Deciphering structure and aggregation in asphaltenes: hypothesis-driven design and development of synthetic model compounds.

Asphaltenes comprise the heaviest and least understood fraction of crude petroleum. The asphaltenes are a diverse and complex mixture of organic and organometallic molecules in which most of the molecular constituents are tightly aggregated into more complicated suprastructures. The bulk properties of asphaltenes arise from a broad range of polycyclic aromatics, heteroatoms, and polar functional groups. Despite much analytical effort, the precise molecular architectures of the material remain unresolved. To understand asphaltene characteristics and reactivity, the field has turned to synthetic model compounds that mirror asphaltene structure, aggregation behavior, and thermal chemistry, including the nucleation of coke. Historically, molecular asphaltene modeling was limited to commercial compounds, offering little illumination and few opportunities for hypothesis-driven research. More recently, however, rational molecular design and modern organic synthesis have started to impact this area. This review provides an overview of commercially available model compounds but is principally focused on the design and synthesis of structurally advanced and appropriately functionalized compounds to mimic the physical and chemical behavior of asphaltenes. Efforts to model asphaltene aggregation are briefly discussed, and a prognosis for the field is offered. A referenced tabulation of the synthetic compounds reported to date is provided.

Enantioselective Synthesis of Polycyclic γ-Lactams with Multiple Chiral Carbon Centers via Ni(0)-Catalyzed Asymmetric Carbonylative Cycloadditions without Stirring.

γ-Lactam derivatives with multiple contiguous stereogenic carbon centers are ubiquitous in physiologically active compounds. The development of straightforward and reliable synthetic routes to such chiral structural motifs in a stereocontrolled manner should thus be of importance. Herein, we report a strategy to construct polycyclic γ-lactam derivatives that contain more than two contiguous stereogenic centers in an enantioselective as well as atom-economic manner. Moreover, we have achieved the first enantioselective synthesis of strigolactam derivative GR-24, a racemic variant of which is a potential seed germination stimulator and plant-growth regulator. A key of the procedure presented here is a nickel(0)/chiral phosphoramidite-catalyzed asymmetric [2+2+1] carbonylative cycloaddition between readily accessible ene-imines and carbon monoxide, which proceeded enantioselectively to furnish up to 90% ee (>99% ee after recrystallization). The results of mechanistic studies, including the isolation of a chiral heteronickelacycle, support that the enantioselectivity on the two contiguous carbon atoms of the γ-lactams is determined during the oxidative cyclization on nickel(0).

Acute toxicity of copper to the larval stage of three species of ambystomatid salamanders.

Copper (Cu) appears to be consistently more toxic to anuran species relative to other vertebrate taxa. There are limited Cu toxicity data for salamanders; of the few studies conducted on salamanders, most examined Cu effects on the embryonic, but not the larval, stage. We performed acute toxicity experiments, to quantify LC50s, on Harrison stage 46 larvae (free swimming hatchlings with egg yolk completely absorbed) of three ambystomatid salamander species. Each LC50 experiment used exposure concentrations of 10, 20, 30, 40, 50, and 60 µg/L with 10 replicates per concentration each containing one larva. We found very high toxicity for all species compared to previously published research on the embryo stage. Specifically, the 4-d LC50s for Ambystoma tigrinum and A. opacum were 35.3 and 18.73 µg/L, respectively. The same Cu concentrations caused similar toxicity to A. talpoideum (LC50 = 47.88 µg/L), but exposures required up to 48 d to elicit the same level of mortality. A time-to-event analysis indicated that time to mortality was significantly affected by Cu concentration. Additionally, for A. talpoideum, we observed that elevated levels of Cu decreased growth rate. Comparisons with previously reported Cu toxicity for embryos suggest that, as with fish, Cu may be more toxic to larval salamander stages than for embryos. Further, our data suggest that Cu is an important environmental contaminant that deserves increased scrutiny on the potential for population-level effects where contamination has occurred in wetlands and streams inhabited by salamanders.

Genomic data detect corresponding signatures of population size change on an ecological time scale in two salamander species.

