Precision determination of absolute neutron flux.

A technique for establishing the total neutron rate of a highly-collimated monochromatic cold neutron beam was demonstrated using an alpha-gamma counter. The method involves only the counting of measured rates and is independent of neutron cross sections, decay chain branching ratios, and neutron beam energy. For the measurement, a target of 10B-enriched boron carbide totally absorbed the neutrons in a monochromatic beam, and the rate of absorbed neutrons was determined by counting 478 keV gamma rays from neutron capture on 10B with calibrated high-purity germanium detectors. A second measurement based on Bragg diffraction from a perfect silicon crystal was performed to determine the mean de Broglie wavelength of the beam to a precision of 0.024%. With these measurements, the detection efficiency of a neutron monitor based on neutron absorption on 6Li was determined to an overall uncertainty of 0.058%. We discuss the principle of the alpha-gamma method and present details of how the measurement was performed including the systematic effects. We also describe how this method may be used for applications in neutron dosimetry and metrology, fundamental neutron physics, and neutron cross section measurements.

Improved determination of the neutron lifetime.

The most precise determination of the neutron lifetime using the beam method was completed in 2005 and reported a result of τ(n)=(886.3±1.2[stat]±3.2[syst]) s. The dominant uncertainties were attributed to the absolute determination of the fluence of the neutron beam (2.7 s). The fluence was measured with a neutron monitor that counted the neutron-induced charged particles from absorption in a thin, well-characterized 6Li deposit. The detection efficiency of the monitor was calculated from the areal density of the deposit, the detector solid angle, and the evaluated nuclear data file, ENDF/B-VI 6Li(n,t)4He thermal neutron cross section. In the current work, we measure the detection efficiency of the same monitor used in the neutron lifetime measurement with a second, totally absorbing neutron detector. This direct approach does not rely on the 6Li(n,t)4He cross section or any other nuclear data. The detection efficiency is consistent with the value used in 2005 but is measured with a precision of 0.057%, which represents a fivefold improvement in the uncertainty. We verify the temporal stability of the neutron monitor through ancillary measurements, allowing us to apply the measured neutron monitor efficiency to the lifetime result from the 2005 experiment. The updated lifetime is τ(n)=(887.7±1.2[stat]±1.9[syst]) s.

The Fundamental Neutron Physics Facilities at NIST.

The program in fundamental neutron physics at the National Institute of Standards and Technology (NIST) began nearly two decades ago. The Neutron Interactions and Dosimetry Group currently maintains four neutron beam lines dedicated to studies of fundamental neutron interactions. The neutrons are provided by the NIST Center for Neutron Research, a national user facility for studies that include condensed matter physics, materials science, nuclear chemistry, and biological science. The beam lines for fundamental physics experiments include a high-intensity polychromatic beam, a 0.496 nm monochromatic beam, a 0.89 nm monochromatic beam, and a neutron interferometer and optics facility. This paper discusses some of the parameters of the beam lines along with brief presentations of some of the experiments performed at the facilities.

Measurement of the Neutron Lifetime by Counting Trapped Protons.

We measured the neutron decay lifetime by counting in-beam neutron decay recoil protons trapped in a quasi-Penning trap. The absolute neutron beam fluence was measured by capture in a thin (6)LiF foil detector with known efficiency. The combination of these measurements gives the neutron lifetime: τ n = (886.8 ± 1.2 ± 3.2) s, where the first (second) uncertainty is statistical (systematic) in nature. This is the most precise neutron lifetime determination to date using an in-beam method.

Multiple enhancer elements mediate induction of c-fos in vascular smooth muscle cells.

Previous work from this and other laboratories has demonstrated that the vasoconstrictor peptide angiotensin II results in hypertrophy of rat aortic smooth muscle cells that is associated with an increase in transcription of the early growth response gene c-fos. To explore the molecular mechanism responsible for c-fos induction in rat aortic smooth muscle cells, we used a series of reporter constructs linked to the chloramphenicol acetyl transferase gene in transient transfection experiments in rat aortic smooth muscle cells. Constructs containing both the serum response element and cAMP response element exhibited a 20-fold increase in chloramphenicol acetyl transferase activity in response to either serum or angiotensin II, whereas no increase was seen in vehicle-treated cells. Mutations in either the serum response element or cAMP response element alone, which have been demonstrated to inactivate these elements in other cell types, had no effect on chloramphenicol acetyl transferase inducibility. In contrast, if both elements were mutated, inducibility was almost abolished. Electrophoretic mobility shift assays with oligonucleotides corresponding to either serum response element or cAMP response element demonstrated that these oligonucleotides are capable of forming specific complexes with proteins from rat aortic smooth muscle cell nuclear extracts. One of the proteins binding to the serum response element is the previously described serum response factor, since it was supershifted by a monospecific antibody. These studies demonstrate that c-fox induction in smooth muscle occurs by a dual mechanism that can activate transcription via the serum response element or cAMP response element. These elements appear to act equally and independently, involving a distinct set of transacting factors.

