In vivo imaging of zebrafish reveals differences in the spinal networks for escape and swimming movements.

Most studies of spinal interneurons in vertebrate motor circuits have focused on the activity of interneurons in a single motor behavior. As a result, relatively little is known about the extent to which particular classes of spinal interneurons participate in different behaviors. Similarities between the morphology and connections of interneurons activated in swimming and escape movements in different fish and amphibians led to the hypothesis that spinal interneurons might be shared by these behaviors. To test this hypothesis, we took advantage of the optical transparency of zebrafish larvae and developed a new preparation in which we could use confocal calcium imaging to monitor the activity of individual identified interneurons noninvasively, while we simultaneously filmed the movements of the fish with a high-speed digital camera. With this approach, we could directly examine the involvement of individual interneurons in different motor behaviors. Our work revealed unexpected differences in the interneurons activated in swimming and escape behaviors. The observations lead to predictions of different behavioral roles for particular classes of spinal interneurons that can eventually be tested directly in zebrafish by using laser ablations or mutant lines with interneuronal deficits.

A confocal study of spinal interneurons in living larval zebrafish.

We used confocal microscopy to examine the morphology of spinal interneurons in living larval zebrafish with the aim of providing a morphological foundation for generating functional hypotheses. Interneurons were retrogradely labeled by injections of fluorescent dextrans into the spinal cord, and the three-dimensional morphology of living cells was reconstructed from confocal optical sections through the transparent fish. At least eight types of interneurons are present in the spinal cord of larval zebrafish; four of these are described here for the first time. The newly discovered cell types include classes of commissural neurons with axons that ascend, descend, and bifurcate in the contralateral spinal cord. Our reexamination of previously described cell types revealed functionally relevant features of their morphology, such as undescribed commissural axons, as well as the relationships between the trajectories of the axons of interneurons and the descending Mauthner axons. In addition to describing neurons, we surveyed their morphology at multiple positions along the spinal cord and found longitudinal changes in their distribution and sizes. For example, some cell types increase in size from rostral to caudal, whereas others decrease. Our observations lead to predictions of the roles of some of these interneurons in motor circuits. These predictions can be tested with the combination of functional imaging, single-cell ablation, and genetic approaches that make zebrafish a powerful model system for studying neuronal circuits.

Postnatal growth curve of the infant with extremely low birth weight who was fed enterally.

The primary objective of this study was the establishment of a postnatal growth curve for the very low-birth-weight infant. Only infants whose size was appropriate for gestational age and whose predominant form of nourishment was enteral were included in the study. Two growth curves were constructed: one for infants weighing less than 900 g (group A, birth weight 799 +/- 79 [SD] g, mean gestational age 26.5 weeks), and one for infants weighing 901 to 1,100 g (group B, birth weight 1,023 +/- 53 [SD] g, mean gestational age 28.5 weeks). Growth was followed over the first 50 postnatal days. Group A infants gained an average of 10.2 g/d overall during the first 50 postnatal days and group B infants gained an average of 17.1 g/d over the same period. Because the major objective of this study was construction of a growth curve for infants weighing less than 900 g, direct comparison is made with the Dancis grid (1948) as this is the only standard for this group. The growth rates of our infants were found to be more than twice that of the original prediction of Dancis.

A prospective study of free bilirubin and other risk factors in the development of kernicterus in premature infants.

To assess the value of free bilirubin (FB) measurements in predicting kernicterus (KI) in sick premature infants, 91 newborns weighing less than 1,500 gm at birth were observed during the first week of like with twice daily FB and total bilirubin determinations. Autopsies were performed on 30 of the 53 infants who died. Seven had KI and 23 did not. There were no differences between infants with and with out KI in the maximum FB level (KI 18.2 +/- 4.5 [SEM] nm/liter, no KI 11.1 +/- 0.9 nm/liter, P not significant) or the total bilirubin level (KI 7.3 +/- 1.3 mg/100 ml, no KI 6.1 +/- 0.5 mg/100 ml, P not significant). In fact, three kernicteric infants had very low maximum FB levels (less than 10 nm/liter). These three infants had prolonged episodes of acidosis, hypoxemia, or hypothermia during the 24 hours preceding their maximum level of FB. Although elevated levels of FB may be predictive of KI in some infants, other factors may make the blood-brain barrier more permeable to low levels of FB. This may limit the clinical applicability of FB measurements.

