neurturin receptor (c-ret, GFRα-1 and α-2) mRNAs in postnatal rat brain: differential regional and temporal expression in hippocampus, cortex and cerebellum

Molecular Brain Research 73 Ž1999. 151–171 www.elsevier.comrlocaterbres

Research report

Localization of GDNFrneurturin receptor žc-ret, GFRa-1 and a-2 / mRNAs in postnatal rat brain: differential regional and temporal expression in hippocampus, cortex and cerebellum Tanya C.D. Burazin, Andrew L. Gundlach

)

The UniÕersity of Melbourne, Clinical Pharmacology and Therapeutics Unit, Department of Medicine, Austin and Repatriation Medical Centre, Heidelberg, Victoria 3084, Australia Accepted 7 July 1999

Abstract Recent studies have identified a multi-component receptor system for the neurotrophic factor, glial cell line-derived neurotrophic factor ŽGDNF. and its homolog, neurturin ŽNTN., comprising the signaling tyrosine kinase, Ret and multiple GPI-linked binding proteins, GDNF family receptor a-1 and a-2 ŽGFRa-1 and GFRa-2.. In the present study the localization of c-ret and GFRa-1 and GFRa-2 mRNAs was assessed in the developing rat brain from postnatal day 4 to 70 by in situ hybridization histochemistry, using specific w35Sx-labeled oligonucleotides. GFRa-1 and GFRa-2 mRNAs were differentially distributed throughout the brain at all ages studied, particularly in cerebral cortex, hippocampus, substantia nigra and regions of the thalamus and hypothalamus — both distributions overlapping but different to that of c-ret mRNA. C-ret mRNA was abundant in areas such as the lateral habenula, reticular thalamic nucleus, substantia nigra pars compacta, cranial motor nuclei, and the Purkinje cell layer of the cerebellum. GFRa-1 mRNA was abundant in dorsal endopiriform nucleus, medial habenula, reticular thalamic nucleus, pyramidal and granule cell layers of the hippocampus, substantia nigra pars compacta and in cranial motor nuclei. GFRa-2 mRNA was highly expressed in many regions including olfactory bulb, lateral olfactory tract nucleus, neocortical layers IV and VI, septum, zona incerta, and arcuate and interpeduncular nuclei. GFRa-2 mRNA was detected in the pyramidal cell layers ŽCA3. of hippocampus at P4 and P7, but was no longer detectable at P14 and beyond, including P70 Žadult.. GFRa-2 mRNA was also detected in Purkinje cells throughout the cerebellum in young postnatal rats, but was enriched in the posterior lobes at P28 and P70. These localization studies support evidence of GDNFrNTN as target-derived and autocrinerparacrine trophic factors in developing brain pathways and earlier suggestions of unique and complex signaling mechanisms for these factors via a family of receptors. Strong expression of GFRa-1 and GFRa-2 mRNAs in adult brain suggests possible non-trophic functions of GDNFrNTN, as described for other neurotrophins, such as brain-derived neurotrophic factor. q 1999 Elsevier Science B.V. All rights reserved. Keywords: GDNF; Neurturin; GPI-linked receptor; GFRa-1 and GFRa-2; Ret-tyrosine kinase; Development; Gene expression; In situ hybridization histochemistry

1. Introduction Glial cell line-derived neurotrophic factor ŽGDNF. was originally isolated based on its ability to support the survival of embryonic, dopaminergic neurons in vitro w30x and was also shown to potently increase survival of embryonic motor neurons w17x — increasing cell number, neurite outgrowth and choline acetyltransferase activity w71x. In

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vivo, GDNF almost completely rescues motor neurons from cell death following axotomy of the facial nerve in newborn rats w69x and following sciatic nerve transection in postnatal mice w39x. In fact, GDNF is now known to promote the survival of a wide variety of neuronal populations in both the central w1,13,24,28,29,33,35,57,66,67 x and peripheral w17,61x nervous systems. A structurally and biologically similar peptide, neurturin ŽNTN., was similarly identified on the basis of its ability to support the survival of rat superior cervical ganglion neurons in culture w27x; and NTN can also promote the survival of sensory neurons of the nodose and dorsal root ganglia w27x, prevent the death of embryonic dopaminergic and spinal

0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 9 . 0 0 2 1 7 - X

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T.C.D. Burazin, A.L. Gundlachr Molecular Brain Research 73 (1999) 151–171

motor neurons in vitro w24x and promote the survival of adult motor and midbrain dopaminergic neurons in vivo w19x. GDNF and NTN have been shown to mediate their actions through a multi-component, trans-membrane signaling receptor system composed of a GPI-linked binding protein ŽGFRa-1 or GFRa-2. and the Ret-tyrosine kinase signaling protein w2,4,22,24,50,55,58,60,62 x. GFRa-1 Žalso known as GDNFR-a . has been identified as the preferential ligand binding component of the multi-component receptor complex for GDNF w22,58x. Similarly, GFRa-2 Žalso known as TrnR-2, NTNR-a, RETL2 and GDNFR-b. is the GPI-linked binding protein of the NTN receptor complex w2,4,24,50,55x. Lower affinity interactions do occur between GFRa-1 and NTN and between GFRa-2 and GDNF, and it has been demonstrated that GFRa-2 and Ret form a multi-component receptor for NTN in embryonic motor neurons in vitro and in a human neuroblastoma cell line w24x. Whether other signaling components interact with GFRa s is as yet unknown. The broad tissue distribution of c-ret and GFRa-1 and GFRa-2 transcripts has been described by several groups using Northern blot analysis and in situ hybridization histochemistry and recently accounts have been published of the regional distribution of GDNF, NTN, GFRa-1 and GFRa-2, and c-ret mRNAs in adult mouse brain w15x and in adult and developing brain and peripheral tissues of the rat w64x. Thus, GFRa-1 and c-ret mRNAs are present in kidney, intestine, lung, dorsal root ganglia and brain w37,55,59,70x. In rat brain, high levels of GFRa-1 and c-ret mRNA are reported in the subiculum, lateral septum, dorsal endopiriform nucleus, habenula, hippocampal CA3 region, substantia nigrarventral tegmental area, cranial motor neurons and associated with the Purkinje layer of the cerebellum w14,59,64x. Similar studies detected GFRa-2 transcripts in brain, spleen, lung, kidney and gastrointestinal tract of adult rat and in fetal brain, intestine, placenta, lung and kidney w62x. In situ hybridization histochemistry was used to visualize GFRa-2 mRNA in the olfactory bulb, neocortex, septum, zona incerta, inferior and superior colliculi, entorhinal cortex and spinal dorsal horn of adult rat brain w24,62x and in spinal motor neurons and a subpopulation of dorsal root and trigeminal ganglia neurons, as well as in the adrenal cortex and capsule, kidney and gut in E17 rat embryos w24,55x. However, in light of the powerful effects of both GDNF and NTN on the survival of brain catecholaminergic w1,30x, cholinergic w66x, sensory, autonomic w5,27,33,61x and motor w17,29,69x neurons, and in an attempt to provide a better basis for further studies of the roles of these neurotrophic factors and their receptors in neuronal development and pathway regulation, in the present study in situ hybridization histochemistry of w35 Sx-labeled oligonucleotide probes was used to provide further details of the regional and cellular distributions of mRNAs encoding the GDNF- and NTN-binding proteins, GFRa-1 and GFRa-2,

and their signaling partner, Ret Žc-ret ., during postnatal development of the rat brain. A preliminary account of this research was presented at the Forum of European Neuroscience in 1998 w7x.