Nicotine-produced relearning deficit in C57BL/6J and DBA/2J mice.

Acute nicotine administration has been shown to influence the acquisition and retention of learning tasks. In order to investigate the many possible behavioral and pharmacological effects of nicotine, a modified 2 X 2 state-dependent learning design was used to assess nicotine's effects on active avoidance learning. Male and female mice of the C57BL/6J (C57) and DBA/2J (DBA) inbred strains were injected with a control solution or with 0.5, 1.0, or 2.0 mg/kg nicotine 5 min before the start of training and, following a 24-h period, 5 min before retraining. Nicotine had no effect on the acquisition of the learning task but, depending on strain and sex, did have an effect on relearning. Relearning in the C57 males was unaffected by nicotine injection, whereas the most prominent effect of nicotine in the C57 females and the DBA males and females was a retrieval deficit. The prevalence of a nicotine-induced retrieval deficit in the present experiment suggests that those mechanisms underlying the retrieval of previously learned information are, in part, mediated or modulated by perturbations within nicotine-sensitive areas of the central nervous system.

Long-term consequences of prenatal cocaine exposure on biogenic amines in the brains of mice: the role of sex.

Prenatal cocaine exposure leads to multiple abnormalities in the mature offspring. We explored the effects of gestational exposure to cocaine on neurotransmitter systems of adult mice. The subjects were the mature offspring of mice (a) prenatally fed cocaine between gestational day (G) 8 and G19, (b) pair-fed chow and water, or fed chow and water ad libitum. The forebrains of the mature offspring were assayed for monoamines and amino acids. Cocaine exposure particularly affected the dopaminergic system and in a sex-specific manner. In males dopamine concentrations were decreased and dopamine turnover was increased, whereas in females dopamine concentrations were increased and turnover was decreased. Neither norepinephrine, the serotonergic system, nor neuroactive amino acids (or their precursors) were affected by cocaine. Thus, in utero exposure to cocaine produces long-lasting, specific defects in the dopaminergic system.

Genetic influences on brain growth restriction induced by development exposure to alcohol.

Genetic factors have been implicated as contributing to the considerable variation in the severity of alcohol-related birth defects in offspring of women who drink heavily during pregnancy. Two animal models of alcohol-related developmental effects incorporated different behavior genetic approaches to examine genetic influences on brain and body growth following alcohol exposure during development. The first, extensively developed in Sprague-Dawley rats, examined the effects of three doses of alcohol administered to two inbred rat strains (MR and M520) via artificial-rearing procedures during the early postnatal brain growth spurt. In both strains, alcohol produced a dose-dependent restriction of brain weight (but not body weight) on postnatal day 10, compared to artificially reared controls. The MR strain was more susceptible to cerebellar growth restriction than the M520 strain, an effect not attributable to strain differences in blood alcohol concentrations. In the second model, pregnant female Long-sleep and Short-sleep mice, selectively bred for differences in initial sensitivity to the hypnotic effects of acute alcohol administration, were intubated with ethanol from gestational days 7-18. Controls included either sucrose or maltose/dextrin intubation controls and non-intubated controls. The LS offspring showed growth deficits and brain weight reductions in adulthood, while the SS offspring were resistant to these detrimental effects of the prenatal alcohol exposure. Thus, differences in either maternal or fetal genotype may contribute to individual differences in the severity of the effects of alcohol exposure during development.

Differential effects of ethanol concentration on blood pH, PCO2 and PO2 in LS and SS mice.

Concentration-dependent effects of ethanol upon behavior and upon physiological regulatory mechanisms have been suggested. In a previous study, we found that the concentration of an acute ethanol injection confounded dose-response relationships for measures of blood pH, PCO2, and PO2. Two lines of mice that differ in CNS sensitivity to the hypnotic effects of ethanol (long sleep, LS; short sleep, SS) have also been found to differ in sensitivity to the physiological depressant effects of this drug. Therefore, we designed the present study to examine how intraperitoneal (IP) injections of varying ethanol concentrations differentially affect blood parameters (pH, PCO2, and PO2) and respiration rate in the LS and SS mouse lines. Different groups of LS female mice were injected IP with 165.0, 198.8, 248.1, or 330.0 mg/ml ethanol at a constant dose of 3.3 g/kg. Groups of SS female mice received 205.0, 247.0, 308.3, or 410.0 mg/ml ethanol at a dose of 4.1 g/kg. Blood parameters and respiration rate were measured at 60 min post-injection. In both the LS and SS mice, increasing concentrations of ethanol caused a progressive decline in respiration rate and blood pH. Blood PCO2 values were greater than control only at the highest ethanol concentration. Concentration-dependent effects of ethanol on blood PO2 values were not found in either line. However, LS PO2 was significantly elevated from the control value at all ethanol concentrations. These results suggest that a dose-response relationship may be obtained by varying ethanol concentration for some physiological measures, but not for others. Thus, attention should be paid to differences in concentration as well as amount of ethanol when dose-response curves are to be constructed.

