The centrally projecting Edinger-Westphal nucleus--I: Efferents in the rat brain.

The oculomotor accessory nucleus, often referred to as the Edinger-Westphal nucleus [EW], was first identified in the 17th century. Although its most well known function is the control of pupil diameter, some controversy has arisen regarding the exact location of these preganglionic neurons. Currently, the EW is thought to consist of two different parts. The first part [termed the preganglionic EW-EWpg], which controls lens accommodation, choroidal blood flow and pupillary constriction, primarily consists of cholinergic cells that project to the ciliary ganglion. The second part [termed the centrally projecting EW-EWcp], which is involved in non-ocular functions such as feeding behavior, stress responses, addiction and pain, consists of peptidergic neurons that project to the brainstem, the spinal cord and prosencephalic regions. However, in the literature, we found few reports related to either ascending or descending projections from the EWcp that are compatible with its currently described functions. Therefore, the objective of the present study was to systematically investigate the ascending and descending projections of the EW in the rat brain. We injected the anterograde tracer biotinylated dextran amine into the EW or the retrograde tracer cholera toxin subunit B into multiple EW targets as controls. Additionally, we investigated the potential EW-mediated innervation of neuronal populations with known neurochemical signatures, such as melanin-concentrating hormone in the lateral hypothalamic area [LHA] and corticotropin-releasing factor in the central nucleus of the amygdala [CeM]. We observed anterogradely labeled fibers in the LHA, the reuniens thalamic nucleus, the oval part of the bed nucleus of the stria terminalis, the medial part of the central nucleus of the amygdala, and the zona incerta. We confirmed our EW-LHA and EW-CeM connections using retrograde tracers. We also observed moderate EW-mediated innervation of the paraventricular nucleus of the hypothalamus and the posterior hypothalamus. Our findings provide anatomical bases for previously unrecognized roles of the EW in the modulation of several physiologic systems.

Solvent immersion imprint lithography.

We present Solvent Immersion Imprint Lithography (SIIL), a technique for polymer functionalization and microsystem prototyping. SIIL is based on polymer immersion in commonly available solvents. This was experimentally and computationally analyzed, uniquely enabling two practical aspects. The first is imprinting and bonding deep features that span the 1 to 100 µm range, which are unattainable with existing solvent-based methods. The second is a functionalization scheme characterized by a well-controlled, 3D distribution of chemical moieties. SIIL is validated by developing microfluidics with embedded 3D oxygen sensors and microbioreactors for quantitative metabolic studies of a thermophile anaerobe microbial culture. Polystyrene (PS) was employed in the aforementioned applications; however all soluble polymers - including inorganic ones - can be employed with SIIL under no instrumentation requirements and typical processing times of less than two minutes.

Planar thin film device for capillary electrophoresis.

Hollow tubular microfluidic channels were fabricated on quartz substrates using sacrificial layer, planar micromachining processes. The channels were created using a bottom-up fabrication technique, namely patterning a photoresist/aluminum sacrificial layer and depositing SiO(2) over the substrate. The photoresist/aluminum layer was removed by etching first with HCl/HNO(3), followed by etching in Nano-Strip, a more stable form of piranha (H(2)SO(4)/H(2)O(2)) stripper. Rapid separation of fluorescently labeled amino acids was performed on a device made with these channels. The fabrication process presented here provides unique control over channel composition and geometry. Future work should allow the fabrication of highly complex and precise devices with integrated analytical capabilities essential for the development of micro-total analysis systems.

Chemical inhibition of nitrification in activated sludge.

