Mild Transient Hypercapnia as a Novel Fear Conditioning Stimulus Allowing Re-Exposure during Sleep

Introduction:Studies suggest that sleep plays a role in traumatic memories and that treatment of sleep disorders may help alleviate symptoms of posttraumatic stress disorder. Fear-conditioning paradigms in rodents are used to investigate causal mechanisms of fear acquisition and the relationship between sleep and posttraumatic behaviors. We developed a novel conditioning stimulus (CS) that evoked fear and was subsequently used to study re-exposure to the CS during sleep.Methods:Experiment 1 assessed physiological responses to a conditioned stimulus (mild transient hypercapnia, mtHC; 3.0% CO2; n = 17)+footshock for the purpose of establishing a novel CS in male FVB/J mice. Responses to the novel CS were compared to tone+footshock (n = 18) and control groups of tone alone (n = 17) and mild transient hypercapnia alone (n = 10). A second proof of principle experiment re-exposed animals during sleep to mild transient hypercapnia or air (control) to study sleep processes related to the CS.Results:Footshock elicited a response of acute tachycardia (30-40 bpm) and increased plasma epinephrine. When tone predicted footshock it elicited mild hypertension (1-2 mmHg) and a three-fold increase in plasma epinephrine. When mtHC predicted footshock it also induced mild hypertension, but additionally elicited a conditioned bradycardia and a smaller increase in plasma epinephrine. The overall mean 24 hour sleep-wake profile was unaffected immediately after fear conditioning.Discussion:Our study demonstrates the efficacy of mtHC as a conditioning stimulus that is perceptible but innocuous (relative to tone) and applicable during sleep. This novel model will allow future studies to explore sleep-dependent mechanisms underlying maladaptive fear responses, as well as elucidate the moderators of the relationship between fear responses and sleep. © 2013 McDowell et al

Properties of double mutants of rhizobium leguminosarum which are defective in the utilization of dicarboxylic acids and sugars

SUMMARY: Rhizobium leguminosarum MNF3201 is a mutant defective in C4 dicarboxylic acid transport (dct). which forms ineffective nodules on pea. The Tn5-induced mutants MNF3041 (ribokinase-negative), MNF3045 (arabinonate dehydratase-negative), MNF3064 (defective in the “Entner-Doudoroff enzymes”) and MNF3070 (pyruvate carboxylase-negative) nodulate peas and fix N2. The carbohydrate-utilization mutations of these strains were each transduced into MNF3201 using phage RL38. The double mutants were defective in the utilization of both C4 dicarboxylic acids and the respective carbohydrates. All the double mutants formed nodules on peas, although these were still ineffective. Thus it appears that R. leguminosarum can use carbon sources other than dicarboxylic acids or common classes of sugars to fuel the nodulation process

Fructose metabolism in wild-type, fructokinase-negative and revertant strains of Rhizobium leguminosarum

Rhizobium leguminosarum accumulates fructose by an active process sensitive to azide, 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. The fructose is not phosphorylated during transport. Sorbose and glucose interfere with fructose uptake. Inside the cell fructose is metabolized via fructose 6-phosphate; there is no evidence for an alternative metabolic route via sorbitol to glucose or via sorbitol 6-phosphate to fructose 6-phosphate. Tn5-induced mutants lacking fructokinase failed to grow on fructose, mannitol or sorbitol and grew slowly on sucrose; growth was normal on all other single carbon sources tested. Growth of these mutants on a range of carbon sources was retarded by added fructose. Revertants which had regained the capacity to utilize fructose all had an unstable fructokinase which could be partially stabilized by fructose

Sugar metabolism and the symbiotic properties of carbohydrate mutants of Rhizobium leguminosarum

Rhizobium leguminosarum metabolizes sugars via the Entner-Doudoroff and pentose phosphate pathways but does not have a functional Embden-Meyerhof pathway. Although some sugar catabolizing enzymes are constitutive, activities of the “Entner-Doudoroff” enzymes vary with the carbon source. Bacteroids have complete pathways for sugar catabolism even though the specific activities of some enzymes, e.g., glucokinase, are lower than in free-living cells. Tn5-induced mutants lacking glucokinase, fructokinase and pyruvate dehydrogenase have been isolated. Although these mutants are unable to utilize sugars, they all nodulate peas and fix N2. The capacity to utilize particular C6 and C12 sugars is apparently not essential for bacteroid development or the establishment of effective N2 fixation

Symbiotic and competitive properties of motility mutants of Rhizobium trifolii TA1

Non-motile mutants of Rhizobium trifolii defective in either flagellar synthesis or function were isolated by transposon Tn5 mutagenesis. they were indistinguishable from motile control strains in growth in both laboratory media and in the rhizosphere of clover roots. When each non-motile mutant was grown together with a motile strain in continuous culture, the numbers of motile and non-motile organisms remained in constant proportion, implying that their growth rates were essentially identical. When inoculated separately onto clover roots, the mutants and wildtype did not differ significantly in the number of nodules produced or in nitrogen fixing activity. However, when mixtures of equal numbers of mutant and wild-type cells were inoculated onto clover roots, the motile strain formed approximately five times more nodules than the flagellate or non-flagellate, non-motile mutants, suggesting that motility is a factor in competition for nodule formation

Pentose metabolism in Rhizobium leguminosarum MNF300 and in cowpea Rhizobium NGR234

L-Arabinose is broken down by Rhizobium leguminosarum MNF300 via 2-oxoglutarate semialdehyde. Enzyme activities in cells grown on succinate, mannitol or arabinose indicated much greater modulation of arabinonate dehydratase, 2-keto-3-deoxyarabinonate dehydratase and 2-oxoglutarate semialdehyde dehydrogenase than of arabinose dehydrogenase or of arabinono-γ-lactonase. In cowpea Rhizobium NGR234, all the enzymes of L-arabinose metabolism except L-arabinono-γ-lactonase were inducible. Assays for such enzymes in snake bean bacteroids indicated that L-arabinose did not reach the bacteroids in large quantities. The Tn5-induced mutant MNF3045 of R. leguminosarum was unable to grow on L-arabinose and accumulated L-arabinono-γ-lactone and L-arabinonate. Product accumulation and enzyme assays suggested that this mutant was defective in L-arabinonate dehydratase. It nodulated peas and the nodules fixed N2, indicating that the supply of L-arabinose is not essential for bacteroid function. Another Tn5-induced mutant of R. leguminosarum, MNF3041, lacked ribokinase and was unable to grow on D-ribose; this mutant was also able to nodulate peas and fix N2

Properties of organic acid utilization mutants of Rhizobium leguminosarum strain 300

Mutants of Rhizobium leguminosarum 300 which were unable to utilize one or more organic acids as growth substrates were obtained by Tn5 mutagenesis. Mutant strain MNF3080 was defective in dicarboxylate transport and was unable to grow on succinate. Strain MNF3085 was defective in phosphoenolpyruvate carboxykinase and hence could not carry out gluconeogenesis. This strain did not grow on pyruvate, succinate, glutamate or arabinose but grew on glucose and on glycerol. Strain MNF3075 was unable to utilize pyruvate; the biochemical lesion in this mutant was not identified. MNF3085 and MNF3075 were symbiotically effective. MNF3080 nodulated peas, but the nodules were ineffective in N2 fixation and displayed morphological abnormalities. These data support previous findings which suggest that utilization of exogenous dicarboxylates is essential for effective nodule development by R. leguminosarum