Acute ethanol effects on blood pH, PCO2, and PO2 in LS and SS mice.

The effects of ethanol on blood pH, PCO2, and PO2 were measured in LS and SS mice in an attempt to ascertain whether these lines of mice, which differ in CNS sensitivity to the behavioral effects of ethanol, also differ in sensitivity to physiological effects of this drug. Long-sleep (LS) female mice were injected intraperitoneally with 1.8, 2.5, 3.3, or 3.8 g/kg ethanol; short-sleep (SS) female mice were administered 2.5, 3.3, 4.1, or 4.7 g/kg. Blood pH, PCO2, and PO2 were assessed at 15, 30, 60, 120, or 180 min after injection of the 2.5 and 4.1 g/kg doses or at 60 min after injection of the 1.8, 2.5, 3.3, 3.8, 4.1, and 4.7 g/kg doses. Opposite effects on blood pH and PCO2 over time were obtained in LS and SS mice at the 2.5 g/kg dose. Acidosis characterized the LS line, whereas alkalosis characterized the SS. The results obtained with SS mice at 4.1 g/kg dose were similar to those obtained with LS mice at the 2.5 g/kg dose. The dose-response curve for the SS mice generated at 60 min post-injection lies to the right of that for the LS mice. The effects of high ethanol doses on SS mice resemble the effects of low doses on LS animals. Thus, the two lines of mice differ in response to the effects of ethanol on these parameters related to respiration. The difference in sensitivity to the respiratory depressant effects of ethanol may contribute to the differences in behavioral sensitivity between the two lines.

Circadian and genetic influences on tissue sensitivity and sleep time to ethanol in LS and SS mice.

Circadian variations in response to ethanol were studied in long-sleep (LS) and short-sleep (SS) mice. Each LS animal received a 2.5 g/kg intraperitoneal ethanol injection, while the SS animals were injected with 4.1 or 5.0 g/kg. Different groups of mice were assessed for sleep time, waking blood alcohol concentration (BAC), and waking brain ethanol concentration (BREC) at 03.00, 09.00, 15.00, or 21.00 hr. Sleep times, waking BACs, and waking BRECs showed circadian variations in the LS mice. SS animals given the 4.1 g/kg dose showed circadian variations for waking BAC and waking BREC, but not for sleep time. The observed variations in the physiological parameters for these animals may have been confounded by a short sleep time so that they reflected circadian variations in drug absorption and/or distribution rather than in CNS sensitivity. SS mice given the 5.0 g/kg dose slept longer than those given the 4.1 g/kg dose and did not show circadian variations for sleep time, waking BAC, or waking BREC. These results suggest both circadian and genetic influences on tissue sensitivity to ethanol.

Fetal alcohol effects in long- and short-sleep mice: activity, passive avoidance, and in utero ethanol levels.

Genetic differences in susceptibility to fetal alcohol effects (FAE) have been suggested by both human and animal studies. The Long-Sleep (LS) and Short-Sleep (SS) mouse lines, selectively bred for differences in ethanol-induced narcosis, provide a model for studying differential alcohol sensitivity in the etiology of FAE. LS and SS mice were intubated with either 2.9 g/kg (20% w/v) ethanol (E) or an isocaloric amount of sucrose (S) twice per day (6 hr apart) on Days 7 through 15 of pregnancy. An untreated control group (C) was maintained for each line. Offspring were fostered to lactating Rockland-Swiss mice at birth. LS offspring prenatally exposed to ethanol exhibited increased open-field activity relative to LS controls, but this effect was due to the overactivity of one litter. Activity for SS mice prenatally exposed to ethanol did not differ from control levels. Ethanol content in blood (280 mg/dl), amniotic fluid (258 mg/dl), and fetal tissue (230 mg/dl) did not differ in similarly treated LS and SS dams. In a second experiment, females were treated from Days 7 through 18 of gestation, and their offspring were tested for either open-field activity or passive avoidance learning. There were no group differences in open-field activity, but LS mice prenatally exposed to alcohol took more trials to reach a passive avoidance criterion than their controls, whereas similarly treated SS mice did not differ from controls. These results suggest that genetically-mediated sensitivity to ethanol influences susceptibility to FAE and that this may be task specific.