2. Experimental procedures 2.1. Animals These studies were carried out with the approval of the Austin and Repatriation Medical Centre Animal Welfare Committee and according to ethical guidelines issued by the National Health and Medical Research Council of Australia. Adult Sprague–Dawley rats were maintained on a 12 h light:dark cycle. Newborn rats were housed in litters with dam until weaning at 21 days postnatal, then allowed access to food and water ad libitum. 2.2. Tissue processing Rats aged 4, 7, 14, 21, 28 and 70 Žadult. days postnatal Ž n s 3 per group. were killed by decapitation, brains were removed, dissected, frozen over liquid N2 and stored at y708C. Coronalrsagittal sections Ž14 mm; n s 2r1 per age group. were cut on a cryostat at y168C and thawmounted onto poly-L-lysine-coated slides. Sections were dehydrated in ethanol Ž70%–100%. and delipidated in 100% chloroform for 10 min to decrease non-specific ‘myelin binding’ of oligonucleotides during hybridization, rinsed and stored in ethanol at 48C w6x. 2.3. Oligonucleotide probes Oligonucleotides were prepared complementary to nucleotides 11–49 and 239–277 of the rat c-ret cDNA w9x, nucleotides 737–775 and 1568–1606 of the rat GFRa-1 cDNA w22x and nucleotides 812–840 and 974–1012 of the rat GFRa-2 cDNA w59x. Screening of the sequences used against gene sequence databases ŽEMBL and Genbank; National Center for Biotechnology Information, NIH Blast Email Network Service. revealed homology Ž87%–100%. only with the appropriate rat, mouse and human c-ret and GFR receptor cDNAs. Oligonucleotides were labeled with a-w35 SxdATP Ž1200 Cirmmol, NEN; AMRAD-Biotech, Melbourne, Australia. using the tailing enzyme terminal deoxynucleotidyl transferase ŽBoehringer Mannheim, Sydney, Australia. to a specific activity of 1–2 = 10 9 dpmrmg. 2.4. In situ hybridization histochemistry In situ hybridization was carried out as described w6,68x. Briefly, sections were incubated overnight at 428C with w35 Sx-labeled probes w68x in hybridization buffer containing 50% formamide, 4 = SSC Ž1 = SSC; 0.15 M NaCl and 15 mM sodium citrate, pH 7.0. and 10% dextran sulphate

T.C.D. Burazin, A.L. Gundlachr Molecular Brain Research 73 (1999) 151–171

with 200 mM dithiothreitol. Slides were washed in 1 = SSC at 558C for 1 h, rinsed in 1 = SSC, 0.1 = SSC, then dehydrated in 70%, 95% ethanol. Sections were apposed to Kodak Biomax film ŽIntegrated Sciences, Melbourne, Australia. for 2–4 weeks and then dipped in photographic emulsion ŽIlford K5, diluted 1:1 with dH 2 O; Ilford-Anitec, Melbourne, Australia. and exposed for 8–20 weeks prior to development and examination. After processing sections were lightly stained with 0.01% thionin and coverslipped. Examination of staining on sections was done under an Olympus Vanox microscope and images were photographed with Kodak Tmax film. The specificity of the hybridization signal for each pair of c-ret, GFRa-1 and GFRa-2 probes used in this study was confirmed by hybridizing each probe separately, with the resultant production of identical patterns of distribution, as well as the combination of probes producing an amplification of signal intensity. The authenticity of hybridization for each pair of oligonucleotides was further determined by the addition of a 100-fold excess of unlabeled probes to eliminate specific hybridization and by comparison of hybridization patterns with reported distributions w59,64,70x. 2.5. Analysis Brain sections from animals of different ages were processed together and all animals of the same age were exposed to the same film to allow some degree of comparison of relative levels of hybridization signal within groups. Relative levels of c-ret, GFRa-1 and GFRa-2 mRNA hybridization reflected in resultant autoradiograms are not directly comparable, however, as experiments were conducted on different days, the relative specific activity of the probes differed slightly and film and photographic exposure times were not identical. The relative clarity of images from P4 to P21 brain sections is somewhat less than images from P28 and P70 sections, presumably due to the differing degrees of myelination and cellular organization at these ages and some distortion of tissues from younger animals during sectioning Žsee Section 3.. Relative abundance of hybridization for individual mRNA species was assessed using X-ray film images and the density of silver grains over neurons in discrete anatomical nuclei on emulsion-coated slides of serial coronal and sagittal sections, and was graded according to the scale of Ž0. no hybridization, equivalent to general tissuerfilm background; Žq. low or weak, scattered labeling in the region; Žqq . moderate levels of even labeling, Žqqq . high levels of labeling of distinct groups of cells in the nucleusrregion, Žqqqq . intense labeling of clustered cells in the nucleusrregion, for example equivalent to the level of c-ret mRNA in the pars compacta of the substantia nigra, GFRa-1 mRNA in the reticular thalamic nucleus or GFRa-2 mRNA in the lateral olfactory tract nucleus Žsee Section 3.. The atlases of Paxinos et al.

153

w40,41x were used as a reference to identify rat brain structures. It is important to note that it is possible, given the limits of sensitivity of in situ hybridization using w35 Sx-labeled oligonucleotide probes, that certain brain regions may contain low levels of mRNA encoding the GDNF receptor components studied, which were not detected in the current study. Non-specific labeling of white matter tracts by the GFRa-2 oligoprobes also limited our ability to study the potential expression of GFRa-2 mRNA in glial elements Žsee also Ref. w65x..

3. Results 3.1. OÕerÕiew of c-ret, GFR a-1 and GFR a-2 mRNA distributions in deÕeloping rat brain The localization and relative regional abundance of c-ret, GFRa-1 and GFRa-2 mRNAs was examined throughout the developing rat brain from postnatal days 4 to 70 ŽTable 1; Figs. 1–8.. The distribution of each mRNA species was heterogeneous and distinct. Predictably, several brain regions contained more than one species in an overlapping fashion, particularly c-ret and GFRa-1 Že.g., substantia nigra pars compacta and reticular thalamic nucleus., while the distribution of GFRa-2 mRNA was often complementary to that of GFRa-1 mRNA as observed in the cortex, thalamus and ventral midbrain. Some brain structures such as the reticular thalamic nucleus exhibited differential temporal expression of particular transcripts, while other areas such as the hippocampal pyramidal layers exhibited transient expression. A relatively restricted c-ret mRNA distribution was detected throughout the rat brain, at all ages studied ŽTable 1; Figs. 1–4.. Intense levels of expression were detected in the substantia nigra and ventral tegmental area, in cranial motor nuclei and associated with the cerebellar Purkinje cell layer. Levels of GFRa-1 mRNA in positively labeled regions were relatively consistent across the ages studied ŽFigs. 1–4.. High levels of labeling were detected in the dorsal endopiriform nucleus, medial Žbut not lateral. habenula, and in the reticular thalamic nucleus ŽTable 1; Figs. 1–6.. GFRa-2 mRNA was more widely distributed throughout rat brain than GFRa-1, at all postnatal ages studied ŽTable 1; Figs. 1–4, 7 and 8.. Intense GFRa-2 mRNA labeling was observed in cortical layer IV and high levels in layer VI ŽFigs. 1–3 and 7.. Intense labeling was also detected in the septum, lateral olfactory tract nucleus and in the interpeduncular nuclei ŽFigs. 1–3.. There was an obvious absence of GFRa-2 mRNA expression in cranial motor nuclei such as the facial nucleus ŽFig. 3., and in the hippocampus from P7 to P70 ŽFigs. 1–4.. The distribution of each of the GDNFrNTN receptor mRNA species in a number of different brain regions throughout postnatal development is described in more detail in the following sections Žsee Table 1; Figs. 1–8..