Conventional aerobic nitrification was adversely affected by single pulse inputs of six different classes of industrially relevant chemical toxins: an electrophilic solvent (1-chloro-2,4-dinitrobenzene, CDNB), a heavy metal (cadmium), a hydrophobic chemical (1-octanol), an uncoupling agent (2,4-dinitrophenol, DNP), alkaline pH, and cyanide in its weak metal complexed form. The concentrations of each chemical source that caused 1 5, 25, and 50% respiratory inhibition of a nitrifying mixed liquor during a short-term assay were used to shock sequencing batch reactors containing nitrifying conventional activated sludge. The reactors were monitored for recovery over a period of 30 days or less. All shock conditions inhibited nitrification, but to different degrees. The nitrate generation rate (NGR) of the shocked reactors recovered overtime to control reactor levels and showed that it was a more sensitive indicator of nitrification inhibition than both initial respirometric tests conducted on unexposed biomass and effluent nitrogen species analyses. CDNB had the most severe impact on nitrification, followed by alkaline pH 11, cadmium, cyanide, octanol, and DNP. Based on effluent data, cadmium and octanol primarily inhibited ammonia-oxidizing bacteria (AOB) while CDNB, pH 11,and cyanide inhibited both AOB and nitrite-oxidizing bacteria (NOB). DNP initially inhibited nitrification but quickly increased the NGR relative to the control and stimulated nitrification after several days in a manner reflective of oxidative uncoupling. The shocked mixed liquor showed trends toward recovery from inhibition for all chemicals tested, but in some cases this reversion was slow. These results contribute to our broader effort to identify relationships between chemical sources and the process effects they induce in activated sludge treatment systems.

Deflocculation effects due to chemical perturbations in sequencing batch reactors.

Toxic shock-induced deflocculation was examined for activated sludge exposed to six different classes of industrially relevant chemical toxins: an electrophilic solvent (1-chloro-2,4-dinitrobenzene, CDNB), a heavy metal (cadmium), a hydrophobic chemical (1-octanol), an uncoupling agent (2,4-dinitrophenol, DNP), alkaline pH, and weakly complexed cyanide. The concentrations required to inhibit respiration by 50% were used to shock sequencing batch reactors (SBRs) containing a nitrifying (10-day solids retention time (SRT)) and a non-nitrifying (2-day SRT) biomass. Effluent total suspended solids (TSS) and soluble potassium were monitored to examine deflocculation caused by a bacterial stress response mechanism called glutathione-gated potassium efflux (GGKE). Reactors were monitored for recovery over a period of 3 SRTs or less. At the concentrations tested, CDNB, cadmium and pH 11 were found to cause significant increases in effluent TSS concentrations and showed elevated levels of potassium. In contrast, octanol, DNP and cyanide did not induce severe deflocculation and showed moderate increases in effluent potassium levels. Recovery of effluent TSS and potassium concentrations to control levels generally did not correlate, supporting the hypothesis that reflocculation requires regrowth of biomass. These results suggest that different chemicals induce deflocculation in SBRs, but deflocculation is not necessarily caused by the GGKE mechanism in all cases.

Temporal and receptor correlates of the estrogen response in sheep.

Uterine blood flow and uterine cytosol and nuclear estrogen receptors were measured at critical times during estradiol-induced vasodilatation in acute anesthetized and chronic conscious sheep preparations at estradiol bolus injection frequencies from 1 to 24 hours. During acute experiments, the uterine blood flow response was muted and cytosol estrogen receptor replenishment did not occur whereas full replenishment occurred in 12 hours in conscious ewes. In ewes treated with daily estradiol that had a stable daily uterine blood flow response, the uterine blood flow response 24 hours after uterine biopsy was similar to the preoperative one. Analysis of the duration of peak uterine blood flow levels and the rate of uterine blood flow descent from peak levels showed that an interval of 18 hours between estradiol injections was necessary for the uterine blood flow response to approximate that observed after 24 hours. These observations suggest: (1) that uterine vascular receptor replenishment is delayed compared with that of the total uterus; (2) that operative stress compromises cytosol estrogen receptor metabolism and possibly nuclear estrogen receptor function; (3) that the delayed maximum uterine blood flow response to estradiol in ewes previously untreated with estradiol is due to a trophic uterine effect of daily estradiol stimulation.

Variable decelerations during nonstress tests are not a sign of fetal compromise.

