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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.

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.

Little is known about the propensity of crocodilians to bioaccumulate trace elements as a result of chronic dietary exposure. We exposed 36 juvenile alligators (Alligator mississippiensis) to one of four dietary treatments that varied in the relative frequency of meals containing prey from coal combustion waste (CCW)-contaminated habitats vs. prey from uncontaminated sites, and evaluated tissue residues and growth rates after 12 mo and 25 mo of exposure. Hepatic and renal concentrations of arsenic (As), cadmium (Cd) and selenium (Se) varied significantly among dietary treatment groups in a dose-dependent manner and were higher in kidneys than in livers. Exposure period did not affect Se or As levels but Cd levels were significantly higher after 25 mo than 12 mo of exposure. Kidney As and Se levels were negatively correlated with body size but neither growth rates nor body condition varied significantly among dietary treatment groups. Our study is among the first to experimentally examine bioaccumulation of trace element contaminants in crocodilians as a result of chronic dietary exposure. A combination of field surveys and laboratory experiments will be required to understand the effects of different exposure scenarios on tissue residues, and ultimately link these concentrations with effects on individual health.

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.

Many metals are acutely toxic to aquatic organisms at high concentrations and for some metals, such as copper (Cu), even low-level chronic contamination may be cause for conservation concern. Amphibian susceptibility to Cu has been examined in only a few species, and susceptibility is highly variable. The lethal and sublethal effects were examined of chronic aqueous Cu exposure on embryonic and larval eastern narrowmouth toads, Gastrophryne carolinensis. Copper levels as low as 10 μg Cu/L reduced embryonic and larval survival. Embryonic survivorship varied within- and between-source populations, with embryos derived from uncontaminated-wetland parents having greater survival at lower Cu levels than embryos from parents from a metal-contaminated constructed wetland. At 30 μg/L, embryos from the contaminated site had greater survival. Overall survival from oviposition to metamorphosis was 68.9% at 0 μg/L and 5.4% at 10 μg/L. Similarly, embryos exposed to ≥50 μg/L demonstrated developmental delays in transition from embryo to free-swimming larva. These results demonstrate a negative population-specific response to environmentally relevant levels of Cu. Environ Toxicol Chem 2015;34:575–582. © 2014 SETAC

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.

Environmental contaminants are implicated in the global decline of amphibian populations. Copper (Cu) is a widespread contaminant that can be toxic at concentrations just above the normal physiological range. In the present study we examined the effects of chronic Cu aqueous exposure on embryos and larvae of southern toads, Anaxyrus (Bufo) terrestris. Measurable levels of Cu were found in larvae, with tissue concentrations up to 27.5 μg Cu/g dry mass. Aqueous concentrations of Cu as low as 10 μg/L significantly reduced survival to the free-swimming stage and no larvae reached metamorphosis at concentrations above 15 μg/L. Clutches from populations with prior Cu exposure had the lowest survivorship. Among several populations there was significant variation in survivorship at different levels of Cu. More data are needed to understand the underlying causes of within- and among-population resilience to anthropogenic stressors.

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.

Understanding the demography of species over recent history (e.g.

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.

Exposure to environmental contaminants contributes to the global decline of amphibian populations. The impacts of organic contaminants on amphibians are well documented. However, substantially less is known concerning the potential effects of metals on amphibian populations. Copper (Cu) is an essential element, but it can be toxic at concentrations only slightly higher than the normal physiological range. The present study examines the effects of chronic Cu exposure on embryos and larvae of southern leopard frogs, Lithobates (Rana) sphenocephalus. Groups of eggs from multiple clutches were collected from two wetlands and exposed to a range of Cu concentrations (0-150 mg/L) until they reached the free-swimming stage, and then individual larvae were reared to metamorphosis. Higher Cu concentrations significantly reduced embryo survival to the free-swimming stage but did not further reduce survival to metamorphosis. Larval period was affected by Cu treatment, but the clutch from which larvae originated (i.e., parentage) explained a higher proportion of the variation. Embryo survival to hatching varied significantly among clutches, ranging from 42.9 to 79.2%. Measurable levels of Cu were found in larvae with body burdens up to 595 mg Cu/g dry mass in the 100 mg/L treatment, and larval Cu body burdens were higher than in metamorphs. The present study also demonstrated that higher initial egg density ameliorated embryo mortality at higher Cu levels and should be accounted for in future studies. Environ. Toxicol. Chem. 2012;31:1587-1594. # 2012 SETAC Keywords—Lithobates (Rana) sphenocephalus Copper Amphibian Ecotoxicology Metal toxicity