Understanding the demography of species over recent history (e.g. <100 years) is critical in studies of ecology and evolution, but records of population history are rarely available. Surveying genetic variation is a potential alternative to census-based estimates of population size, and can yield insight into the demography of a population. However, to assess the performance of genetic methods, it is important to compare their estimates of population history to known demography. Here, we leveraged the exceptional resources from a wetland with 37 years of amphibian mark-recapture data to study the utility of genetically based demographic inference on salamander species with documented population declines (Ambystoma talpoideum) and expansions (A. opacum), patterns that have been shown to be correlated with changes in wetland hydroperiod. We generated ddRAD data from two temporally sampled populations of A. opacum (1993, 2013) and A. talpoideum (1984, 2011) and used coalescent-based demographic inference to compare alternate evolutionary models. For both species, demographic model inference supported population size changes that corroborated mark-recapture data. Parameter estimation in A. talpoideum was robust to our variations in analytical approach, while estimates for A. opacum were highly inconsistent, tempering our confidence in detecting a demographic trend in this species. Overall, our robust results in A. talpoideum suggest that genome-based demographic inference has utility on an ecological scale, but researchers should also be cognizant that these methods may not work in all systems and evolutionary scenarios. Demographic inference may be an important tool for population monitoring and conservation management planning.

Integrating copper toxicity and climate change to understand extinction risk to two species of pond-breeding anurans.

Chemical contamination is often suggested as an important contributing factor to amphibian population declines, but direct links are rarely reported. Population modeling provides a quantitative method to integrate toxicity data with demographic data to understand the long-term effects of contaminants on population persistence. In this study we use laboratory-derived embryo and larval toxicity data for two anuran species to investigate the potential for toxicity to contribute to population declines. We use the southern toad (Anaxyrus terrestris) and the southern leopard frog (Lithobates sphenocephalus) as model species to investigate copper (Cu) toxicity. We use matrix models to project populations through time and quantify extinction risk (the probability of quasi-extinction in 35 yr). Life-history parameters for toads and frogs were obtained from previously published literature or unpublished data from a long-term (>35 yr) data set. In addition to Cu toxicity, we investigate the role of climate change on amphibian populations by including the probability of early pond drying that results in catastrophic reproductive failure (CRF, i.e., complete mortality of all larval individuals). Our models indicate that CRF is an important parameter for both species as both were unable to persist when CRF probability was >50% for toads or 40% for frogs. Copper toxicity alone did not result in significant effects on extinction risk unless toxicity was very high (>50% reduction in survival parameters). For toads, Cu toxicity and high probability of CRF both resulted in high extinction risk but no synergistic (or greater than additive) effects between the two stressors occurred. For leopard frogs, in the absence of CRF survival was high even under Cu toxicity, but with CRF Cu toxicity increased extinction risk. Our analyses highlight the importance of considering multiple stressors as well as species differences in response to those stressors. Our models were consistently most sensitive to juvenile and adult survival, further suggesting the importance of terrestrial stages to population persistence. Future models will incorporate multiple wetlands with different combinations of stressors to understand if our results for a single wetland result in a population sink within the landscape.

Patterns of amphibian infection prevalence across wetlands on the Savannah River Site, South Carolina, USA.

Amphibian diseases, such as chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) and ranaviral disease caused by ranaviruses, are often linked to global amphibian population declines, yet the ecological dynamics of both pathogens are poorly understood. The goal of our study was to determine the baseline prevalence, pathogen loads, and co-infection rate of Bd and ranavirus across the Savannah River Site (SRS) in South Carolina, USA, a region with rich amphibian diversity and a history of amphibian-based research. We tested over 1000 individuals, encompassing 21 amphibian species from 11 wetlands for both Bd and ranavirus. The prevalence of Bd across individuals was 9.7%. Using wetland means, the mean (±SE) Bd prevalence was 7.9 ± 2.9%. Among toad species, Anaxyrus terrestris had 95 and 380% greater odds of being infected with Bd than Scaphiopus holbrookii and Gastrophryne carolinensis, respectively. Odds of Bd infection in adult A. terrestris and Lithobates sphenocephalus were 75 to 77% greater in metal-contaminated sites. The prevalence of ranavirus infections across all individuals was 37.4%. Mean wetland ranavirus prevalence was 29.8 ± 8.8% and was higher in post-metamorphic individuals than in aquatic larvae. Ambystoma tigrinum had 83 to 85% higher odds of ranavirus infection than A. opacum and A. talpoideum. We detected a 4.8% co-infection rate, with individuals positive for ranavirus having a 5% higher occurrence of Bd. In adult Anaxyrus terrestris, odds of Bd infection were 13% higher in ranavirus-positive animals and odds of co-infection were 23% higher in contaminated wetlands. Overall, we found the pathogen prevalence varied by wetland, species, and life stage.