An examination of 908 fetal heart rate tests of 418 consecutive patients revealed brief variable decelerations in more than 50.7% of the patients. Although an association existed with nuchal cord location found at delivery, no association existed between these variable decelerations and fetal heart rate decelerations during labor, low Apgar scores at birth, or birth weight. We find no evidence to suggest that these brief variable decelerations are a sign of fetal compromise or an indication for obstetric intervention.

Catecholamine responses in fetal lambs subjected to hemorrhage.

We monitored plasma epinephrine and norepinephrine responses to hemorrhage of 20% of estimated blood volume in 11 chronically instrumented, unanesthetized fetal lambs. In addition, we performed control experiments--blood sampling but no hemorrhage--in five fetuses. Arterial blood gases, pH, mean arterial pressures, heart rates, and plasma norepinephrine and epinephrine levels were similar in both groups in the resting state. Arterial blood gases and pH did not change significantly during the experimental period in either group. Mean arterial pressure, heart rate, and plasma norepinephrine and epinephrine levels did not change in the control group during the experimental period. Hemorrhage was associated with a significant decrease in fetal mean arterial pressure, 39.8 +/- 1.2 to 29.6 +/- 2.1 mm Hg, and heart rate, 186 +/- 8 to 146 +/- 6 bpm (p less than 0.01 in both cases). There was a significant increase in plasma norepinephrine, 664.9 +/- 91.6 to 1384.8 +/- 216.7 pg/ml (p less than 0.02) and epinephrine, 224.6 +/- 43.6 to 681.7 +/- 199.0 pg/ml (p less than 0.01) with hemorrhage. These results demonstrate significant catecholamine responses to hypovolemia in the fetal lamb.

A vasopressin antagonist blocks the norepinephrine and epinephrine responses to hemorrhage in the fetus.

In 8 chronically cannulated fetal lambs between 119 and 127 days gestation the resting plasma norepinephrine concentration was 528 +/- 77 pg X ml-1 and the resting plasma epinephrine concentration 159 +/- 42 pg X ml-1. Hemorrhage of 20% of estimated blood volume at 2% per min produced a 2.1-fold increase in plasma norepinephrine levels and a 3.4-fold increase in plasma epinephrine levels when the animals were pretreated with an injection of saline (1 ml). Plasma catecholamine levels returned toward control values following return of the shed blood. In contrast, hemorrhage of these animals following pretreatment with an antagonist of the pressor effect of vasopressin did not cause an increase in fetal plasma catecholamine levels. Thus, vasopressin may mediate the sympathetic responses to volume depletion in the fetus.

Use of avidin-biotinylated horseradish peroxidase complex for visualization of spirochetes.

The use of avidin-biotinylated peroxidase as a simple technique for light microscopic visualization of spirochetes is described. The three major genera of spirochetes--Treponema, Borrelia, and Leptospira--were stained with the avidin complex.

Vasopressin is important for restoring cardiovascular homeostasis in fetal lambs subjected to hemorrhage.

To determine if the posterior pituitary hormone vasopressin is important for maintaining fetal cardiovascular homeostasis during hypovolemic stress, in seven chronically catheterized fetal lambs we induced hemorrhage of 20% of estimated blood volume in the presence and in the absence of a potent antagonist to the pressor effects of vasopressin. The study was a paired crossover design with at least 48 hours separating experiments in the same animal. Injection of the vasopressin antagonist did not alter basal fetal heart rate or arterial blood pressure, but hemorrhage of 2% of estimated fetal blood volume per minute for 10 minutes produced a greater fall in blood pressure (13 +/- 2 versus 10 +/- 2 torr, p less than 0.05) when the blocker was present than when it was absent. Arterial blood pressure remained below control levels longer following hemorrhage when the fetuses were pretreated with the antagonist (49.7 +/- 6 versus 26.6 +/- 6 minutes, p less than 0.01), and the integrated fall in arterial blood pressure with hemorrhage was greatest (283 +/- 53 versus 169 +/- 57 mm Hg . min p less than 0.01) when the blocker was used. The fall in heart rate following hemorrhage was similar with and without blocker pretreatment. These results indicate that vasopressin plays a physiologic role in blood pressure regulation in fetal lambs during periods of hypovolemia.