T.C.D. Burazin, A.L. Gundlachr Molecular Brain Research 73 (1999) 151–171

154

Table 1 Distribution and relative expression levels of c-ret, GFRa-1 and GFRa-2 mRNAs in developing and adult rat brain Brain Area

Postnatal Age P4

Olfactory bulb, glomerular layer Olfactory bulb, granule cell layer Cortical layer IV Cortical layer V Cortical layer VI Tenia tecta Caudate putamenr nucleus accumbens Dorsal endopiriform nucleusr claustrum Septum Diagonal band, vertical, horizontal Hippocampus, pyramidal layer, dorsal Hippocampus, pyramidal layer, ventral Dentate gyrus, granule cell layer Reticular thalamic nucleus Habenula, medial Habenula, lateral Dorsolateral geniculate nucleus Anterior hypothalamic area Arcuate nucleus Zona incerta Supramammillary nucleus Substantia nigra, compacta Substantia nigra, reticulata Substantia nigra, lateralis Ventral tegmental area Interpeduncular nucleus Central gray Raphe nuclei Superior colliculus, superficial layers Inferior colliculus Tegmental nucleus, dorsal, ventral Motor trigeminal nucleus Facial nucleus Nucleus ambiguus Hypoglossal nucleus Spinal trigeminal nucleus Cerebellum, Purkinje cell layer Cerebellum, granule cell layer

P7

P14

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Relative density of labeling was assessed independently by two investigators and categorized according to the scale: 0, no hybridization equivalent to tissuerfilm background; q, scattered labeling in the region; qq, moderate levels of labeling in the region; qqq, higher levels of labeling of distinct groups of cells in the nucleusrregion; qqqq, heavy, intense labeling of clustered cells in a nucleusrregion. ND, not determined. n s 2–3 rats in each age group. a In P4–P7 animals at the level of X-ray film autoradiograms it is not possible to clearly differentiate the Purkinje and granule cell layers and therefore values given represent an aggregate level of expression.

T.C.D. Burazin, A.L. Gundlachr Molecular Brain Research 73 (1999) 151–171

P21

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156

T.C.D. Burazin, A.L. Gundlachr Molecular Brain Research 73 (1999) 151–171

Fig. 1. Localization of c-ret, GFRa-1 and GFRa-2 mRNAs in P4 rat brain by in situ hybridization of w35 Sx-labeled oligonucleotide probes. X-ray film autoradiograms illustrate hybridization signal associated with ŽA,B. c-ret, ŽC,D. GFRa-1 and ŽE,F. GFRa-2 mRNA in coronal sections. Images reveal the relative and differential distribution of each transcript at the level of ŽA,C,E. hippocampus, reticular thalamic nucleus, hypothalamus and cortex and ŽB,D,F. substantia nigra, ventral tegmental area and superior colliculus. Scale bar s 1.5 mm.

3.2. Distribution of c-ret, GFR a-1 and GFR a-2 mRNAs in rat brain at postnatal day 4 3.2.1. c-ret mRNA At P4, intense labeling of c-ret mRNA was detected in the ventral tegmental area, substantia nigra, ventral hippocampus ŽFig. 1B. and cranial motor nuclei ŽTable 1.. Moderate to high levels of c-ret mRNA were detected in the glomerular layer of the olfactory bulb, the dorsal hippocampus ŽCA3. and the reticular thalamic nucleus ŽFig. 1A, Fig. 4A.. Low to moderate levels of c-ret mRNA were observed in tenia tecta, septum and diagonal band, arcuate nucleus, zona incerta, and throughout the remaining thalamus ŽFig. 1A,B; Table 1.. Low levels of labeling were observed in the medial and lateral habenula, anterior hypothalamic area, the superior and inferior colliculi, subiculum and entorhinal cortex, while c-ret mRNA was not detected in the dentate gyrus ŽFig. 4A., or the caudate putamenrnucleus accumbens ŽTable 1..

mRNA. Cranial motor neurons including those in the motor trigeminal, facial and hypoglossal nuclei, all displayed intense GFRa-1 mRNA labeling at P4. High to intense levels of GFRa-1 mRNA were detected in the substantia nigra pars compacta and ventral tegmental area and superficial layer of the superior colliculus ŽFig. 1D.. Moderate to high levels of GFRa-1 mRNA were detected in the dorsal endopiriform nucleus, lateral and medial septal nuclei, zona incerta, geniculate nuclei, anterior hypothalamic area including the paraventricular hypothalamic nucleus, arcuate and dorsal raphe nuclei, external cortex of the inferior colliculus, pontine tegmental nuclei and in the spinal trigeminal nucleus. Low levels were detected in the neocortex, the pyramidal and granule cell layers of the dorsal hippocampusrdentate gyrus ŽFig. 1C, Fig. 4C. and in the olfactory bulb, tenia tecta and diagonal band. GFRa-1 mRNA was not detected in the caudate putamenrnucleus accumbens, interpeduncular nucleus, or central gray at this age ŽTable 1..

3.2.2. GFR a-1 mRNA Intense GFRa-1 mRNA labeling was detected in the reticular thalamic nucleus and over the very densely packed neurons in the medial habenula ŽFig. 1C, Fig. 4C.. In contrast, the lateral habenula was almost devoid of GFRa-1

3.2.3. GFR a-2 mRNA At P4, intense GFRa-2 mRNA labeling was observed in the piriform and cingulate cortex and throughout layer IV of neocortex ŽFig. 1E, Fig. 7B., in the supramammillary and arcuate nuclei and in the interpeduncular nucleus.

T.C.D. Burazin, A.L. Gundlachr Molecular Brain Research 73 (1999) 151–171 Fig. 2. Localization of c-ret, GFRa-1 and GFRa-2 mRNAs in P21 rat brain. X-ray film autoradiograms illustrate the levels and distribution of hybridization signal associated with ŽA,B. c-ret, ŽC,D. GFRa-1 and ŽE,F. GFRa-2 mRNAs. Note the differential distribution of the transcripts in ŽA,C,E. dorsal endopiriform nucleus and lateral olfactory tract nucleus and in ŽB,D,F. the dorsolateral geniculate nucleus, subiculum, substantia nigrarventral tegmental area and interpeduncular nucleus. Scale bar s1.5 mm.

