Physiological homeostasis is essential for organism survival. Highly responsive neuronal networks are involved but constituent neurons are just beginning to be resolved. To query brain serotonergic neurons in homeostasis, we used a synthetic GPCR (Di)-based neuronal silencing tool, mouse RC∷FPDi, designed for cell type-specific, ligand (clozapine-N-oxide, CNO)-inducible and reversible suppression of action potential firing. In mice harboring Di-expressing serotonergic neurons, CNO administration by systemic injection attenuated the chemoreflex that normally increases respiration in response to tissue CO2 elevation and acidosis. At the cellular level, CNO suppressed firing rate increases evoked by CO2/acidosis. Body thermoregulation at room temperature was also disrupted following CNO triggering of Di; core temperatures plummeted, then recovered. This work establishes that serotonergic neurons regulate life-sustaining respiratory and thermoregulatory networks, and demonstrates a noninvasive tool for mapping neuron function.

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Recent large-scale mutagenesis screens have made the zebrafish the first vertebrate organism to allow a forward genetic approach to the discovery of developmental control genes. Mutations can be cloned positionally, or placed on a simple sequence length polymorphism (SSLP) map to match them with mapped candidate genes and expressed sequence tags (ESTs). To facilitate the mapping of candidate genes and to increase the density of markers available for positional cloning, we have created a radiation hybrid (RH) map of the zebrafish genome. This technique is based on somatic cell hybrid lines produced by fusion of lethally irradiated cells of the species of interest with a rodent cell line. Random fragments of the donor chromosomes are integrated into recipient chromosomes or retained as separate minichromosomes. The radiation-induced breakpoints can be used for mapping in a manner analogous to genetic mapping, but at higher resolution and without a need for polymorphism. Genome-wide maps exist for the human, based on three RH panels of different resolutions, as well as for the dog, rat and mouse. For our map of the zebrafish genome, we used an existing RH panel and 1,451 sequence tagged site (STS) markers, including SSLPs, cloned candidate genes and ESTs. Of these, 1,275 (87.9%) have significant linkage to at least one other marker. The fraction of ESTs with significant linkage, which can be used as an estimate of map coverage, is 81.9%. We found the average marker retention frequency to be 18.4%. One cR3000 is equivalent to 61 kb, resulting in a potential resolution of approximately 350 kb.

SUMMARY Despite the well-established role of serotonin signaling in mood regulation, causal relationships between serotonergic neuronal activity and behavior remain poorly understood. Using a pharmacogenetic approach, we find that selectively increasing serotonergic neuronal activity in wild-type mice is anxiogenic and reduces floating in the forced-swim-test, while inhibition has no effect on the same measures. In a developmental mouse model of altered emotional behavior, increased anxiety and depression-like behaviors correlate with reduced dorsal raphe and increased median raphe serotonergic activity. These mice display blunted responses to serotonergic stimulation and behavioral rescues through serotonergic inhibition. We furthermore identify opposing consequences of dorsal versus median raphe serotonergic neuron inhibition on floating behavior, together suggesting that median raphe hyperactivity increases anxiety, while a low dorsal/median raphe serotonergic activity ratio increases depression-like behavior. Thus we find a critical role of serotonergic neuronal activity in emotional regulation and uncover opposing roles of median and dorsal raphe function.