Effects of metal and predator stressors in larval southern toads (Anaxyrus terrestris).

Natural and anthropogenic stressors typically do not occur in isolation; therefore, understanding ecological risk of contaminant exposure should account for potential interactions of multiple stressors. Realistically, common contaminants can also occur chronically in the environment. Because parental exposure to stressors may cause transgenerational effects on offspring, affecting their ability to cope with the same or novel environmental stressors, the exposure histories of generations preceding that being tested should be considered. To examine multiple stressor and parental exposure effects we employed a 2 × 2 × 2 factorial design in outdoor 1000-L mesocosms (n = 24). Larval southern toads (Anaxyrus terrestris), bred from parents collected from reference and metal-contaminated sites, were exposed to two levels of both an anthropogenic (copper-0, 30 µg/L Cu) and natural (predator cue - present/absent) stressor and reared to metamorphosis. Toads from the metal-contaminated parental source population were smaller at metamorphosis and had delayed development; i.e., a prolonged larval period. Similarly, larval Cu exposure also reduced size at metamorphosis and prolonged the larval period. We, additionally, observed a significant interaction between larval Cu and predator-cue exposure on larval period, wherein delayed emergence was only present in the 30-µg/L Cu treatments in the absence of predator cues. The presence of parental effects as well as an interaction between aquatic stressors on commonly measured endpoints highlight the importance of conducting multistressor studies across generations to obtain data that are more relevant to field conditions in order to determine population-level effects of contaminant exposure.

Scalable, chromatography-free synthesis of alkyl-tethered pyrene-based materials. Application to first-generation "archipelago model" asphaltene compounds.

In this paper, we report a highly efficient, scalable approach to the total synthesis of conformationally unrestricted, electronically isolated arrays of alkyl-tethered polycyclic aromatic chromophores. This new class of modular molecules consists of polycyclic aromatic "islands" comprising significant structural fragments present in unrefined heavy petroleum, tethered together by short saturated alkyl chains, as represented in the "archipelago model" of asphaltene structure. The most highly branched archipelago compounds reported here share an architecture with first-generation dendrimeric constructs, making the convergent, chromatography-free synthesis described herein particularly attractive for further extensions in scope and applications to materials chemistry. The syntheses are efficient, selective, and readily adaptable to a multigram scale, requiring only inexpensive, "earth-abundant" transition-metal catalysts for cross-coupling reactions and extraction and fractional crystallization for purification. This approach avoids typical limitations in cost, scale, and operational practicality. All of the archipelago compounds and synthetic intermediates have been fully characterized spectroscopically and analytically. The solid-state structure of one archipelago model compound has been determined by X-ray crystallography.

Development of an on-animal separation-based sensor for monitoring drug metabolism in freely roaming sheep.

The development of an on-animal separation-based sensor that can be employed for monitoring drug metabolism in a freely roaming sheep is described. The system consists of microdialysis sampling coupled to microchip electrophoresis with electrochemical detection (MD-ME-EC). Separations were accomplished using an all-glass chip with integrated platinum working and reference electrodes. Discrete samples from the microdialysis flow were introduced into the electrophoresis chip using a flow-gated injection approach. Electrochemical detection was accomplished in-channel using a two-electrode isolated potentiostat. Nitrite was separated by microchip electrophoresis using reverse polarity and a run buffer consisting of 50 mM phosphate at pH 7.4. The entire system was under telemetry control. The system was first tested with rats to monitor the production of nitrite following perfusion of nitroglycerin into the subdermal tissue using a linear probe. The data acquired using the on-line MD-ME-EC system were compared to those obtained by off-line analysis using liquid chromatography with electrochemical detection (LC-EC), using a second microdialysis probe implanted parallel to the first probe in the same animal. The MD-ME-EC device was then used on-animal to monitor the subdermal metabolism of nitroglycerin in sheep. The ultimate goal is to use this device to simultaneously monitor drug metabolism and behavior in a freely roaming animal.

Terrestrial distribution of pond-breeding salamanders around an isolated wetland.