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158 T.C.D. Burazin, A.L. Gundlachr Molecular Brain Research 73 (1999) 151–171 Fig. 3. Localization of c-ret, GFRa-1 and GFRa-2 mRNAs in P70 rat brain. X-ray film autoradiograms illustrate the hybridization signal associated with ŽA–D. c-ret, ŽE–H. GFRa-1 and ŽI–L. GFRa-2 mRNAs. Images reveal the differential level of hybridization in ŽA,B,E,F,I,J. forebrain ŽC,G,K. midbrain nuclei and ŽD,H,L. brainstem. Scale bar s1.5 mm.

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Fig. 4. Differential distribution of c-ret, GFRa-1 and GFRa-2 mRNAs in hippocampus of postnatal day 4 and 70 rat brain. Re-photographed X-ray film images illustrate the levels of ŽA,B. c-ret, ŽC,D. GFRa-1 and ŽE,F. GFRa-2 mRNAs in hippocampus of ŽA,C,E. postnatal day 4 and ŽB,D,F. postnatal day 70 rat brain. ŽG–I. High-power brightfield ŽG. and corresponding darkfield ŽH. micrographs of emulsion-dipped sections reveal the distribution of GFRa-2 mRNA-associated silver grains over individual neurons in the dentate gyrus and CA3 region of the hippocampus. Scale bar ŽA,C,E. s 35 mm; ŽB,D,F. s 500 mm; ŽG–H. s100 mm; ŽI. s10 mm.

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Fig. 5. Differential distribution of c-ret, GFRa-1 and GFRa-2 mRNAs in subdivisions of the habenula at postnatal day 70. Low-power brightfield micrographs ŽA,D. and corresponding darkfield images of ŽB. c-ret, ŽC. GFRa-1 and ŽE. GFRa-2 mRNA-associated silver grains reveal distinct c-ret mRNA expression in the LHb and strong GFRa-1 mRNA expression in the MHb compared to the relative absence of GFRa-2 in either structure. Scale bar s 200 mm.

High levels of GFRa-2 mRNA labeling were detected in the granule layer of olfactory bulb, tenia tecta, reticular thalamic nucleus, superficial gray layers of the superior colliculus and dorsal and ventral tegmental nuclei. Moderate to high levels of GFRa-2 mRNA were detected in the lateral septum, diagonal band, central gray, dorsal raphe nucleus, presubiculum ŽFig. 1F. and inferior colliculus. Low levels were observed in the cranial motor, cuneiform and spinal trigeminal nuclei, as well as in the cerebellum ŽFig. 8A. and caudate putamenrnucleus accumbens ŽTable 1.. GFRa-2 mRNA was expressed in the hippocampus at P4, particularly in the pyramidal layer of the CA2–CA3 region at levels higher than at all subsequent ages ŽFig. 1E, Fig. 4E,G–I..

mRNA were detected in the pyramidal cell layer of the hippocampus, reticular thalamic nucleus and zona incerta, while lower levels were present in the dentate gyrus, medial and lateral habenula and arcuate nucleus. At P14, intense labeling of c-ret mRNA was detected in the lateral habenula and subdivisions of the substantia nigra, consistent with P4 and P7 ŽTable 1.. Moderate to high levels of c-ret mRNA labeling were observed in the Purkinje and granule cell layers of the cerebellum and low to moderate levels were detected in the ventral hippocampus and dentate gyrus. Scattered low levels of c-ret mRNA were detected in regions such as the septum and central gray and c-ret mRNA was not detected in the endopiriform nucleus or caudate putamen.

3.3. Distribution of c-ret, GFR a-1 and GFR a-2 mRNAs at postnatal day 7 and 14

3.3.2. GFR a-1 mRNA The distribution and relative intensity of GFRa-1 mRNA at P7 and P14 was also largely equivalent to that observed at P4 ŽTable 1.. High to intense labeling of GFRa-1 mRNA was observed in the dorsal endopiriform nucleus including the dorsal extension into the claustrum and cortical layer VI, in the septum, medial habenula,

3.3.1. c-ret mRNA The distribution and relative levels of c-ret mRNA in P7 and P14 brain were largely consistent with those observed at P4 ŽTable 1.. At P7, moderate levels of c-ret

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Fig. 6. Similar distribution of c-ret and GFRa-1 mRNAs in the adult substantia nigra. Brightfield ŽA,C. and corresponding darkfield micrographs of near–adjacent, emulsion-dipped sections reveal the distribution of ŽB. c-ret and ŽD. GFRa-1 mRNA-associated silver grains over cells in the SNC. Scale bar s 100 mm.

reticular thalamic and supramammillary nuclei and substantia nigra. Low to moderate levels of labeling were detected in the tenia tecta, islands of Calleja, vertical and horizontal diagonal band, paraventricular hypothalamic nucleus, zona incerta, arcuate and dorsolateral geniculate nuclei and in both the dorsal and ventral divisions of the hippocampusrdentate gyrus. GFRa-1 mRNA was in very low abundance in all layers of the neocortex at P7 and P14. At P14, low to moderate labeling of GFRa-1 mRNA was observed over, or adjacent to, the Purkinje cell layer of the cerebellum. 3.3.3. GFR a-2 mRNA Most regions positive for GFRa-2 mRNA at P7 and P14 were previously observed at P4 ŽTable 1; Fig. 8.. High to intense GFRa-2 mRNA labeling was detected in the cingulate cortex, cortical layers IV and VI, septum, arcuate and ventromedial hypothalamic nuclei, zona incerta and interpeduncular nucleus. Moderate to high levels of GFRa-2 mRNA labeling were present in the glomerular cell layer of the olfactory bulb, reticular thalamic nucleus, the anterior hypothalamic area and in the spinal trigeminal nucleus, while weak, scattered labeling was observed in the geniculate nuclei and medial and lateral habenula

ŽTable 1.. In contrast to the expression of GFRa-2 mRNA seen at P4, only moderate to low levels were detected in the ventral hippocampus and no GFRa-2 mRNA was detected in the dorsal or ventral dentate gyrus. Moderate to high levels of labeling were detected over the Purkinje layer of the cerebellum at P7 and P14 ŽFig. 8B,C.. GFRa-2 mRNA appeared to be at peak levels in the cingulate and frontal cortices, and in the granule cell layer of the olfactory bulb at P14. GFRa-2 mRNA was not detected in cranial motor nuclei at P14, or any later age. 3.4. Distribution of c-ret, GFR a-1 and GFR a-2 mRNAs at postnatal day 21 and 28 3.4.1. c-ret mRNA Generally, the pattern of c-ret mRNA labeling did not differ from earlier ages, with high to intense labeling in substantia nigra, ventral tegmental area and cranial motor nuclei ŽFig. 2A,B.. More concentrated c-ret mRNA labeling was detected over the Purkinje cell layer of the cerebellum at P21 and P28 than P14, with weak, scattered labeling in the granule cell layer ŽTable 1.. Moderate to high levels of c-ret mRNA were detected in the lateral habenula, higher than at P4. No c-ret mRNA was detected

162 T.C.D. Burazin, A.L. Gundlachr Molecular Brain Research 73 (1999) 151–171 Fig. 7. Localization of GFRa-2 mRNA in the cerebral cortex by in situ hybridization of w35 Sx-labeled oligonucleotide probes. Single or composite brightfield and darkfield images reveal the distribution of GFRa-2 mRNA-associated silver grains over cells in the Cg and Fr of ŽA–B. postnatal day 4 and ŽC–D. postnatal day 70 rats. Low- and high-power magnification images reveal GFRa-2 mRNA over individual neurons in layer IV of the adult cortex ŽE–G.. Scale bar ŽA–B. s150 mm; ŽC–D. s180 mm; ŽE–F. s100 mm; ŽG. s10 mm.

