Glial-cell-line-derived neurotrophic factor (GDNF), a recently cloned new member of the transforming growth factor-beta superfamily, promotes survival of cultured fetal mesencephalic dopamine neurons and is expressed in the developing striatum. There have, however, been no reports about effects of GDNF in situ. We have used the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces parkinsonian symptoms in man, to determine whether GDNF might exert protective or regenerative effects in vivo in the adult nigrostriatal dopamine system in C57/B1 mice. GDNF injected over the substantia nigra or in striatum before MPTP potently protects the dopamine system, as shown by numbers of mesencephalic dopamine nerve cell bodies, dopamine nerve terminal densities and dopamine levels. When GDNF is given after MPTP, dopamine levels and fibre densities are significantly restored. In both cases, motor behaviour is increased above normal levels. We conclude that intracerebral GDNF administration exerts both protective and reparative effects on the nigrostriatal dopamine system, which may have implications for the development of new treatment strategies for Parkinson's disease.

Dopamine (DA) neurotransmission has been implicated in several neurological and psychiatric disorders. The dopamine transporter (DAT) is highly expressed in dopaminergic neurons of the ventral mesencephalon and regulates neurotransmission by transporting DA back into the presynaptic terminals. To mediate restricted DNA recombination events into DA neurons using the Cre/loxP technology, we have generated a knockin mouse expressing Cre recombinase under the transcriptional control of the endogenous DAT promoter. To minimize interference with DAT function by preservation of both DAT alleles, Cre recombinase expression was driven from the 3' untranslated region (3'UTR) of the endogenous DAT gene by means of an internal ribosomal entry sequence. Crossing this murine line with a LacZ reporter showed colocalization of DAT immunocytochemistry and beta-galactosidase staining in all regions analyzed. This knockin mouse can be used for generating tissue specific knockouts in mice carrying genes flanked by loxP sites, and will facilitate the analysis of gene function in dopaminergic neurons.

Retinoic acid (RA), a retinoid metabolite, acts as a gene regulator via ligand-activated transcription factors, known as retinoic acid receptors (RARs) and retinoid X receptors (RXRs), both existing in three different subtypes, alpha, beta and gamma. In the intracellular regulation of retinoids, four binding proteins have been implicated: cellular retinol binding protein (CRBP) types I and II and cellular retinoic acid binding protein (CRABP) types I and II. We have used in situ hybridization to localize mRNA species encoding CRBP- and CRABP I and II as well as all the different nuclear receptors in the developing and adult rat and mouse central nervous system (CNS), an assay to investigate the possible presence of RA, and immunohistochemistry to also analyse CRBP I- and CRABP immunoreactivity (IR). RXRbeta is found in most areas while RARalpha and -beta and RXRalpha and -gamma show much more restricted patterns of expression. RARalpha is found in cortex and hippocampus and RARbeta and RXRgamma are both highly expressed in the dopamine-innervated areas caudate/putamen, nucleus accumbens and olfactory tubercle. RARgamma could not be detected in any part of the CNS. Using an in vitro reporter assay, we found high levels of RA in the developing striatum. The caudate/putamen of the developing brain showed strong CRBP I-IR in a compartmentalized manner, while at the same time containing many evenly distributed CRABP I-IR neurons. The CRBP I- and CRABP I-IR patterns were closely paralleled by the presence of the corresponding transcripts. The specific expression pattern of retinoid-binding proteins and nuclear retinoid receptors as well as the presence of RA in striatum suggests that retinoids are important in many brain structures and emphasizes a role for retinoids in gene regulatory events in postnatal and adult striatum.

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Cloning strategies were used to identify a gene termed glial cell line-derived neurotrophic factor receptor-␤ (GDNFR-␤) related to GDNFR-␣. In situ hybridization was then used to map cellular expression of the GDNF-related trophic factor neurturin (NTN) and GDNFR-␤ mRNA in developing and adult mice, and comparisons with GDNFR-␣ and RET were made. Neurturin is expressed in postnatal cerebral cortex, striatum, several brainstem areas, and the pineal gland. GDNFR-␤ mRNA was more widely expressed in the developing and adult CNS, including cerebral cortex, cerebellum, thalamus, zona incerta, hypothalamus, brainstem, and spinal cord, and in subpopulations of sensory neurons and developing peripheral nerves. NTN colocalized with RET and GDNFR-␣ in ureteric buds of the developing kidney. The circular muscle layer of the developing intestines, smooth muscle of the urether, and developing bronchiolae also expressed NTN. GDNFR-␤ was found in myenteric but not submucosal intestinal plexuses. In developing salivary glands NTN had an epithelial expression, whereas GDNFR-␤ was expressed in surrounding tissue. Neurturin and GDNFR-␤ were present in developing sensory organs. In the gonads, NTN appeared to be expressed in Sertoli cells and in the epithelium of the oviduct, whereas GDNFR-␤ was expressed by the germ cell line. Our findings suggest multiple roles for NTN and GDNFR-␤ in the developing and adult organism. Although NTN and GDNFR-␤ expression patterns are sometimes complementary, this is not always the case, suggesting multiple modi operandi of GDNF and NTN in relation to RET and the two binding proteins, GDNFR-␣ and GDNFR-␤.

The recently cloned, distant member of the transforming growth factor 13

Glial-cell-line-derived neurotrophic factor (GDNF) is a distant member of the transforming growth factor-beta family and has potent neurotrophic effects on several classes of neurons including dopamine neurons and motoneurons. Here, we have used in situ hybridization to describe the development of the cellular expression of GDNF mRNA pre- and postnatally. Consistent with dopaminotrophic activity, GDNF mRNA is expressed in the developing basal ganglia and the olfactory tubercle. It is also found in a thalamic nucleus, in neurons of the substantia innominata, in the developing Purkinje neurons and the developing locus coeruleus area, and in trigeminal brainstem nuclei. In the spinal cord, neuronal expression is found in Clarke's column. GDNF mRNA is also expressed in the dorsal horns during development. Additional GDNF mRNA expression in the head region includes the carotid body, the retina, the vibrissae, the inner ear, the ear canal, and epithelium in the nasal cavity. Prominent expression is also found in the developing teeth. The widespread expression of GDNF in developing skeletal muscle is consistent with trophic activity on alpha-motoneurons. The smooth muscle layers of the gastrointestinal tract are also strongly positive. A very strong signal is found in the outer mesenchyme of the developing metanephric kidney. We conclude that GDNF mRNA is expressed in many different cellular systems inside and outside the central nervous system during development, suggesting multiple functions of GDNF in the developing organism.

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