The finding of orexin/hypocretin deficiency in narcolepsy patients suggests that this hypothalamic neuropeptide plays a crucial role in regulating sleep/wakefulness states. However, very little is known about the synaptic input of orexin/hypocretin-producing neurons (orexin neurons). We applied a transgenic method to map upstream neuronal populations that have synaptic connections to orexin neurons and revealed that orexin neurons receive input from several brain areas. These include the amygdala, basal forebrain cholinergic neurons, GABAergic neurons in the preoptic area, and serotonergic neurons in the median/paramedian raphe nuclei. Monoamine-containing groups that are innervated by orexin neurons do not receive reciprocal connections, while cholinergic neurons in the basal forebrain have reciprocal connections, which might be important for consolidating wakefulness. Electrophysiological study showed that carbachol excites almost one-third of orexin neurons and inhibits a small population of orexin neurons. These neuroanatomical findings provide important insights into the neural pathways that regulate sleep/wakefulness states.

In this paper, there was an error in the description of the fusion protein used in the transgenic construct. We described the construct as TTC::GFP in the text and in Figure 1A. The order of the components of the fusion protein was wrong. The fusion protein used in this study should be described as GFP::TTC (the tetanus toxin C-terminal fragment is fused to the C terminus of GFP). Although this correction does not affect the data or the conclusions of the paper, the authors would like to apologize to readers who have been misled by these mistakes. The authors also wish to correct the affiliations of Natsuko Tsujino and Yoshimasa Koyama as listed above.

The authors note that due to a printer's error, on page 4460, Fig. 2 appears incorrectly in part. The middle and bottom panels were transposed. The corrected figure and its legend appear below. MGAS5005∆ccpA comp∆ccpA

Background: Extracellular endosulfatases Sulf1 and Sulf2 hydrolyze 6-O-sulfate in heparan sulfate. Results: Disaccharide analysis showed that 2-O-, 6-O-, and N-trisulfated disaccharide units in heparan sulfate were increased to different degrees in different organs in Sulf1 and Sulf2 knock-out mice. Conclusion: Sulfs generate organ-specific sulfation patterns of heparan sulfate. Significance: This may indicate differences in activity between Sulf1 and Sulf2 in vivo.

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Summary: Transgenic mice ubiquitously expressing enhanced green fluorescent protein (EGFP) are useful as marker lines in chimera experiments. We established a new embryonic stem (ES) cell line (named B6G-2) from a C57BL/6 blastocyst showing ubiquitous EGFP expression. Undifferentiated B6G-2 cells showed strong green fluorescence and mRNAs of pluripotent marker genes. B6G-2 cells were transferred into a C57BL/6 blastocyst to generate a germline chimera, the progeny of which inherited ubiquitous EGFP expression. Mice derived completely from B6G-2 cells were also developed from the ES cells; these were tetraploid chimeras. The established B6G-2 cells were shown to be pluripotent and to be capable of differentiating into cells of all lineages.

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