In the course of study examining the role of these inhibitory neurotransmission on the PCD of MNs, we found that the treatment of strychinine, a glycine receptor antagonist, advanced the onset of PCD in chick embryos. Within a discrete period of the embryonic development (E5~E8), for instance, approximately 50% motoneurons (MNs) undergo PCD in the chick embryo. PCD of MNs initiates with provisional synaptic connections with target muscles, and neurotrophic hypothesis explains the principle of developmental PCD: There was a competition among MN to acquire sufficient survival factors, and the limited amounts of available target-derived neurotrophic signals determine the extent of PCD [3-5]. In consistent with this hypothesis, surgical removal or addition of target muscles in chick embryos accordingly resulted in the augmentation or reduction of the PCD, respectively [2,6]. However, neurotrophic hypothesis does not explain how PCD initiates. Before their target muscle innervation, young MNs do not undergo PCD although they never be able to obtain target-derived signals. Considering that surgical removal of target does not modify the onset timing of PCD, it appears that the onset of PCD may be independent to the target-derived signals. Some paracrine/autocrine signals or cell-autonomous modifications may be involved in the onset of MN PCD, but molecular mechanisms and responsible extracellular factors are largely unknown. Neuronal activity during the embryonic development plays significant role in the innervations and maturation of synaptic circuits, via modulation of the growth cone guidance and formation of synapses [7-9]. During the early development, inhibitory neurotransmitters such as glycine and GABA act as excitatory signals and trigger neuronal depolarization [10-12]. In addition, several excitatory or inhibitory neurotransmissions directly or indirectly affect the PCD of MNs [13-15]. In the course of study examining the role of these inhibitory neurotransmission on the PCD of MNs, we found that the treatment of strychinine, a glycine receptor antagonist, advanced the onset of PCD in chick embryos. This new observation may trigger new insight how the onset of the MN PCD is modulated during the development. == MATERIALS AND METHODS == == Animals and treatments == Fertilized chicken eggs were obtained from Pulmuone Co. (Korea). Eggs were incubated in humidified incubator at 38. Stage of chick embryo was identified according to the Hamilton-Hamburger’s criteria [3]. Strychnine (300 g in 100 l Saline, Sigma S8753, St. Louis, MO), Ha966 (300 g in 100 l Saline, Tocris 0281, Ellisville, Missouri) or L-701324 (300 g in 100 l Saline, Tocris 0907, Ellisville, Missouri) were applied twice with 12 hours interval on E3, E4 or E5, and sacrificed 12 hours after last treatment. == Histology == Immunohistochemical analyses were performed as previously reported [16]. Briefly, trunk tissues were isolated from embryos and immersion-fixed with 4% paraformaldehyde overnight. Tissues were then transferred in 30% sucrose, sectioned (7 m) and attached on a gelatin-coated slide glass. After blocking the sections with PBS Melanocyte stimulating hormone release inhibiting factor containing 3% BSA and 0.1% Triton-X100, activated caspase-3 antibody (1:500; Cell signaling Technology, Beverly, Rabbit polyclonal to SAC MA) was applied overnight. After several washes with PBS, Alexa488-conjugated donkey anti-rabbit antibody was applied Melanocyte stimulating hormone release inhibiting factor for 30 min. Subsequently, sections were washed, counterstained with Hechest33342, mounted and observed with a confocal microscope (Zeiss LSM510, Goettingen, Germany). == RT-PCR == Total RNAs (1 g) purified from lumbar spinal cords of chick embryo were reverse-transcribed with reverse transcriptase, oligo (dT) primer and RNasin (Promega). An aliquot of the synthesized cDNA was subjected to PCR amplification with specific primers for the target genes. Primer sets for Glycine receptor alpha-1 (5′-AGA Melanocyte stimulating hormone release inhibiting factor GCC CAT TCC TCC CTC CC-3′ as 5′-primer and 5′-GGC AGA TCG TGC TGC TGC TT-3′ as 3′-primer), Glycine receptor alpha-2 (5′-CCA GCC AGA GTT GCA CTG GG-3′ as 5′-primer and 5′-AGG AAG GCG AGT GGG AAT GC-3′ as 3′-primer), Glycine receptor alpha-3 (5′-CAG ACA GCG CAA GAT CCC GT-3′ as 5′-primer and 5′-TGA GAT CCA TTG GGC Melanocyte stimulating hormone release inhibiting factor AGG ACA-3′ as 3′-primer), Glycine receptor alpha-4 (5′-GGA GCG GCA GAT GGG CTA TT-3′ as 5′-primer and 5′-CAG CCC GTA GCC TCG GAA GT-3′ as 3′-primer), Glycine receptor beta (5′-CAC AGC GCT GCA AGA TGC AA-3′ as 5′-primer and 5′-TGC CTT GGC AAT CTT GGC TT-3′ as 3′-primer) and GAPDH (5′-GCT CCC TCA GCT GAT GCC CCC-3′ as 5′-primer and 5′-AGG GAT GAC TTT CCC CAC AGC C-3′ as 3′-primer) were used. To obtain semi-quantitative data, unsaturated range of PCR amplification cycles were determined, as 30 (Glycine receptor isoforms) or 25 (GAPDH) cycles. Amplified PCR.