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Fig. 8. Localization and differential expression of GFRa-2 mRNA in rat cerebellum from postnatal day 4 to 70. ŽA,B–E. X-ray film autoradiograms of parasaggital sections reveal GFRa-2 mRNA-containing Purkinje cells of the cerebellum from P7 to P70, with particularly prominent expression in the posterior lobes at P28 and P70. ŽF–H. High-power images reveal GFRa-2 mRNA-associated silver grains over individual Purkinje cells in lobule 10 of the cerebellum at P70. Inhibition of authentic hybridization by the presence of a 100-fold excess of unlabeled probes Ž100=s. is demonstrated in X X X X sections at postnatal days 4 and 70 ŽA ,E .. Scale bar ŽA,A . s 500 mm; ŽB–D. s1 mm; ŽE,E . s 500 mm; ŽF,G. s100 mm; ŽH. s 5 mm.

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in the dorsal hippocampus at P21, in contrast to the moderate to high labeling intensity seen at P4 to P14, and very low levels of c-ret mRNA were detected throughout the rostro-caudal extent of the hippocampusrdentate gyrus ŽFig. 2B.. 3.4.2. GFR a-1 mRNA At P21 and P28, the distribution and relative levels of GFRa-1 mRNA observed were also similar to that at earlier ages ŽTable 1; Fig. 2C,D.. Moderate to high levels of labeling were detected in the dorsal endopiriform nucleus, reticular thalamic nucleus, dorsolateral geniculate nucleus, subiculum, raphe nuclei and low to moderate levels were detected in the zona incerta and dorsal and ventral hippocampusrdentate gyrus ŽFig. 2C,D.. Within the cerebellum, low to moderate labeling was associated with the Purkinje cell-granule cell layer interface. 3.4.3. GFR a-2 mRNA The relative intensity of GFRa-2 mRNA labeling detected in the reticular thalamic nucleus declined considerably during postnatal development, with high to intense GFRa-2 mRNA labeling evident at P4, high labeling at P7 and P14 and lower levels at P21 to P28 ŽTable 1; Fig. 1E, Fig. 2E.. Relative levels of GFRa-2 mRNA in the supramammillary nucleus and substantia nigra at P21 and P28 were comparable to the levels detected at P4 to P14. Regions that displayed moderate to high levels of GFRa-2 mRNA at P21rP28 included the olfactory bulb and horizontal limb of the diagonal band ŽTable 1., while the relative abundance of GFRa-2 mRNA declined from high to low–moderate levels in tenia tecta. The lateral habenula displayed moderate levels of labeling at P28 ŽTable 1.. In contrast to P4 and P7, very low GFRa-2 mRNA labeling was observed in the pyramidal layers and dentate gyrus of the hippocampus at P21 and P28. Low levels of GFRa-2 mRNA were observed in the granule cell and molecular layers of the cerebellum at this age, whereas high levels of GFRa-2 mRNA were associated with the Purkinje cell layer, particularly in the posterior lobes ŽFig. 8D.. 3.5. Distribution of c-ret, GFR a-1 and GFR a-2 mRNAs at postnatal day 70 (adult) 3.5.1. c-ret mRNA The distribution and relative intensity of c-ret mRNA expression at P70 was similar to that at younger ages ŽTable 1; Fig. 3A–D, Fig. 5B, Fig. 6A,B.. All cranial motor neurons continued to display intense labeling, as did the Purkinje cell layer of the cerebellum ŽFig. 3D. and the presumed, dopaminergic cells in the substantia nigra pars compacta ŽFig. 3C, Fig. 6B.. Moderate levels of c-ret mRNA were observed in the lateral habenula ŽFig. 4B, Fig. 5B., compared to low levels detected at P4 ŽFig. 4A. and strong labeling was present in cells of the gigantocellular and intermediate reticular nuclei of the medulla ŽFig. 3D..

C-ret mRNA was at low levels in the hippocampus and dentate gyrus at P70 ŽFig. 3B,C, Fig. 4B.. 3.5.2. GFR a-1 mRNA At P70, GFRa-1 mRNA levels were relatively more concentrated in the dorsal endopiriform nucleus than at earlier ages ŽFig. 3E.. Intense labeling was maintained in adult motor neurons ŽFig. 3H.. High levels were detected in the lateral and compact regions of the substantia nigra and the ventral tegmental area ŽFig. 3G, Fig. 6D., moderate levels in the raphe and dorsolateral geniculate nuclei and throughout the superior colliculus ŽFig. 3G., with low levels over the Purkinje cell layer of the cerebellum ŽTable 1; Fig. 3H.. 3.5.3. GFR a-2 mRNA GFRa-2 mRNA levels remained high at P70 in cortical layer IV including the cingulate and frontal cortices and in layer VI of neocortex ŽFig. 3I–K, Fig. 7C–G., and moderate in the mitral and granule cell layer of the olfactory bulb ŽTable 1.. In contrast to GFRa-1 mRNA, high levels of GFRa-2 mRNA labeling was detected in the Purkinje cell layer of restricted lobes of the cerebellum ŽFig. 8E.. The spinal trigeminal nucleus displayed low–moderate GFRa-2 mRNA labeling at P70, but the cranial motor nuclei were all devoid of labeling ŽTable 1; Fig. 3L.. All regions of the hippocampus including the dorsal and ventral pyramidal layers and dentate gyrus had very low levels or were devoid of GFRa-2 mRNA labeling ŽFig. 3J,K, Fig. 4F; Table 1.. At P70, low to moderate labeling was detected in the diagonal bandrventral pallidum and moderate to high labeling persisted in the lateral septum ŽFig. 3I.. GFRa-2 mRNA was not detected in the dorsal endopiriform nucleus or in piriform cortical regions, below the rhinal fissure ŽFig. 3I,J., but was expressed in the entorhinal cortex and subiculum ŽFig. 3K..