Alcohol absorption rate affects hypothermic response in mice: evidence for rapid tolerance.

Three studies were conducted to examine how absorption rate affects rapid tolerance to ethanol-induced hypothermia. Two procedures for administering alcohol were used to vary the rates of ethanol absorption: a 40-min intravenous (IV) infusion and the more rapidly absorbed intraperitoneal (IP) injection. Hypothermic responses of Long-Sleep (LS), Short-Sleep (SS), and DBA/2JIbg (DBA) mice were examined. Although IV ethanol infusions produced similar or higher blood ethanol levels (BELs) compared to IP administration, the degree of hypothermia was significantly greater with the more rapidly absorbed IP administration. It was concluded that the rate at which ethanol increases in the blood influences the degree of hypothermia: the greater the rate of blood ethanol increase, the greater the hypothermic effect. Rapid development of tolerance to ethanol may explain why a slow increase in BEL produces less hypothermia than a rapid increase in BEL. The IV route provides a precise method of drug administration for the study of acute tolerance.

Dose-related growth deficits in LS but not SS mice prenatally exposed to alcohol.

Genetically based alcohol sensitivity may influence the severity of alcohol-related birth defects. To examine this question, measures of growth and survival were examined in offspring of the alcohol sensitive Long-Sleep (LS) and alcohol-resistant Short-Sleep (SS) mouse lines following prenatal ethanol exposure. Pregnant LS and SS mice received an ethanol dose of either 6 or 8 g/kg/day from days 7 through 18 of pregnancy. Control groups received a maltose-dextran solution made isocaloric to the 8 g/kg/day dose. Ethanol and maltose-dextrin solutions were administered as split doses, 6 h apart, via gavage. Nonintubated lab chow control groups were also included for both mouse lines. Offspring were fostered at birth to lactating mice of an outbred stock. Pregnancy was longer for ethanol-treated LS dams compared to maltose-dextrin and lab chow LS control groups, whereas pregnancy length for ethanol-treated SS dams was similar to SS controls. Prenatal ethanol exposure resulted in dose-related growth deficits in LS but not in SS litters. Line differences in postnatal growth deficits in response to prenatal alcohol exposure suggest maternal or fetal alcohol sensitivity influence alcohol-related birth defects.

Alcohol-related birth defects in long- and short-sleep mice: postnatal litter mortality.

Alcohol sensitivity may influence the severity of alcohol-related birth defects (ARBD). To examine this hypothesis, pregnancy outcome and offspring development were examined in alcohol-sensitive Long-Sleep (LS) mice and alcohol-resistant Short-Sleep (SS) mice following prenatal ethanol exposure. Dams were intragastrically intubated twice per day (6 hr apart) with either 4.5 g/kg (20% w/v) ethanol (E) or an isocaloric amount of sucrose (S) on days 7 through 18 of pregnancy. An untreated control group (C) was maintained for each line. Results showed litter mortality at 10 days of age was greater for LS-E litters compared to both LS-S and LS-C litters. Litter mortality for SS-E litters did not differ from either SS-S or SS-C litters. Maternal weight gain, blood ethanol levels, and birth weight deficits were similar for ethanol-exposed LS and SS groups. These results suggest genetically based alcohol sensitivity influences the severity of ARBD.

Ethanol teratogenesis in the C57BL/6J, DBA/2J, and A/J inbred mouse strains.

Research has shown variations in susceptibility to alcohol-related birth defects in humans. Genetic differences are one reason for this variability. This study compared three inbred mouse strains to determine whether they differ in their susceptibilities to ethanol teratogenesis because previous studies have generated conflicting data. Pregnant C57BL/6J (B6), DBA/2J (D2), and A/J (A) dams were intubated intragastrically with either an acute dose of ethanol (5.8 g/kg) or an isocaloric amount of maltose-dextrine on day 9 of pregnancy. Litters were removed on day 18 of pregnancy and examined for gross, soft-tissue, and skeletal malformations. Results showed that ethanol-exposed B6 litters had a higher percentage of digit (19%), kidney (24%), and skeletal (32%, mostly vertebral) malformations than their maltose-exposed controls (7% or below). Prenatal exposure to ethanol increased skeletal (68%, both rib and vertebral) malformations for A litters when compared to their maltose-exposed controls (4%), but did not increase digit or kidney malformations. Ethanol-exposed D2 litters did not differ from maltose-exposed controls. Maternal blood ethanol levels did not differ among the B6, D2, and A strains. These results provide additional evidence suggesting a genetic component to ethanol teratogenesis.

