Expert Industry Secrets Over VAV2 Unveiled
Fish were sampled every 4?hr throughout the first cycle under LD and DD conditions. Under both conditions, nptx2b mRNA levels in the LH were highest during the day and lowest during the night (p Lonafarnib the number of SYP-EGFP puncta reach their maximum level ( Figures 2L and 2M). Rhythmic nptx2b expression was not restricted solely to the LH. In the habenula, nptx2b mRNA levels were high during the night (CT11�CCT19) and low during the day (CT23�CCT7) under DD (p VAV2 of synaptic plasticity in these neurons, we first analyzed the effect of nptx2b on synaptic density. We were unable to pursue a loss-of-function strategy, as no nptx2b?/? mutants are available and antisense morpholino approaches are inefficient at the developmental stage of fish in our experiments (7 dpf). Further, NPTX2b loss could be compensated by the effects of other pentraxins such as NPTX2a and NPTX1 (see Discussion). Therefore, we pursued a gain-of-function strategy and generated a stable transgenic line overexpressing nptx2b under the control of the hcrt promoter ( Figure?5D). This promoter drives a strong, arrhythmic expression specifically in HCRT neurons ( Faraco et?al., 2006?and?Appelbaum et?al., 2009). To further validate that the HCRT promoter is arrhythmic, we show that, in adult zebrafish, hcrt mRNA levels are arrhythmic with a slight increase toward the end of the night (CT23, Figure?S5). These data match well with mammalian studies that show that hcrt mRNA expression is arrhythmic ( St��tz et?al., 2007). Both pre- and postsynaptic density structures were monitored with SYP-EGFP ( Figure?5) and PSD95-EGFP ( Figure?S6), respectively, as NPTX2 has Tyrosine Kinase Inhibitor Library cost been shown to act both pre- and postsynaptically in?vitro ( O'Brien et?al., 1999). PSD95 is a validated postsynaptic marker of excitatory synapses in mammals and zebrafish ( Ebihara et?al., 2003, Niell et?al., 2004?and?Livneh et?al., 2009). To study the function of NPTX2b in a single HCRT neuron, we used transient expression ( Figure?5A), taking advantage of mosaic effects obtained after injections of the hcrt:SYP-EGFP or hcrt:PSD95-EGFP construct at the one-cell stage of stable hcrt:NPTX2b transgenic or wild-type sibling embryos.