The cell suspension, containing 95% neurons, was collected by aspiration, and the number of cells was counted inside a hemocytometer

The cell suspension, containing 95% neurons, was collected by aspiration, and the number of cells was counted inside a hemocytometer. and Barde, 1996). With regard to neuronal survival, however, the loss of trkC function is definitely less dramatic than that of NT3, and detailed analyses with neurons isolated from (?/?) mice have exposed that in the absence of trkC, NT3 uses trkA and trkB instead (Davies et al., 1995). Although amazingly useful for analyzing the relevance of neurotrophin receptor genes, such studies do not define the various molecular components participating in neurotrophin binding at the surface of neurons. studies, notably with PC12 cells, possess helped to define the molecular nature of high-affinity specific NGF binding sites (Chao et al., 1986; Radeke et al., 1987; Hempstead et al., 1991). These studies led to the conclusion that p75NTR, expressed at appropriate levels, increases the ability of the NGF receptor trkA to detect low concentrations of NGF (Benedetti et al., 1993; Barker and Shooter, 1994) and confers improved specificity of NGF binding for trkA (Davies et al., 1993; Lee et al., 1994). With regard to neurotrophins other than NGF, only a few studies to date possess examined the molecular nature of neurotrophin binding sites on neurons. With BDNF and NT3, previous cross-linking experiments have recognized trkB and trkC as components of high-affinity receptors on embryonic neurons (Escandn et al., 1993; Rodrguez-Tbar et al., 1993; Escandn et al., 1994), but ACTN1 the participation of p75 in the formation of such sites, if any, remains unclear. The chick sympathetic neurons are one human population of neurons that are amenable to receptor composition analyses. Like dissociated chick retinal cells, used previously for related purposes (Rodrguez-Tbar et al., 1993), they can be isolated in adequate LY223982 figures. Sympathetic neurons can be analyzed both after acute dissociation and in cell tradition after they have formed long processes. This is of interest, because Total RNA was from sympathetic ganglia and neuronal cell cultures, with use of the RNEasy kit (Qiagen, Hilden, Germany), and treated with DNase. One microgram of total RNA was reverse-transcribed with 0.5 g of oligo-dT12C18 (Pharmacia, Piscataway, NJ) and Superscript II Reverse Transcriptase (Life Sciences, Hialeah, FL) for 1 hr at 45C. Transcripts of 30C60 ng total RNA were amplified (94, 62, 72C, 30 min each) with 0.1 m primer (observe below), 0.33 mm dNTP (Pharmacia), and 0.4 l (2 U) Ampli-(Perkin-Elmer, Emeryville, CA) in a total volume of 60 l. SYBR Green (Biomol, Plymouth Achieving, PA) stained amplificates were analyzed by 1% agarose gel electrophoresis.amplificates were transferred onto Hybond N membrane (Amersham-Buchler, Braunschweig, Germany) and hybridized against a32P-labeled full-length and kinase truncation transcripts (Garner and Large, 1994) for 24C26 cycles with 5-AGCCCCACCAATGACAAAATGC-3 and 5-CTCCCAGAGGATCACCCCG-3, chickkinase deletion transcripts (Garner and Large, 1994) for 34C36 cycles with 5-CCAATAAGCCTCCCTGGACATTCC-3 and 5-CCCTGTTATCTGGTGACAGAAAACC-3, and chick (Large et al., 1989) for 31C33 cycles with 5-CCTGCCTGGACAGTGTGAC-C-3 and 5-TCTGCCAGGGTGGTGGCC-3. Amplified fragments were isolated from agarose gels and sequenced with the amplification primers on a sequencing automate (Applied Biosystems 373A; Applied Biosystems, Foster City, CA). Recombinant mouse NT3 and BDNF produced in a Vaccinia virus-based manifestation system were utilized for iodination (G?tz et al., 1992). Chinese Hamster Ovary (CHO)-derived human recombinant protein was utilized for radiolabeling of NGF. For biological assays and inhibition of binding, neurotrophins from the following sources were used: mouse NGF from salivary glands of adult male mice (purified relating to Suda et al., 1978), recombinant human being NGF (Genentech, San Francisco, CA), Vaccinia-derived mouse recombinant BDNF, human being recombinant BDNF indicated in CHO cells (Genentech), Vaccinia-derived mouse recombinant NT3, and human LY223982 being recombinant NT4/5 (CHO, Genentech). The rabbit polyclonal serum (Chex) directed against the extracellular website of chick p75NTR was a gift from Drs. Weskamp and Reichardt. Fertilized chicken eggs were from numerous commercial sources. For the preparation of sympathetic chains, embryos were staged relating to Hamburger and Hamilton (1951) and E11 (stage 37/38) animals were removed from the egg, washed in PBS, and dissected under a stereomicroscope. Paravertebral sympathetic chains were taken from the thoracolumbar portion of the embryos, freed from connective cells, and collected in ice-cold PBS. The chains were treated with 0.1% trypsin (Worthington, LY223982 Freehold, NJ) at 37C in PBS for 15C20 min. The ganglia were washed once in PBS and resuspended in Hams F14 (Existence Sciences) supplemented with 10% horse serum (Boeh-ringer Mannheim) and 5% LY223982 fetal calf serum (Existence Sciences). Chains were dissociated by mild trituration inside a siliconized Pasteur pipette. The cell suspension was then filtered through a 40 m nylon mesh (Falcon) and incubated on plastic cell culture dishes (Nunc, LY223982 Dannstadt, Germany).