Human and animal studies have converged to suggest that caffeine consumption prevents memory deficits in aging and Alzheimer’s disease through the antagonism of adenosine A2A receptors (A2AR). To test if A2AR activation in hippocampus is actually sufficient to impair memory function and to begin elucidating the intracellular pathways operated by A2AR, we have developed a chimeric rhodopsin-A2AR protein (optoA2AR), which retains the extracellular and transmembrane domains of rhodopsin (conferring light responsiveness and eliminating adenosine binding pockets) fused to the intracellular loop of A2AR to confer specific A2AR signaling. The specificity of the optoA2AR signaling was confirmed by light-induced selective enhancement of cAMP and phospho-MAPK (but not cGMP) levels in HEK293 cells, which was abolished by a point mutation at the C-terminal of A2AR. Supporting its physiological relevance, optoA2AR activation and the A2AR agonist CGS21680 produced similar activation of cAMP and phospho-MAPK signaling in HEK293 cells, of pMAPK in nucleus accumbens, of c-Fos/pCREB in hippocampus and similarly enhanced long-term potentiation in hippocampus. Remarkably, optoA2AR activation triggered a preferential phospho-CREB signaling in hippocampus and impaired spatial memory performance while optoA2AR activation in the nucleus accumbens triggered MAPK signaling and modulated locomotor activity. This shows that the recruitment of intracellular A2AR signaling in hippocampus is sufficient to trigger memory dysfunction. Furthermore, the demonstration that the biased A2AR signaling and functions depend on intracellular A2AR loops, prompts the possibility of targeting the intracellular A2AR interacting partners to selectively control different neuropsychiatric behaviors.

We evaluate the operational MODIS Leaf Area Index (LAI) product using field-sampled data collected at five sites in southern Africa in March 2000. One site (Mongu, Zambia) was sampled monthly throughout the year. All sites were along the International Geosphere Biosphere Programme's (IGBP) Kalahari Transect, which features progressively lower annual precipitation, and hence, lower vegetation productivity, from north to south. The soils are consistently sandy. At each site, we sampled the vegetation overstory along three 750-m transects using the Tracing Radiation and Architecture in Canopies (TRAC) instrument. The resulting plant area index values were adjusted with ancillary stem area data to estimate LAI. Despite some instrument characterization and production issues in the first year of MODIS operations, our results suggest the first-year MODIS LAI algorithm correctly accommodates structural and phenological variability in semiarid woodlands and savannas, and is accurate to within the uncertainty of the validation approach used here. Limitations of this study and its conclusions are also discussed. D

Immunofluorescence (IF) imaging using antibodies is widely used in biological and clinical laboratories to map the distribution of proteins in situ. A typical IF protocol involves labeling specific targets with primary antibodies followed by signal amplification using secondary antibodies targeting the primary antibodies. This poses a significant challenge for multiplexed imaging as primary antibodies from different species or different subclasses (in the case of mouse antibodies) have to be used for the same sample. Unfortunately, most of the validated antibodies are produced either from mouse or from rabbit, hindering flexible antibody choices. Primary antibodies directly conjugated with fluorophores can be used to bypass the secondary antibody species issues. However, lack of signal amplification from secondary antibodies restricts this approach to the visualization of high abundance targets. In order to address the abovementioned challenges, we integrated a DNA-based signal amplification method, Hybridization Chain Reaction (HCR) 1-4 , with IF to achieve high quality multiplexed imaging using same species primary antibodies (Figure 1).In HCR, a single DNA initiator sequence will trigger an assembly of a large linear DNA structure by iterative HCR fluorescence hairpin opening 1-4 . We directly conjugated HCR-initiator DNA sequences to primary antibodies through covalent chemical modification 5 . We first tested the labeling specificity of DNA-modified primary antibodies by staining the antibodies in cultured BSC1 cells followed by HCR amplification. Surprisingly, we observed strong nonspecific signals, especially inside the nucleus (Figure 2 and Supplementary Figure 1), which has not been observed previously using DNA-conjugated secondary antibodies 6,7 . We decided to optimize the antibody staining protocol to reduce nonspecific binding. Sheared sperm DNA has previously been used to block nonspecific binding of DNA-conjugated antibodies 8 . We showed that addition of 0.5mg/ml of sheared sperm DNA during blocking and antibody incubation could reduce but not completely abolish nonspecific nuclear signal (Figure 2 and Supplementary Figure 1). In order to further suppress nonspecific signals, we tested the addition of dextran sulfate in the antibody incubation buffer. High concentration (10% -20%) of dextran sulfate has been used as a crowding reagent in DNA and RNA FISH experiments 9,10 . Empirically, we noticed that some primary antibodies failed to bind to their targets in 10% dextran sulfate solution (data not shown). We therefore performed a titration experiment to determine the minimal concentration of dextran sulfate. The result showed a good suppression of nonspecific nuclear signal could be achieved with as low as 0.02% dextran sulfate (Supplementary Figure 2). We used 0.05% or 0.1% dextran sulfate plus 0.5mg/ml sheared sperm DNA for all following experiments. Similar to conventional immunostaining, we also noticed for some antibodies the antibody concentration also had a modest effect on the nonspecif...