Diminished bilirubin binding by the serum of pregnant women.

Enzymatic peroxidase oxidation was used to study albumin-bilirubin binding in the serum of pregnant women, their term infants, and nonpregnant women. When the bilirubin titration curves were corrected for differing albumin concentrations in the three groups, the bilirubin binding of pregnant women was inferior to that of control women but better than that of their infants. Diminished maternal binding could not be explained by altered affinity. This suggests that the function of albumin during pregnancy is impaired by a factor common to both fetal and maternal circulations that appears to act by decreasing the number of available bilirubin binding sites.

Influence of gestational age on cord serum bilirubin binding studies.

The effect of gestational age on bilirubin binding was studied using cord serum from 22 preterm infants and 13 term infants and serum from 17 adults. Using the peroxidase oxidation method, a bilirubin titration curve was obtained by adding bilirubin to serum and measuring the apparent unbound bilirubin concentration. The resultant curve was analyzed using the least-squares fit of the empiric equation Y = aXb. After correction for albumin concentration by plotting the apparent unbound bilirubin concentration against the molar ratio of total bilirubin/albumin, term and preterm infants had identical titration curves, which remained inferior to that of adults. In addition, the apparent primary bilirubin association constant Ka'1 was similar for all infants but was two to three times less than that for adults. We conclude that bilirubin binding by cord serum is equivalent regardless of gestational age. However, adult serum binds bilirubin qualitatively better than does serum from infants of all gestational ages. We suggest that the adverse effect of prematurity on bilirubin binding noted in previous studies may have reflected postnatal complications rather than gestational age as such.

Bilirubin binding in premature infants from birth to 3 months.

The effect of postnatal age on serum bilirubin binding measurements was studied prospectively in extremely premature infants of 25-28 weeks' gestation. Serum was obtained from 10 infants at birth, 2-4 days of age, 1 week, 2 weeks, 4 weeks, 8 weeks, and 10-13 weeks. Using peroxidase oxidation, the apparent unbound bilirubin concentration (AUBC) was measured and plotted versus the molar ratio of total bilirubin:albumin (R) using the empiric power curve AUBC = aRb. The apparent unbound bilirubin concentration at bilirubin:albumin ratio 0.6 was used to compare relative binding ability among specimens. This value, as well as the apparent association constants, showed dramatic deterioration after birth, which persisted without improvement until 8 weeks of age. This pattern of recovery correlated in general with the resolution of clinical problems. Binding values equivalent to adult serum were achieved by 10-13 weeks. This study emphasises that diminished bilirubin binding by the sera of premature infants can persist for a prolonged period.

The effect of hematin on bilirubin binding in bilirubin-enriched neonatal cord serum.

It has been suggested that the serum of infants with ABO hemolytic disease contains hematin which might interfere with bilirubin binding to albumin, increasing the risk of kerincterus. Using the horseradish peroxidase assay, we investigated this possibility. Cord serum from healthy term infants was enriched with bilirubin to a bilirubin/albumin molar ratio of 0.6, and then varying amounts of hematin were added. Hematin had a significant adverse effect on bilirubin binding when present in half to equimolar ratios with bilirubin. This high concentration of hematin does not appear to be present in serum from infants with ABO hemolytic disease and no difference was seen when comparing the dilute and the undilute peroxidase assays. These data suggest that hematin can alter bilirubin binding in neonatal serum but is not os physiologic importance for infants with ABO-isoimmune hemolysis.

Epaxial muscle function in trotting dogs.