4. Discussion GDNF and NTN have been reported to exhibit potent and selective neurotrophic influences on various populations of neurons in rat brain via interaction with the GDNF family a-receptors and the tyrosine kinase receptor, Ret Žsee Section 1.. This in situ hybridization histochemical study compared the expression of c-ret, GFRa-1 and GFRa-2 receptor mRNAs during postnatal development of the rat brain to further establish any patterns of overlapping or complimentary expression of the different components of the GDNF signaling system in particular brain regions or pathways, and to determine whether expression patterns altered significantly over the postnatal period. Using oligonucleotide probes, c-ret, GFRa-1 and GFRa-2 mRNAs were localized in discrete regions of postnatal rat brain and the distribution patterns seen in adult brain were

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generally in good agreement with results published prior to, or during the course of, our studies w14,24,37, 55,59,62,64,70x. While generally, small changes, if any, were detected in the relative abundance of each mRNA species across the postnatal time-course, some examples of differential temporal expression during development were observed, in regions including the hippocampal formation. Further examples of the particular labeling patterns of c-ret, GFRa-1 and GFRa-2 mRNAs within key structures are discussed below, in relation to the distribution of GDNF and NTN mRNAs and possible functional interactions of the components of the GDNF receptor system and their ligands. 4.1. Distribution of GDNF and NTN mRNA in deÕeloping and adult rat brain In this and several previous studies, GFRa mRNAs were localized in areas of the brain in which GDNF- and NTN-responsive neurons are located Žsee Section 1. and consistent with the target-derived nature of these trophins, GDNF and NTN mRNAs are expressed in areas that are anatomical targets for responsive neurons. These areas have been demonstrated using several methods to be widely distributed in adult rat and mouse brain and include the striatumrnucleus accumbens, cingulate- and neo-cortex, hippocampus, septum, thalamus, hypothalamus, midbrain, brainstem and cerebellum w1,11,15,38,42,53,59x. More specifically and relevant to the current work, studies using radioactive and non-radioactive in situ hybridization have described somewhat contrasting patterns of strong GDNF mRNA labeling in several thalamic nuclei including the ventromedial and ventrolateral nuclei, in scattered cells of the granule cell layer of the cerebellum and the internal granule layer of the olfactory bulb w59x; and hippocampal pyramidal and dentate gyrus granule cells, medial septumrdiagonal band, medial habenula, nigral dopamine cells, Purkinje cells and cranialrspinal motor neurons w42x, respectively. In postnatal brain, Nosrat et al. w37x described highest levels of GDNF mRNA in striatum, principal sensory and motor trigeminal nuclei, and lower levels in hippocampus, cortex Žapart from a strong and distinct layer of cells in the adjacent subplate region. and in the cerebellum, although cerebellar expression is reportedly absent after postnatal day 10 w11x. Similarly, NTN mRNA was detected in adult and developing striatum, and developing cingulate and neo-cortex, septum, dentate gyrus and brainstem in the mouse w19,64x. Notably, the developing pineal gland contained high levels of NTN mRNA. In a more recent study, strong NTN mRNA expression was also observed in these areas and in the anteromedial and anteroventral thalamic nuclei and the paraventricular and supraoptic nuclei of adult brain w15x. Using more energetic w33 Px-labeled oligonucleotide probes, we have also examined the localization of GDNF mRNA throughout the developing and adult rat brain Žw8x;

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unpublished observations.. Consistent with similar early studies, GDNF mRNA was observed in only a few rat brain regions throughout the postnatal period, including the caudate putamen, mediodorsal and anteroventral thalamic nuclei, septum and spinal trigeminal nucleus, with weak, scattered labeling in the dentate gyrus, hippocampus and across the cerebellar layers at P4 and P7. GDNF mRNA was no longer detected in the dentate gyrus or hippocampus at P14 and thereafter. Moderate levels of GDNF mRNA persisted at P21–P28 in areas such as the inferior colliculus, but in contrast to the relatively high levels of GDNF receptor mRNAs present in adult brain, no GDNF mRNA was detected in the substantia nigra, or hippocampusrdentate gyrus at this age, using these oligonucleotide probes Žunpublished observations; see below.. 4.2. Differential c-ret, GFR a-1 and GFR a-2 mRNA expression in deÕeloping and adult rat brain GFRa-1 and GFRa-2 and c-ret transcripts were differentially expressed in several brain areas. For instance, GFRa-1 and GFRa-2 mRNA had markedly different distributions in areas including the cortex, developing hippocampus and thalamic nuclei. Adult substantia nigra-ventral tegmental area, reticular thalamic nucleus and cranial motor nuclei, all expressed GFRa-1 and c-ret mRNA, but not GFRa-2 mRNA. GFRa-1 mRNA was strongly expressed in the medial habenula, while lower levels of c-ret and GFRa-2 mRNA were detected in both the medial and lateral habenula. Notably, GFRa-2 mRNA levels were high in the interpeduncular nucleus which has reciprocal projections torfrom the habenula Žmedial.. GFRa-1 mRNA was detected in endopiriform nucleus, dentate gyrus and CA1–CA3 pyramidal layers of the hippocampus at all ages studied, whereas GFRa-2 and c-ret mRNAs were low and variable across development in these areas and low in adult brain. These and similar results Žsee below. suggest the existence of different modes of interaction between the Ret protein and the GFRa s — namely interactions in the same cell in regions that respond to GDNFrNTN, or interaction of Ret with receptors in the target neurons Žso-called cisrtrans interaction. w59,70x. In the olfactory bulb, specific patterns of mRNA expression for GDNF and its receptors have been previously reported, with expression of c-ret and GFRa-2 in the glomerular layer and GDNF and GFRa-2 in the granule cell layer of the adult brain. GFRa-1 mRNA was observed in the external and internal plexiform layers, while GFRa-2 was expressed in the mitral cell layer w59,60x. Thus, these authors suggested that GDNF molecules may mediate paracrine and reciprocal trophic actions between the different neuronal populations of the olfactory bulb w60x. In the present study, as early as P4, GFRa-2 mRNA transcripts were also detected within the lateral olfactory tract nu-

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cleus, an ‘amygdaloid’ region that has direct projections to and from the olfactory bulb. GDNF mRNA was also detected in the lateral olfactory tract nucleus at postnatal day 14 Žunpublished observations., suggesting that GDNF ŽNTN. trophic mechanisms function throughout the extended olfactory system. In the neocortex, low to negligible levels of c-ret and GFRa-1 mRNA were detected at most ages studied Žexcept for layer VI at P4–P14., whereas strong GFRa-2 mRNA expression was detected throughout the rostrocaudal extent of the neocortex in an anatomically striking pattern in layers IV and VI. Another recent study also reported that layer IV cortical neurons express very high levels of GFRa-2 mRNA and that cortical layer VI expressed GFRa-2 transcripts, while layers I–III did not w70x. These findings are consistent with the idea that incoming axons from corticothalamic projections are promoted to grow into the cortex across layers VI and V to end in layer IV, where the highest level of GFRa-2 expression is observed Žw70x; present study.. In the thalamus, c-ret and GFRa-1 mRNAs were detected within the reticular thalamic nucleus at all postnatal ages and were strongly expressed in the adult Žsee also Refs. w59,70x.. In contrast, we detected GFRa-2 mRNA in this area at P4 and P7 Žsee also Ref. w70x., but levels declined to near undetectable by P70. Interestingly, expression of GFRa-2 mRNA is reportedly maintained in the reticular nucleus of the adult mouse brain w15,64x. Both GFRa-1 and GFRa-2 mRNAs Žbut not c-ret . are expressed at low–moderate levels throughout development in the geniculate nuclei Žsee also Ref. w15x.. Unlike many other regions of the thalamus, the predominately GABAergic neurons in the reticular thalamic nucleus do not express GDNF or NTN mRNA Žunpublished observations; w1,15,53,59,64x., but importantly this region is reciprocally connected with all other thalamic nuclei w43x and presumably neurons in this area obtain the appropriate trophic factor via retrograde transport from these other regions, including the anteromedial, anteroventral, ventromedial and ventrolateral nuclei which contain GDNF andror NTN mRNAs Žproteins?. w15,59x. In the hippocampal formation c-ret and GFRa-2 mRNAs were strongly expressed in CA3 pyramidal cells at P4, while GFRa-1 mRNA was only weakly abundant. Weak scattered GDNF mRNA expression was detected in the dentate gyrus and hippocampus at P4 and P7 which was not evident at P14 or later postnatal ages Žunpublished observations; w42,59x.. By adulthood, c-ret and GFRa-2 mRNAs were undetectable, while GFRa-1 mRNA was prominent in the dentate gyrus and all CA fields of the hippocampus. In fact, GFRa-2 mRNA was no longer evident in the hippocampus by P14. In vivo studies have indicated that cholinergic neurons of the basal forebrain w66x, as well as thalamic and hippocampal neurons w31x, benefit from treatment with GDNF prior or subsequent to experimental lesioning. Is it presumed therefore, that the