Terrestrial habitats surrounding isolated wetlands are a critical resource for many pond-breeding amphibian species, yet few studies have examined the terrestrial distribution of post-metamorphic juveniles and adults. We used an encircling drift fence at a breeding pond in conjunction with partial fences at 90, 172, and 332 m from the wetland to estimate the terrestrial distribution of adult marbled salamanders (Ambystoma opacum; four breeding seasons) and mole salamanders (A. talpoideum; two seasons), as well as the dispersion of newly metamorphosed A. opacum (one summer). For newly metamorphosed A. opacum, 79% emigrated < 90 m from the wetland, and 8% moved beyond 172 m; movement distance was unrelated to body size. Distribution of adult A. opacum varied among years, with an average of 28% (range 23-31%) occurring beyond 172 m in all years. Averaged across two years, 51% of adult A. talpoideum occurred beyond 172 m. Lognormal models provided a good fit to both the juvenile and adult ambystomatid distributions, and parameters differed between age classes, sexes, species, and years within species. For adult A. opacum a buffer radius of 300 m or 340 m, depending on the year, is estimated to include 95% of adults; for A. talpoideum the estimate is 464 m or 501 m. A reanalysis of distribution data for seven ambystomatid species shows that a previous estimate of a 164-m radius to protect 95% of a population underestimates the needed buffer radius by 185 m. Because our study wetland requires a nearly 500 m wide radius to protect 95% of its ambystomatid adults, preservation of similar communities may require much more surrounding terrestrial habitat than previously thought.

Development and optimization of an integrated PDMS based-microdialysis microchip electrophoresis device with on-chip derivatization for continuous monitoring of primary amines.

An all-PDMS on-line microdialysis-microchip electrophoresis with on-chip derivatization and electrophoretic separation for near real-time monitoring of primary amine-containing analytes is described. Each part of the chip was optimized separately, and the effect of each of the components on temporal resolution, lag time, and separation efficiency of the device was determined. Aspartate and glutamate were employed as test analytes. Derivatization was accomplished with naphthalene-2,3,-dicarboxyaldehyde/cyanide (NDA/CN(-)), and the separation was performed using a 15-cm serpentine channel. The analytes were detected using LIF detection.

Microdialysis sampling coupled to microchip electrophoresis with integrated amperometric detection on an all-glass substrate.

The development of an all-glass separation-based sensor using microdialysis coupled to microchip electrophoresis with amperometric detection is described. The system includes a flow-gated interface to inject discrete sample plugs from the microdialysis perfusate into the microchip electrophoresis system. Electrochemical detection was accomplished with a platinum electrode in an in-channel configuration using a wireless electrically isolated potentiostat. To facilitate bonding around the in-channel electrode, a fabrication process was employed that produced a working and a reference electrode flush with the glass surface. Both normal and reversed polarity separations were performed with this sensor. The system was evaluated in vitro for the continuous monitoring of the production of hydrogen peroxide from the reaction of glucose oxidase with glucose. Microdialysis experiments were performed using a BASi loop probe with an overall lag time of approximately five minutes and a rise time of less than 60 seconds.

Maternal transfer of contaminants and reduced reproductive success of southern toads ( Bufo [Anaxyrus] terrestris ) exposed to coal combustion waste.

Bioaccumulation of contaminants and subsequent maternal transfer to offspring are important factors that affect the reproductive success of wildlife. However, maternal transfer of contaminants has rarely been investigated in amphibians. We examined maternal transfer of trace elements in southern toads ( Bufo[Anaxyrus] terrestris) residing in two locations: (1) an active coal combustion waste (CCW) disposal basin and adjacent 40-ha floodplain contaminated with CCW over 35 years ago and (2) an uncontaminated reference site. Our study is among the few to document tissue concentration-dependent maternal transfer of contaminants and associated adverse effects in amphibians. We found that females collected from the CCW-contaminated area had elevated concentrations of Ni, Se, and Sr; these females also transferred elevated levels of Cu, Pb, Se, and Sr to their eggs compared to females from the reference site. Overall reproductive success, estimated as a function of clutch size and offspring viability, was reduced by 27% in clutches collected from parents from the contaminated site compared to the reference site. Offspring viability negatively correlated with female and/or egg concentrations of Se and Ni. Reproductive success negatively correlated with Se and Cu concentrations in females, and Se concentrations in eggs. Our study highlights how exposure to CCW can negatively affect amphibian reproduction.

Effects of two stressors on amphibian larval development.

