosine hydroxylase immunohistochemistry, associated with GDNF in situ hybridization, show that the majority of dopaminergic neurons express GDNF. The widespread expression of GDNF throughout the adult brain suggests that its administration in Parkinson's disease should be restricted to the altered structures, in order to avoid possible deleterious side effects.