Noradrenergic Neurons in the Locus Coeruleus TrkA, Transport NGF, and Respond to NGF Christopher S. von Bartheld,’ Mark Bothwell’ ‘Department of Physiology of Developmental Biology,
Received July I, 1994; revised Sept. 21, 1994; accepted Sept. 27, 1994. We thank Dr. H. Tanaka (Kumamoto University, Japan) for the gift of p75 antibody and Dr. Ron Lindsay (Regeneron) for the gift of BDNF and NT-3. We also thank Dr. Ed Rubel (University of Washington) for generous permission to use microscopic equipment. Mr. Anders BBckstrijm kindly shared sequence information, and Dr. David Holtzman kindly shared data prior to publication. We thank Dr. Anton Reiner for critical comments on the manuscript. This study was supported by NIH Grants HD 29177 (C.S.V.B.) and NS 30305 (M.B.), German Science Fobndation Grant 489/1-l (A.S.), the Medical Faculty of Uppsala University (R.W.), and the Swedish Natural Science Research Council (B-BU 04024-313) (T.E.). Correspondence should be addressed to Christopher S. van Bartheld, Department of Physiology and Biophysics SJ-40, University of Washington, Seattle, WA 98195. “Present address: Department of Anatomy and Cell Biology, -. University of Heidelberg, Germany. bPresent address: Department of Molecular Biology, Karolinska Institute, CMB Stockholm, Sweden. Copyright 0 1995 Society for Neuroscience 0270.6474/95/152225-15$05.00/O
of Birds Express
and Biophysics, University of Washington, Seattle, Washington Biomedical Center, Uppsala University, Uppsala, Sweden
The chicken locus coeruleus contains a population of noradrenergic neurons which express the neurotrophin receptor ~75 (von Battheld and Bothwell, 1992). To determine which neurotrophin may regulate the development of noradrenergic neurons in the chicken locus coeruleus, expression of trk receptors, retrograde transport of neurotrophins, and responses to NGF were examined. P7Bexpressing noradrenergic neurons were found to project to the basal forebrain. They transport radio-iodinated NGF after injections into this target. The retrograde transport of NGF is specific to the noradrenergic neuronal population as evidenced by double labeling with antibodies against dopamine+-hydroxylase. The same neuronal pop ulation expresses trkA receptor mRNA. The size of noradrenergic neurons in the locus coeruleus proper, but not in the nucleus subcoeruleus, is significantly increased after injections of NGF into the telencephalon, consistent with the hypothesis that target-derived NGF provides trophic support. Noradrenergic coeruleus neurons are rescued from toxic effects of 6-hydroxydopamine injected into the telencephalon when NGF is injected into the midbrain. NGF has no rescue effect when it is coinjected with 6-hydroxydopamine into the telencephalon. In explant or dissociated cultures, noradrenergic coeruleus neurons do not respond to elevated levels of NGF with increased neurite outgrowth. Taken together, these results suggest that NGF plays a role in the development and maintenance of noradrenergic coeruleus neurons in the chick brain. The data also support our previous conclusion that major species differences exist between birds (chicken) and mammals with regard to
98195 and *Department
trophic regulation of presumptive homologous neuronal populations. [Key words: locus coeruleus, noradrenaline, NGF, trkA, NGF receptor, Oil, chick embryo, retrograde transport, neurotrophin, dopamineg -hydroxylase, 6-hydroxydopamine, neuroprotection, in situ hybridization]
Several types of neuronshave been characterized on the basis of morphological and functional criteria. Neuronsof the “reticular type” are believed to serve integrative functions in the brain (Ramon-Moliner and Nauta, 1966; Hobson and Brazier, 1980; Mesulam et al., 1989) and are known for their remarkableregenerative capacities(Bjorklund et al., 1975). Prototypical neurons of the reticular type are those of the locus coeruleus.The noradrenergiccoeruleusprojections to the forebrain have been implicated in memory processing (Kety, 1970; Stephenson, 1991), and particularly in attentional aspectsof learning (Mason, 1984). Neuronal populations of the reticular type maintain high levels of the neurotrophin receptor ~7.5in the mature brain, for example, the cholinergic neuronsof the basalforebrain (Hefti, 1986; Thoenen et al., 1987; Olson, 1993) and the locus coeruleus (von Bartheld et al., 1991a; von Bartheld and Bothwell, 1992). These data suggestthe possibility that neurotrophic factors releasedby the targetselicit sprouting and plasticity of locus coeruleus neurons. Trophic factors thus may be involved in mechanismsunderlying integrative functions, including synaptic plasticity and processesrelated to memory (Purves, 1988;Thoenen, 1991). Recently, we have shown that noradrenergic neuronsin the locus coeruleusof the posthatchchicken maintain high levels of