In parallel with a renewed interest in nuclear power and its possible environmental impacts, a new environmental radiation protection system calls for environmental indicators of radiological stress. However, because environmental stressors seldom occur alone, this study investigated the combined effects of an ecological stressor (larval density) and an anthropogenic stressor (ionizing radiation) on amphibians. Scaphiopus holbrookii tadpoles reared at different larval densities were exposed to four low irradiation dose rates (0.13, 2.4, 21, and 222 mGy d(-1)) from (137)Cs during the sensitive period prior to and throughout metamorphosis. Body size at metamorphosis and development rate served as fitness correlates related to population dynamics. Results showed that increased larval density decreased body size but did not affect development rate. Low dose rate radiation had no impact on either endpoint.

Climate change correlates with rapid delays and advancements in reproductive timing in an amphibian community.

Climate change has had a significant impact globally on the timing of ecological events such as reproduction and migration in many species. Here, we examined the phenology of reproductive migrations in 10 amphibian species at a wetland in South Carolina, USA using a 30 year dataset. We show for the first time that two autumn-breeding amphibians are breeding increasingly later in recent years, coincident with an estimated 1.2°C increase in local overnight air temperatures during the September through February pre-breeding and breeding periods. Additionally, two winter-breeding species in the same community are breeding increasingly earlier. Four of the 10 species studied have shifted their reproductive timing an estimated 15.3 to 76.4 days in the past 30 years. This has resulted in rates of phenological change that range from 5.9 to 37.2 days per decade, providing examples of some of the greatest rates of changing phenology in ecological events reported to date. Owing to the opposing direction of the shifts in reproductive timing, our results suggest an alteration in the degree of temporal niche overlap experienced by amphibian larvae in this community. Reproductive timing can drive community dynamics in larval amphibians and our results identify an important pathway by which climate change may affect amphibian communities.

The potential role of nitric oxide synthase in survival and regeneration of magnocellular neurons of hypothalamo-neurohypophyseal system.

Previous investigations from this laboratory have demonstrated that hypophysectomy induces up-regulation of neuronal nitric oxide synthase (nNOS) in magnocellular neurons of the mammalian hypothalamo-neurohypophyseal system (HNS). Accompanied by this upregulation of nNOS, both neuronal regeneration and degeneration are also observed in this system following hypophysectomy. The specific aim of this study was to determine the potential role of nNOS upregulation in neuronal survival and regeneration after hypophysectomy in the adult Sprague-Dawley (SD) rat by using a competitive nitric oxide synthase blocker, N(G)-nitrol-L: -arginine methyl ester (L: -NAME). We found that L: -NAME treatment effectively blocked the regeneration of magnocellular neurons of the rodent hypothalamus as observed in the lumen of the third cerebral ventricle following hypophysectomy. However, L: -NAME had no effect on the survival of magnocellular neurons in the supraoptic (SON) and paraventricular (PVN) nuclei after hypophysectomy. These results suggest that the induced increase of nNOS expression enhance the regenerative ability of magnocellular neurons of the HNS following hypophysectomy.

Delayed effects and complex life cycles: How the larval aquatic environment influences terrestrial performance and survival.

Species with complex life cycles are susceptible to environmental stressors across life stages, but the carryover and latent effects between stages remain understudied. For species with biphasic life histories, such as pond-breeding amphibians, delayed effects of aquatic conditions can influence terrestrial juveniles and adults directly or indirectly, usually mediated through fitness correlates such as body size. We collected adult southern toads (Anaxyrus terrestris) from 2 source populations-a natural reference wetland and a metal-contaminated industrial wetland-and exposed their offspring to 2 aquatic stressors (a metal contaminant, copper [Cu], and a dragonfly predator cue) in outdoor mesocosms (n = 24). We then reared metamorphs in terraria for 5 mo to examine delayed effects of early life stage environmental conditions on juvenile performance, growth, and survival. Larval exposure to Cu, as well as having parents from a contaminated wetland, resulted in smaller size at metamorphosis-a response later negated by compensatory growth. Although Cu exposure and parental source did not affect larval survival, we observed latent effects of these stressors on juvenile survival, with elevated Cu conditions and metal-contaminated parents reducing postmetamorphic survival. Parental source and larval Cu exposure affected performance at metamorphosis through carryover effects on body size but, 1 mo later, latent effects of parental source and larval predator exposure directly (i.e., not via body size) influenced performance. The carryover and latent effects of parental source population and aquatic Cu level on postmetamorphic survival and juvenile performance highlight the importance of conducting studies across life stages and generations. Environ Toxicol Chem 2018;37:2660-2669. © 2018 SETAC.