septal Žcholinergic. projections into the hippocampus mature at P4–P7 and that our results are consistent with the importance of the GDNF family of ligands and receptors in the developmental establishment and maturation of this and other pathways Že.g., from regions such as the mammillary nuclei w16x which contain high levels of GFRa-2 mRNA.. The significance of the substantial expression of GFRa-1 in adult hippocampus is still unknown, however, the increased expression of GDNF and GFRa-1 and GFRa-2 mRNAs in the hippocampus after kainate administration is related to excitatory stimuli and suggests that GDNF signaling may play a role in the maintenance of hippocampal structurerfunction, and post-activation plasticity w20,60x Žsee below.. In the habenula, very high levels of GFRa-1 mRNA were consistently observed in the medial nucleus Žsee also Ref. w14x., whereas GFRa-2 and c-ret mRNA levels in this area and the adjacent lateral region were generally low, although c-ret mRNA was present in moderate to high levels from P14 to P70 in lateral habenula Žsee also Ref. w59x.. At present it is unclear whether GDNF Žor NTN. are expressed in the habenula, as reports of both high and negligible mRNA levels in this region can be found in the literature, although different methods of detection were used in these studies w15,42,59,64x. Interestingly though, the habenula is thought to innervate the pineal gland w45,46x and the pineal expresses high levels of NTN Žand GDNF. during postnatal development w53,64x. The habenula also sends efferent pathways to various brain regions with a particularly prominent projection from the medial habenula to the interpeduncular nucleus w18,54x, which, in turn, was found to contain very high densities of GFRa 2 mRNA expression, but unknown levels of GDNFrNTN. The lateral habenula projects to a more diverse range of structures than the medial nucleus, including the substantia nigra pars compacta, the raphe nuclei and, importantly, several thalamic fields w18,54x, that are known to express GDNF. In the basal ganglia, c-ret and GFRa-1 mRNAs Žbut not GFRa-2. were very strongly expressed in the substantia nigra pars compacta and ventral tegmental area throughout postnatal development, relative to a lack of expression in the dopaminergic terminal regions in the caudate putamen, nucleus accumbens and olfactory tubercle Žbut see Refs. w19,37x.. However, in line with the target-derived nature of GDNF trophic actions on midbrain dopaminergic neurons, GDNF mRNA has been consistently detected throughout the developing and adult caudate putamen, nucleus accumbens, olfactory tubercle and segments of the globus pallidus w15,38,42,53,59x. Furthermore, GDNF mRNA has also been reported in the pars compacta and lateralis of the substantia nigra w42x. NTN is also expressed in developing basal ganglia, both in ventral midbrain and forebrain regions w19,64x. In the cerebellum, c-ret and GFRa-1 mRNAs were detected in the immature granulerŽPurkinje. cell layer,

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while GFRa-2 mRNA was present in presumed Purkinje cells from P4 to P70. Higher levels of GFRa-2 mRNA were detected in Purkinje cells in the posterior vermis and hemispheres than in the anterior vermis in mature animals, in line with previous findings on other transcripts from this and other laboratories Žsee Refs. w47,49x and references therein.. Such spatial differences are consistent with Purkinje cells in the posterior lobules being derived from different precursor cell populations, with apparent resultant differences in phenotype w3x. In an earlier study also using oligonucleotide probes, strong labeling of both GFRa-1 and c-ret mRNAs was reported in a narrow band at the external edge of the granule cell layer over cells directly adjacent to Purkinje cells, but these authors failed to detect mRNA for either of these GDNF receptors in Purkinje cells of adult animals, whereas GFRa-1, c-ret and GDNF mRNAs were detected in the deep cerebellar nuclei w59x. In the same study, GDNF expression was also reported in scattered cells of the granule cell layer. Similarly, in adult mouse, only GFRa-2 was expressed in Purkinje cells, while c-ret and GFRa-1 were expressed in adjacent, presumed basket or glial cells w15x. GFRa-2 mRNA was also expressed in granule cells and all three receptor transcripts were detected in deep cerebellar nuclei of mouse. Using RT–PCR, postnatal expression of GDNF in cerebellum was reported to be high at P0 and detectable in the adult, but absent at P10 w11x. GDNF has been shown to have potent and specific effects on Purkinje cells in mixed cultures of embryonic cerebellum w35x, being reportedly a very potent survival and differentiation factor for cultured Purkinje cells, presumably due to a specific target-derived trophic factor mechanism Ži.e., in vivo, GDNF is derived from deep cerebellar nuclei, the targets of Purkinje cell axons, or is secreted by granule cells w11,35x.. These results are consistent with the localization data described. Prominent GFRa-1 and c-ret mRNA expression is detected in all cranial motor nuclei across all ages studied, whereas GFRa-2 mRNA expression is absent, consistent with a more prominent role for GFRa-1-linked signaling than GFRa-2 in the development and survival of motor neurons w8,39,69x Žsee below.. 4.3. Regulation of GDNFr NTN receptor component expression in experimental models To better understand the possible role Žs . of GDNFrNTN-GFR systems in the adult brain, attempts have been made to examine the regulation of GFRa-1 and a-2 expression. For example, Trupp et al. w60x reported that although expressed below detection levels in adult hippocampus, GFRa-2 mRNA expression was induced by kainic acid treatment, which also augments GFRa-1 and c-ret expression w44,59x, and while GFRa-2 mRNA was increased in certain thalamic nuclei, GFRa-1 expression was not w60x. These and other similar findings suggest