mRNA for the neurotrophin receptor p75 (von Bartheld and Bothwell, 1992). Most actionsof the neurotrophinsare mediated by a different classof receptors,tyrosine kinasereceptors(trks). NGF acts selectively on trkA, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) on trkB, and neurotrophin3 (NT-3) on trkC. The ~75 receptors, which bind all four neurotrophins with similar affinities, may interact with the various trk receptorsto enhanceligand specificity and to facilitate signal transduction (Bothwell, 1991, 1995; Chao, 1992; Meakin and Shooter, 1992). Expressionof the ~75 receptor suggeststhat one of the neurotrophinsmay serve as a trophic factor for the avian coeruleusneurons. We now have investigated the potential neurotrophic regulation of noradrenergiccoeruleusneuronsin the developing chicken. We have concentratedon late embryonic stages,whentargets of the locus coeruleusneuronsare accessiblein ovo, and up to the age of hatching when the noradrenergic locus coeruleus
of NGF and NGF
plays an important role in memory processing and imprinting (Davies et al., 1985). This phenomenon has been studied extensively in hatchling chicks (Andrew, 1991; Stephenson, 1991). The aim of the present study is to clarify if coeruleus neurons possess specific, signal-transducing receptors (trks) for neurotrophins, and to test if neurotrophins have effects on coeruleus neurons. We report that the noradrenergic population of the locus coeruleus in chicken embryos heavily expresses the trkA receptor in addition to the p75 neurotrophin receptor, and that the noradrenergic coeruleus neurons retrogradely transport radio-iodinated NGF from the basal forebrain. Administered NGF caused a significant increase in the size of noradrenergic coeruleus neurons in vivo and protected these neurons from toxic effects of 6-hydroxydopamine. These data are consistent with the hypothesis that NGF plays an important role in the development and maintenance of noradrenergic neurons in the chicken locus coeruleus. Preliminary results of this study have been presented in abstract form (Bothwell et al., 1993; von Bartheld et al., 1994b).
Chick embryos Fertilized chicken eggs were purchased from H&N International (Redmond, WA) or Waldens Poultry (Borje, Sweden). They were incubated in a force-draft incubator at 37-38°C and 60-70% humidity. All animals were staged according to Hamburger and Hamilton (1951). Stages are indicated as days of incubation. A total of about 330 embryos and 3 hatched, 2 week old chicks were used. In situ hybridization Embryos at ages E5, E9, and El8 were frozen over liquid nitrogen and stored at -80°C until used. Serial transverse sections (10 pm) through the head or brain (E18) were cut on a cryostat and thaw mounted onto poly+lysine coated slides (50 pg/ml). The sections were air dried and stored at -80°C prior to use. To detect mRNA for trk receptors in tissue sections, synthetic oligonucleotide probes complementary to isolated chicken trkA, trkB and trkC cDNAs were used (Scandinavian Gene Synthesis, Koping, Sweden). The oligonucleotide sequence for chicken trkA is 5’-CGG ATG GCT CCT CGG CGT GAA GCT GAA AGG GTT ATC CAT GAA GCG GCC-3’, corresponding to amino acid residues 376391 in the human trkA sequence (Backstrom et al., unpublished observations). The oligonucleofide sequence for the kinase-containina chicken trkB is 5’-GGG CAG CAT GGT GTG ACC CCC AAC CCT ?+TA GTA GTC TGT GCT GTA CAC-3’. The sequence for the kinase-containing chicken trkC probe is 5’-AGC GGG TCA CCA TCA CCA CAC ACA CCA TAG AAC TTG ACG ATG TGC TCA-3’ (for the non-kinase-specific trkC oligosequence and other details, see Williams et al., 1993, 1994). The oligonucleotide probes (50 ng) were labeled at the 3’-end with deoxyadenosine 5’[email protected]
(Amersham, Arlington Heights, IL) using terminal deoxynucleotidyl transferase (Promega, Madison, WI) to a specific activity of approximately 1 X 10’ cpm/pg. The probes were purified on Nensorb columns (Du Pont, Wilmington, DE) prior to use. Hybridization was performed at 42°C for approximately 15 hr in a humidified chamber with 100 pl of hybridization cocktail. The hybridization cocktail contained 50% formamide, 4 X SSC, 10% dextran sulfate, 0.5 mg/ml yeast tRNA, 0.06 M dithiothreitol, and 0.1 mg/ml sonicated salmon sperm DNA. As a control for the specificity, control hybridization solutions included the addition of unlabeled probe at 100X excess. In addition, controls for cross-hybridization have been performed with 100X excess of each of the other two unlabeled probes included in the hybridization solution. After hybridization, the slides were washed for 15 min in 1 X SSC and 0.05% sarcosyl, three times for 15 min each in 0.5 X SSC at 55°C and twice for 1 min each in cold, RNase-free water. The sections were dehydrated in ethanol, air dried, and coated with Kodak NTB-2 p...