A subpopulation of reactive astrocytes at affected neuronal perikarya after hypophysectomy in adult rats.

Intermediate filaments (IFs) of nestin and vimentin are expressed in immature astrocytes. In this study, we examined the re-expression of these early glial traits in rat reactive astrocytes in affected neuronal perikarya in supraoptic (SON) and paraventricular (PVN) nuclei induced by hypophysectomy. Double-labeling immunofluorescence confocal laser microscopy demonstrated that by 7 days post-lesion, both nestin and vimentin were present intensely in hypertrophied GFAP-IR reactive astrocytes in the area of hypophysectomized magnocellular neurons in SON and PVN, while nestin and vimentin are absent in the normal or sham-operated animals. As the gliotic reaction progressed, the morphology of nestin or vimentin-positive reactive astrocytes in SON but not PVN changed from stellate form at 7 days to thin and elongated shape, morphologically compatible with radial glia during development, at 14 days post-lesion. By 28 days post-lesion, while vimentin-IR persisted in reactive astrocytes in SON and PVN, nestin-IR could hardly be detected. The spatiotemporal pattern of nestin-IR and/or vimentin-IR in reactive astrocytes suggests astrocytes attempt to revert to a more primitive glia form indicated by changes in morphology and phenotype following hypophysectomy, which may contribute to neuronal trophism and plasticity in the lesioned HNS favoring neuronal maintenance and fiber outgrowth.

The response of magnocellular neurons of the hypothalamo-neurohyphyseal system to hypophysectomy, nitric oxide synthase expression as well as survival and regeneration in developing vs. adult rats.

This study examined the age-related changes in nitric oxide synthase immunoreactivity (NOS-IR), survival and regeneration of magnocellular neurons in the hypothalamo-neurohypophyseal system (HNS) in rats following hypophysectomy. In adult animal, hypophysectomy induced a significant increase in NOS-IR in the supraoptic (SON), paraventricular nuclei (PVN) and median eminence (ME) by 3 days post-lesion. NOS sustained an increased level until 2 weeks after hypophysectomy and then returned to normal control level. In contrast, at postnatal day 7 (PN7), no obvious increase in NOS-IR was observed in the SON, PVN and ME following the injury compared with age-matched controls. At PN14, the same injury induced an increase in NOS-IR in SON, PVN and ME but the increase was more transient with peak NOS-IR at 3 days and returning to the corresponding control level at 1 week after hypophysectomy. In contrast to a striking age-dependent alteration in NOS-IR in the SON and PVN, hypophysectomy induced substantial degeneration of arginine vasopressin (AVP) and oxytocin (OT) neurons in the SON and PVN in both immature and adult rats and there was no obvious difference in neuronal survival after the same injury among these three groups of different ages by quantitative analysis. Following hypophysectomy, a large number of fibers were observed in the contact zone of the median eminence and the adjacent lumen of the third cerebral ventricle (V3) in adult rats, whereas few fibers could be found in the lumen of the V3 in the immature rats after the same injury. Relationships between NOS induction and magnocellular neuronal survival and regeneration were discussed.

Developmental changes of nitric oxide synthase expression in the rat hypothalamoneurohypophyseal system.

The present study investigated the immunohistochemical localization of neuronal nitric oxide synthase (nNOS) in the hypothalamoneurohypophyseal system (HNS) of the developing rats on postnatal day 1 (PN1), 7 (PN7), 14 (PN14), 21 (PN21), and the adult rats. The nNOS-positive neurons were not discernable in the supraoptic nucleus (SON), the paraventricular nucleus (PVN), and the median eminence (ME) at PN1 and PN7. A few neurons positive for nNOS were first detected at PN14. At PN21, the nNOS-positive cells in SON and PVN rapidly increased in number. The pattern of nNOS expression at this stage approached that of the adult. Moreover, the increase of nNOS expression in the SON and PVN during the postnatal period was accompanied by the maturation of arginine vasopressin (AVP) and oxytocin (OT) neurons as indicated by the number and size of OT or AVP neurons in the SON and PVN. The patterns of AVP versus OT expression also reached that of the adult by the end of the third postnatal week. The time course of the change in nNOS expression coincided with the maturation of AVP and OT neurons in the HNS and suggested that NO synthesized by conversion of NOS is involved in the modulation of activity of neurons in the SON and PVN of the HNS.