We treated neurons with IIK7 or DMSO at 72?h and examined axon-neurite development 72?h later (144?h in total) (Supplementary Number 4aCc). molecular characteristics of the hippocampal neurons dendrite The MT2 receptor is essential for axon development To determine the role of the MT2 receptor in axon differentiation, we 1st applied an study by electroporation with MT2-specific shRNA (Supplementary Number 2a and b), we found that neuronal migration (P0) and axon outgrowth (P3) were significantly retarded by MT2 knockdown (Number 2a, boxes 1 and 2). The SMI312 (a specific axonal marker previously used in an study23) immunoreaction was almost diminished in MT2?/? mice, whereas strong SMI312 immunoreactivity was recognized in the IZ region with enriched axon bundles in C3H mice (Number 2b, boxes 1C4). Recent studies have revealed that many cortical neurons initiate axon outgrowth during radial migration, before they reach Rabbit polyclonal to MMP9 the CP.24, 25 Our data strongly suggests that downregulation of MT2 signals arrests early axonal development. Open in a separate window Number 2 MT2 receptor is essential for axon development. (a) Rats embryos were electroporated with mU6-MT2-shRNA-pUbi-EGFP (si-MT2) or scrambled one (Ssi-MT2) at E16 and the slices were prepared at postnatal day time 0 (P0) or day time 3 (P3). MZ, marginal zone; CP, corticalplate; SP, subplate; IZ, intermediate zone; SVZ, NB-598 hydrochloride subventricular zone; ML, midline. Pub=200?DMSO. (h) Main neurons from E18 rat hippocampus were transfected with shRNA of MT2 (si-MT2) or MT1 (si-MT1) or the vector plasmid (siV) before plating. IIK7 (10?siV We then used high affinity MT2 receptor selective antagonists, 4P-PDOT or K185 and MT2 shRNA for studies. In rat hippocampal neuron cultures, inhibition of the MT2 receptor by 4P-PDOT or K185 significantly inhibited formation and outgrowth of the axon: ~41.7% of the neurons experienced no axon and only ~3.7% of them grew multiple axons after 4P-PDOT treatment, and ~42.5% of the neurons experienced no axon and ~3.5% neuron developed multiple axons after K185 treatment (Figures 2c and d); 4P-PDOT and K185 treatment also reduced axon size with an increased dendrite size and unchanged total neurite quantity (Numbers 2eCg). With specific shRNA focusing on (Supplementary Number 2a and b), we found that knockdown of the MT2 receptor, but not the MT1 receptor, decreased axon formation and outgrowth with an increase in dendrite size, and the deficits of axon development were not rescued by IIK7, a specific MT2 receptor agonist (Numbers 2hCl). These data demonstrate that suppression of the MT2 receptor arrests axonogenesis, indicating an essential part for MT2 receptor in axon development. MT2 receptor activation promotes axon outgrowth We next investigated whether activation of the MT2 receptor promotes axon outgrowth and axon-dendrite differentiation by incubation of rat hippocampal neuron cultures with MT2 agonists, melatonin (MEL) or IIK7 (the concentration of IIK7 was chosen relating to a dose-dependent experiment, see Supplementary Number 3). In the DMSO control group, ~81.5% neurons developed a single-axon with NB-598 hydrochloride 8.9% multiple-axon and 9.6% no-axon neurons (Figures 3a and b). Activation of the MT2 receptor by MEL or IIK7 improved the space of solitary axon projections without influencing dendrite size and neurite quantity, and ~40.6 or 41.2% of MEL-treated or IIK7-treated neurons developed multiple axons (Figures 3aCe), indicating the sufficiency of MT2 receptor activation in axon differentiation. The effect of MT2 receptor activation is unique since only blockage of the MT2 but not MT1 receptor compromised the IIK7 facilitation of the formation and outgrowth of multiple axons (Numbers 3fCj). Furthermore, only overexpression of the rat MT2 receptor but not MT1 receptor improved axon formation and outgrowth (Numbers 3kCo), consistent with the specific effect of MT2 in axon development. Open in a separate window Number 3 Activation of MT2 receptor promotes practical axon formation. (a) Dissociated rat hippocampal neurons (E18) were treated with MT2 receptor agonists, MEL or IIK7, or DMSO at 4 hrs after plating. 72hrs after the treatment the cultures were co-stained with Tau-1 (reddish) and MAP2 (green) (DMSO. (f) Dissociated rat hippocampal neurons (E18) were treated with IIK7 plus antibodies against the MT1 receptor (Cat No. sc-13179, Santa Cruz Biotech.; IgG+IIK7. (k) Rat cultured neurons were transfected with rat MT2 (rMT2) or MT1 (rMT1) or vector (pcDNA) before plating and the images were captured at 72?h after plating (pcDNA Neurons generally do not develop a fresh axon after 72?h in tradition, a time point defined as the maintenance phase of polarity.20 To explore whether activation of the MT2 receptor still encourages axon formation in the NB-598 hydrochloride maintenance phase of neuronal development in cultured rat neurons, we indicated.