One of the features that distinguish mammals from other groups of terrestrial vertebrates is the structure and relative size of their epaxial muscles. Yet we have only a superficial understanding of the role these muscles play in locomotion. To address their locomotor function, we recorded the electrical activity of the iliocostalis, longissimus dorsi and multifidus muscles of trotting dogs. Activity was monitored at both lumbar and thoracic sites. To develop and evaluate hypotheses of epaxial muscle function, we quantified footfall patterns and sagittal trunk kinematics from high-speed videos, and the magnitude and orientation of ground reaction forces from force-plate recordings. All three epaxial muscles tended to exhibit a double-bursting (biphasic) activity pattern, with the exception of the iliocostalis muscle at the thoracic site (which was uniphasic). In general, a large burst of activity in each muscle occurred during the second half of the support phase of the ipsilateral hindlimb, and was active for an average of 30% of the locomotor cycle. A smaller burst of activity occurred during the second half of the support phase of the contralateral hindlimb, and was active for an average of 15% of the locomotor cycle. Analysis of ground reaction forces and sagittal trunk kinematics led us to the hypothesis that the epaxial muscles do not directly stabilize the trunk against the vertical and horizontal components of the ground reaction force. Instead, the epaxial muscles appear to counteract the tendency of the trunk to rebound (flex) in the sagittal plane during the latter half of the support phase. This hypothesis of epaxial muscle function was supported by loading experiments performed on the longissimus dorsi muscle in the lumbar region.

Modification of the colorimetric assay for chloramphenicol in the presence of bilirubin.

We recently found that bilirubin interferes with the colorimetric chloramphenicol assay of Kakemi et al. (K. T. Kakemi, T. Arita, and S. Ohashi, Yakugaku Zasshi 82:342-345, 1962). Levels of serum bilirubin alone (4 to 6 mg/dl) resulted in apparent concentrations of chloramphenicol which appear to be in the therapeutic range. Concentrations of serum bilirubin greater than 8 mg/dl resulted in levels of apparent chloramphenicol associated with toxicity (>50 mug/ml). Small amounts of activated charcoal added to the isoamyl acetate extraction step of the assay eliminated this interference by bilirubin.

Influence of intravenous fat emulsion on serum bilirubin in very low birthweight neonates.

Thirty nine very low birthweight neonates (with a birth weight of 820 to 1500 g and gestation of 27 to 34 weeks) who required total parenteral nutrition were randomly assigned to one of three regimens of administration of fat emulsion for a period of eight days. Groups 1 and 2 received the emulsion at a constant rate over 24 and 16 hours, respectively, beginning with a daily dosage of 1 g/kg and increasing daily by 1 g/kg to a maximum of 4 g/kg. Group 3 received the emulsion at a constant rate of 4 g/kg a day over 24 hours. Plasma concentrations of free fatty acids and serum concentrations of total bilirubin, apparent unbound bilirubin, and albumin were measured at regular intervals. Effects of the three regimens on serum bilirubin measurements were determined. The regimen of fat infusion and rate of infusion seemed to have no effect on serum concentrations of total and apparent unbound bilirubin, although there was a trend towards greater variability in apparent unbound concentrations with the intermittent regimen.

Fetal cystic hygroma and Turner's syndrome.

Large nuchal cystic hygromas were observed in five second-trimester aborted fetuses at autopsy. Two female fetuses with generalized edema were karyotyped as 45,X. One of these was the twin of a 46,XX normal female sibling. The association of generalized edema with large nuchal cystic hygromas was seen only in these two fetuses and represents strong phenotypic evidence of Turner's syndrome. However, the absence of hydrops was not a reliable indicator of normal karyotype. One fetus without generalized edema was karyotyped as 47,XY, +21, inv(9). The remaining cases had normal karyotypes. Placental histology was not useful in discriminating monosomy X from other conditions, but placental tissue culture was important in obtaining a cytogenetic diagnosis. Karyotyping is recommended in all cases of fetal cystic hygroma.