An evaluation of the role of ethanol clearance rate in the differential response of long-sleep and short-sleep mice to ethanol.

Ethanol (ETOH) clearance rates were determined in Long-Sleep (LS) and Short-Sleep (SS) mice, which were selectively bred for differential soporific response to ETOH. Determination of blood ethanol levels at 45 min intervals following administration of 3.8 g/kg IP indicated that SS mice clear ETOH at a faster rate. Repeatedly sampled mice of each line cleared ETOH more slowly than those where samples were taken only once. A second experiment utilized pairs of LS and SS mice matched on body weight. These were treated with one of several doses of ETOH, and duration of loss of the righting reflex measured for one member of the pair. Blood ETOH levels were determined in samples taken from both members of the pair when the tested member regained the righting reflex. LS mice had small but consistently higher blood ETOH levels in these pairs. The magnitude of the differences between the LS and SS mice in these experiments indicate that clearance rate differences can account for only a small portion of the differences in behavioral responses of these mice to ETOH.

Responses to ethanol challenge in long- and short-sleep mice prenatally exposed to alcohol.

Individual sensitivity to alcohol may influence the severity of functional deficits due to prenatal alcohol exposure. To examine this hypothesis, Long-Sleep (LS) and Short-Sleep (SS) mice, selectively bred for differences in ethanol-induced narcosis, were intubated with either 2.9 g/kg ethanol (E) or an isocaloric amount of sucrose (S) twice per day on days 7 through 15 of pregnancy. An untreated control group (C) was maintained for each line. Offspring were fostered to lactating Rockland-Swiss mice at birth. Males and females from each litter were challenged with an acute dose of ethanol (3.8 g/kg) at 30 days of age. Measures of sleep time duration, waking blood ethanol concentrations (BEC), rectal temperatures, heart rate, and ethanol clearance were obtained to examine whether the acute effects of ethanol are altered by prenatal alcohol exposure. Prenatal alcohol exposure did not differentially affect responses to ethanol challenge within either genotype. Ethanol-induced hypothermia, heart-rate depression, and sleep time did differ between genotypes, with LS more affected than SS mice. Ethanol clearance rates were faster for SS than LS mice. These results suggest postnatal pharmacological responses to acute ethanol challenge are not altered by prenatal alcohol exposure in LS and SS mice. Prenatal alcohol-exposed offspring of both mouse genotypes showed lower average heart rate responses than controls, suggesting this measure may be a sensitive indicator of prenatal alcohol effects in mice.

A comparison of ethanol absorption and narcosis in long- and short-sleep mice following intraperitoneal or intragastric ethanol administration.

Blood ethanol concentrations (BEC) were determined in Long-Sleep (LS) and Short-Sleep (SS) mice during a 30 min period following ethanol (ETOH) administration. Absorption of ETOH was rapid and followed a similar time course in the two lines after intraperitoneal (IP) administration of 3.8 or 4.5 g/kg. Following intragastric (IG) administration, slower absorption and lower peak BECs were noted in both lines, but in LS mice this effect was more pronounced. The two routes of administration were not effective in altering duration of loss of the righting reflex (LRR), or waking BECs following 4.5 g/kg ETOH. LS mice had the expected longer LRR durations and lower BECs at waking than did SS mice. Differences in absorption rate and peak BEC are concluded to be unrelated to ETOH neurosensitivity in these mice.

Ethanol teratogenesis in mice selected for differences in alcohol sensitivity.

Long-Sleep (LS) and Short-Sleep (SS) mice, selectively bred for differences in ethanol-induced narcosis, were administered ethanol (2.9, 4.0, 4.5, or 5.0 g/kg) twice per day during the period of organogenesis. On gestation day 18, the dams were sacrificed and the uterine horns were examined for live, dead, and resorbed fetuses. Live fetuses were weighed and assessed for either skeletal or soft tissue anomalies. The 5.8 g/kg/day dose had no effect on prenatal mortality, litter size, body weight, or number of physical anomalies in either line. However, the alcohol-sensitive LS mice exposed to ethanol doses of 8.0 g/kg/day or more evidenced decreased body weights while weights for the alcohol-insensitive SS mice differed from controls at only the highest dose tested. The incidence of skeletal variants was increased in the LS mice exposed to the 10 g/kg/day ethanol dose. These results indicate genetically-mediated alcohol sensitivity increases susceptibility to some of the fetotoxic effects of in utero alcohol exposure.