We conducted a mesocosm study to assess the individual and interactive effects of previous maternal exposure and larval exposure to trace element-laden sediments on southern toads (Bufo terrestris). Previous maternal exposure to coal combustion wastes (CCW) reduced larval survival to metamorphosis up to 57% compared to larvae of unexposed females. Larvae reared on CCW accumulated significant concentrations of trace elements resulting in extended larval periods, reduced growth rates, and reduced mass at metamorphosis. However, the effects were dependent on age of sediments, suggesting the effects of contaminants from CCW may be partially ameliorated over time through the reduced bioavailability of trace elements in aged CCW. Most importantly, maternal exposure to contaminants coupled with larval exposure to fresh CCW interacted to reduce survival to metamorphosis by 85% compared to reference conditions. Our study yields further evidence that disposal of CCW in aquatic basins potentially creates ecological traps for some amphibian populations.

Contaminants often occur as mixtures in the environment, but investigations into toxicity usually employ a single chemical. Metal contaminant mixtures from anthropogenic activities such as mining and coal combustion energy are widespread, yet relatively little research has been performed on effects of these mixtures on amphibians. Considering that amphibians tend to be highly sensitive to copper (Cu) and that metal contaminants often occur as mixtures in the environment, it is important to understand the interactive effects that may result from multiple metals. Interactive effects of Cu and zinc (Zn) on amphibians have been reported as antagonistic and, conversely, synergistic. The goal of our study was to investigate the role of Zn in Cu toxicity to amphibians throughout the embryonic developmental period. We also considered maternal effects and population differences by collecting multiple egg masses from contaminated and reference areas for use in four experiments across three species. We performed acute toxicity experiments with Cu concentrations that cause toxicity (10–200 μg/L) in the absence of other contaminants combined with sublethal concentrations of Zn (100 and 1000 μg/L). Our results suggest very few effects of Zn on Cu toxicity at these concentrations of Zn. As has been previously reported, we found that maternal effects and population history had significant influence on Cu toxicity. The explanation for a lack of interaction between Cu and Zn in this experiment is unknown but may be due to the use of sublethal Zn concentrations when previous experiments have used Zn concentrations associated with acute toxicity. Understanding the inconsistency of amphibian Cu/Zn mixture toxicity studies is an important research direction in order to create generalities that can be used to understand risk of contaminant mixtures in the environment.

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.

In organisms that have complex life cycles, factors in the larval environment may affect both larval and adult traits. For amphibians, the postmetamorphic transition from the aquatic environment to terrestrial habitat may be a period of high juvenile mortality. We hypothesized that lipid stores at metamorphosis may affect an animal's success during this critical transition period. We examined variation in total lipid levels among years and sites in recently metamorphosed individuals of two pond-breeding salamander species, the marbled salamander (Ambystoma opacum) and the mole salamander (A. talpoideum), with limited data for one anuran species (southern leopard frog, Rana sphenocephala). Lipid levels were allometrically related to body size and ranged from 1.9 to 23.8% of body dry mass. The two salamander species differed in lipid allocation patterns, with A. opacum apportioning a higher percentage of total lipid reserves into fat bodies than A. talpoideum. Species differences in lipid allocation patterns may primarily reflect that large metamorphs will mature as one-year olds, and, regardless of species, will alter lipid compartmentalization accordingly. We used mark-recapture data obtained at drift fences encircling breeding ponds for 13 A. opacum cohorts to estimate the proportion of postmetamorphic individuals that survived to breed (age 1-4) and the mean age at first reproduction. Regression models indicated that size-corrected lipid level at metamorphosis (i.e., lipid residuals), and to a lesser extent rainfall following metamorphosis, was positively related to adult survival. Snout-vent length at metamorphosis was negatively related to age at first reproduction. We suggest that lipid stores at metamorphosis are vital to juvenile survival in the months following the transition from aquatic to terrestrial habitat, and that a trade-off shaped by postmetamorphic selection in the terrestrial habitat exists between allocation to energy stores versus structural growth in the larval environment.

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 directly 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 introduction of nitroglycerin into the subdermal tissue using a linear probe. The data acquired using the on-line MD-ME-EC system was compared to that obtained off-line analysis by 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.

