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Here we give names to three new species of Paraburkholderia that can remain in symbiosis indefinitely in the spores of a soil dwelling eukaryote, Dictyostelium discoideum. The new species P. agricolaris sp. nov., P. hayleyella sp. nov., and P. bonniea sp. nov. are widespread across the eastern USA and were isolated as internal symbionts of wild-collected D. discoideum. We describe these sp. nov. using several approaches. Evidence that they are each a distinct new species comes from their phylogenetic position, average nucleotide identity, genome-genome distance, carbon usage, reduced length, cooler optimal growth temperature, metabolic tests, and their previously described ability to invade D. discoideum amoebae and form a symbiotic relationship. All three of these new species facilitate the prolonged carriage of food bacteria by D. discoideum, though they themselves are not food. Further studies of the interactions of these three new species with D. discoideum should be fruitful for understanding the ecology and evolution of symbioses.

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The circadian clock in the mammalian hypothalamic suprachiasmatic nucleus (SCN) is entrained by the ambient light/dark cycle, which differentially acts to cause the clock to advance or delay. Light-induced changes in the rhythmic expression of SCN clock genes are believed to be a critical step in this process, but how the two entrainment modalities--advances vs. delays--engage the molecular clockwork remains incompletely understood. We investigated molecular substrates of photic entrainment of the clock in the SCN by stably entraining hamsters to T cycles (non-24-h light/dark cycles) consisting of a single 1-h light pulse repeated as either a short (23.33-h) or a long (24.67-h) cycle; under these conditions, the light pulse of the short cycle acts as "dawn," whereas that of the long cycle acts as "dusk." Analyses of the expression of the photoinducible and rhythmic clock genes Period 1 and 2 (Per1 and Per2) in the SCN revealed fundamental differences under these two entrainment modes. Light at dawn advanced the clock, advancing the onset of the Per1 mRNA rhythm and acutely increasing mRNA transcription, whereas light at dusk delayed the clock, delaying the offset of the Per2 mRNA rhythm and tonically increasing mRNA stability. The results suggest that the underlying molecular mechanisms of circadian entrainment differ with morning (advancing) or evening (delaying) light exposure, and such differences may reflect how entrainment takes place in nocturnal animals under natural conditions.

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Previous anatomical and physiological studies have implicated the lateral habenula, and especially its medial division (LHbM), as a candidate component of the circadian timing system in rodents. We assayed lateral habenula rhythmicity in rodents using c-FOS immunohistochemistry and found a robust rhythm in immunoreactive cell counts in the LHbM, with higher counts during the dark phase of a light-dark (LD) cycle and during subjective night in constant darkness. We have also observed an obvious asymmetry of c-FOS expression in the LHbM of behaviorally "split" hamsters in constant light, but only during their active phase (when they were running in wheels). Locomotor activity rhythms appear to be regulated by the suprachiasmatic nucleus (SCN) via multiple output pathways, one of which might be diffusible while the other might be neural, involving the lateral habenula.

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"Splitting" of circadian activity rhythms in Syrian hamsters maintained in constant light appears to be the consequence of a reorganized SCN, with left and right halves oscillating in antiphase; in split hamsters, high mRNA levels characteristic of day and night are simultaneously expressed on opposite sides of the paired SCN. To visualize the splitting phenomenon at a cellular level, immunohistochemical c-Fos protein expression in the SCN and brains of split hamsters was analyzed. One side of the split SCN exhibited relatively high c-Fos levels, in a pattern resembling that seen in normal, unsplit hamsters during subjective day in constant darkness; the opposite side was labeled only within a central-dorsolateral area of the caudal SCN, in a region that likely coincides with a photo-responsive, glutamate receptor antagonist-insensitive, pERK-expressing cluster of cells previously identified by other laboratories. Outside the SCN, visual inspection revealed an obvious left-right asymmetry of c-Fos expression in the medial preoptic nucleus and subparaventricular zone of split hamsters killed during the inactive phase and in the medial division of the lateral habenula during the active phase (when the hamsters were running in their wheels). Roles for the dorsolateral SCN and the mediolateral habenula in circadian timekeeping are not yet understood.

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Lead treatment via drinking water for 3 to 6 weeks at 250 ppm was found to significantly decrease the number of spontaneously active dopamine (DA) neurons in both the substantia nigra and ventral tegmental area that were recorded using standard extracellular electrophysiological recording techniques. Lead exposure did not affect the discharge rate or discharge pattern of these DA neurons. No significant decrease in the number of tyrosine hydroxylase immunopositive cells was detected in lead-treated animals relative to controls even though the length of lead exposure was extended beyond that of the electrophysiological studies. The significant lead-induced decrease in spontaneously active cells observed in the electrophysiological studies was, therefore, not due to cell death. An acute drug challenge with the DA receptor agonist apomorphine at a dose known to hyperpolarize midbrain DA neurons (50 mug/kg i.v.) was used to determine whether hyperpolarization would normalize the number of spontaneously active DA neurons. The results suggest that depolarization inactivation was most likely not the cause for this lead effect. The D(1) receptor agonist SKF-38393 [1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol] was iontophoretically applied to type I nucleus accumbens (Nacb) neurons. The results demonstrated that type I Nacb neurons have a significantly lower basal discharge rate in lead-treated animals relative to controls and that the Nacb DA D(1) receptors were significantly less sensitive to SKF-38393 in the lead-treated animals. Therefore, lead exposure decreases DA neuron impulse flow presynaptically and decreases DA D(1) receptor sensitivity postsynaptically in the nucleus accumbens.

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