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regionally- and receptor-specific, activity-regulated trophic responses that could play a role in neuronal plasticity Žsee also Ref. w25x.. Furthermore, in a model involving facial nerve axotomy, levels of both c-ret and GFRa-1 mRNAs were increased in the ipsilateral facial nucleus 1 and 3 days after unilateral facial nerve crush and resection w8x, while GDNF and GFRa-2 mRNAs were not detected in normal or injured facial motor neurons Žunpublished observations.. Similar studies have also shown an up-regulation of c-ret and GFRa-1 mRNA expression in spinal motor neurons and increased GFRa-1 mRNA expression in Schwann cells in sciatic nerve following sciatic nerve transection, along with increases in GDNF expression in the distal nerve and axotomized muscle w36,59x. These data suggest a specific and unique mechanism of trophic support involving an insoluble gradient of GDNF coupled to GFRa-1 for utilization by regenerating motor neurons via Ret signaling w59x. In a more ‘physiological model’ that does not involve neuronal damage, we have also examined the possible regulation of GFR-a 2 expression by cortical spreading depression, a benign, depolarizationrdepression stimulus that is associated with considerable cortical neurochemical plasticity w51,52x. Spreading depression has been shown to produce a protein synthesis-dependent protection of cortical neurons against a delayed, subsequent ischemic episode. However, unlike another retrogradely transported neurotrophic factor, BDNF and its receptor, trk B w26,51x, GDNF and GFRa-2 mRNA levels were not altered at any time examined from 1 h to 4 weeks after spreading depression ŽBurazin, Shen and Gundlach, unpublished observations.. However, GDNF has been reported to diminish the volume of cortical infarction in young and aged rats after middle cerebral artery occlusion w23,63x and a 10-min global forebrain ischemia induced elevated expression of GDNF in dentate gyrus, GFRa-1 throughout hippocampus and GFRa-2 in parietal cortex w25x. These findings suggest that the up-regulation of these trophic factor ligandrreceptor systems is associated more with pathological, rather than physiological perturbations. In this regard, a recent report revealed high levels of NTN mRNA in magnocellular neurons of the hypothalamic paraventricular and supraoptic nuclei w15x and we are currently exploring the possible regulation of NTN and GFRa mRNAs after osmotic stimulation of these cells in salt-loaded rats Žcf. Refs. w10,48x.. 4.4. Future directions — new members of the GDNF-like trophic factor and GFR a family It will be important to further examine the regulation of GFR receptor expression using other experimental models and to look for regulation of GFRs, along with Ret, at the protein level using antibodies that are specific for each

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receptor subtype. In addition, a third member of the GFRa family has been isolated with sequence homology to both GFRa-1 and GFRa-2 and has been termed GFRa-3 w21,32,60,65x. GFRa-3 mRNA was strongly expressed in mouse embryonic tissues at E11–E19 w21,65x, including Schwann cells in the peripheral nervous system and peripheral ganglia and nerves, after which its expression decreased to become very low in adult animals w65x. GFRa-3 mRNA, examined by Northern blot hybridization, was detected in adult rat kidney, spleen, lung, liver and heart, but was undetectable in adult rat brain w21x. In a separate study however, GFRa-3 mRNA expression was reported in the adult rat olfactory tubercle, cerebellum and hippocampus using sensitive riboprobes w32x. Unlike GFRa-2, GFRa-3 does not appear to bind GDNF directly, but was capable of crosslinking GDNF to Ret when both receptors were coexpressed in COS cells w60x. More recently, a fourth member of this receptor family — GFRa-4 — has been cloned that is more closely related to GFRa-1 and GFRa-2 than GFRa-3, and is reported to be expressed in distinctive patterns in brain and other tissues in the developing chicken w56x. Persephin is a novel neurotrophic factor that is 40% identical to GDNF and NTN that supports the survival of motor neurons in culture and in vivo following sciatic nerve axotomy w34x, and it has recently been shown to be the preferred ligand for the functional complex of GFRa-4 and Ret tyrosine kinase w12x. The current study did not investigate the postnatal distribution of NTN mRNA, since only human and mouse sequences were available at the time and oligonucleotide probes seem to be commonly insensitive at detecting what are apparently very low levels of these trophic factor transcripts Žsee Ref. w64x.. Nonetheless it will be of interest to further compare the relative distributions of this family of rare, but potent ligands, with their more abundant, multicomponent receptors, once all the appropriate molecular tools become more widely available Žsee e.g., Refs. w15,19,32x.. 5. Conclusions C-ret, GFRa-1 and GFRa-2 mRNAs have an overlapping andror complementary distribution throughout the CNS and are found in several brain regions that either express GDNF and NTN mRNAs or project to such areas. Strong GFRa-1 and GFRa-2 mRNA expression in normal adult rat brain suggests additional non-trophic functions of GDNF and NTN systems in the CNS. These and previous findings provide strong evidence that GDNF and NTN act as target-derived trophic agents and also act in an autorparacrine fashion. The results are also consistent with GFRa-1 and GFRa-2-independent actions of Ret Žsee new ligands and receptors above., and possible GFRa-1 and GFRa-2 signaling via receptors other than Ret. It has been noted previously that GDNF has a more

pronounced biological effect on lesionedrdamaged neurons than on intact neurons and the limited regulation of GDNF expression levels suggests that GDNF, ŽNTNrpersephin.-related trophic circuits may be dynamically regulated predominately at the level of the multi-component receptors in the CNS w59x. 6. Abbreviations used in figures 7

Facial nucleus

IPN

100x

100-fold excess L1-6 of unlabeled probes CA1–3 CA1–3 pyramidal LHb layers of hippocampus Cb Cerebellum LOT cc

Corpus callosum

Cg CG

Cingulate cortex Central gray

CPu

Caudate putamen

DC

Dorsal cochlear nucleus Dorsal cortex of the inferior colliculus Dorsal endopiriform nucleus Dentate gyrus

DCIC

DEn

DG DLG

Dorsolateral geniculate nucleus Fr Frontal cortex GFRa-1 GDNF family receptor, a-1 GFRa-2 GDNF family receptor, a-2 Gr Granule cell layer of the cerebellum Hb Habenula

Interpeduncular nucleus Cortical layers I–VI Lateral habenula

Lateral olfactory tract nucleus LS Lateral septal nucleus MHb Medial habenula Mol Molecular layer of the cerebellum MS Medial septal nucleus P4–70 postnatal days 4–70 Pir Piriform cortex

Pk

S

Purkinje cell layer of the cerebellum Reticular thalamic nucleus Subiculum

SC SN

Superior colliculus Substantia nigra

SNC

Substantia nigra, compacta Substantia nigra, reticulata

Rt

SNR

SuG

Hip

Hippocampus

VTA

Hyp IC

Hypothalamus Inferior colliculus

wm

Superficial gray layer of superior colliculus Ventral tegmental area White matter tract

T.C.D. Burazin, A.L. Gundlachr Molecular Brain Research 73 (1999) 151–171

Acknowledgements w16x

This research was supported by a Fellowship grant to ALG from the National Health and Medical Research Council ŽNHMRC. of Australia and grants from the Sylvia and Charles Viertel Foundation and the Austin and Repatriation Medical Research Foundation. TCDB is the recipient of a NHMRC Australia Dora Lush ŽBiomedical. Postgraduate Research Scholarship.

w17x

w18x w19x

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