Despite their potential vulnerability to contaminants from exposure at multiple life stages, amphibians are one of the least studied groups of vertebrates in ecotoxicology, and research on radiation effects in amphibians is scarce. We used multiple endpoints to assess the radiosensitivity of the southern toad (Anaxyrus [Bufo] terrestris) during its pre-terrestrial stages of development -embryonic, larval, and metamorphic. Toads were exposed, from several hours after oviposition through metamorphosis (up to 77 days later), to four low dose rates of 137Cs at 0.13, 2.4, 21, and 222 mGy d-1, resulting in total doses up to 15.8 Gy. Radiation treatments did not affect hatching success of embryos, larval survival, or the length of the larval period. The individual family variation in hatching success of embryos was larger than the radiation response. In contrast, newly metamorphosed individuals from the higher dose-rate treatments had higher mass and mass/length body indices, a measure which may relate to higher post-metamorphic survival. The increased mass and index at higher dose rates may indicate that the chronic, low dose rate radiation exposures triggered secondary responses. Additionally, the increases in growth were linked to a decrease in DNA damage (as measured by the Comet Assay) in red blood cells at a dose rate of 21 mGy d-1 and a total dose of 1.1 Gy. In conclusion, the complex effects of low dose rates of ionizing radiation may trigger growth and cellular repair mechanisms in amphibian larvae.

Despite the continuing loss of wetland habitats and associated declines in amphibian populations, attempts to translate wetland losses into measurable losses to ecosystems have been lacking. We estimated the potential productivity from the amphibian community that would be compromised by the loss of a single isolated wetland that has been protected from most industrial, agricultural, and urban impacts for the past 54 years. We used a continuous drift fence at Ellenton Bay, a 10-ha freshwater wetland on the Savannah River Site, near Aiken, South Carolina (U.S.A.), to sample all amphibians for 1 year following a prolonged drought. Despite intensive agricultural use of the land surrounding Ellenton Bay prior to 1951, we documented 24 species and remarkably high numbers and biomass of juvenile amphibians (360,000 individuals;1,400 kg) produced during one breeding season. Anurans (17 species) were more abundant than salamanders (7 species), comprising 96.4% of individual captures. Most (95.9%) of the amphibian biomass came from 232095 individuals of a single species of anuran (southern leopard frog[Rana sphenocephala]). Our results revealed the resilience of an amphibian community to natural stressors and historical habitat alteration and the potential magnitude of biomass and energy transfer from isolated wetlands to surrounding terrestrial habitat. We attributed the postdrought success of amphibians to a combination of adult longevity (often5 years), a reduction in predator abundance, and an abundance of larval food resources. Likewise, the increase of forest cover around Ellenton Bay from20% in 1951 to60% in 2001 probably contributed to the long-term persistence of amphibians at this site. Our findings provide an optimistic counterpoint to the issue of the global decline of biological diversity by demonstrating that conservation efforts can mitigate historical habitat degradation.

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.

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. © 2005 Wiley-Liss, Inc.

The creation of wetlands, such as urban and industrial ponds, has increased in recent decades, and these wetlands often become enriched in pollutants over time. One metal contaminant trapped in created wetlands is copper (Cu(2+)). Copper concentrations in sediments and overlying water may affect amphibian species that breed in created wetlands. The authors analyzed the Cu concentration in dried sediments from a contaminated wetland and the levels of aqueous Cu released after flooding the sediments with different volumes of water, mimicking low, medium, and high pond-filling events. Eggs and larvae of Ambystoma opacum Gravenhorst, a salamander that lays eggs on the sediments in dry pond beds that hatch on pond-filling, were exposed to a range of Cu concentrations that bracketed potential aqueous Cu levels in created wetlands. Embryo survival varied among clutches, but increased Cu levels did not affect embryo survival. At Cu concentrations of 500 µg/L or greater, however, embryos hatched earlier, and the aquatic larvae died shortly after hatching. Because Cu concentrations in sediments increase over time in created wetlands, even relatively tolerant species such as A. opacum may be affected by Cu levels in the posthatching environment.

We report a host gender bias in haemogregarine infection characteristics in the American alligator (Alligator mississippiensis) at the Savannah River Site, South Carolina, USA. Prevalence and severity in female alligators was higher than it was in males. The reason for this pattern is not clear.

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.

This investigation deals with the histochemical and scanning electron microscopic (SEM) correlates that depict regeneration of the neurohypophyseal system that may be nitric oxide dependent following hypophysectomy in the rodent hypothalamus. NOS histochemistry and correlative SEM were employed to establish the rates of regrowth and appearance of NOS-positive supraoptic (SON) and paraventricular (PVN) neurites and their cell bodies following hypophysectomy. NOS activity increased significantly in SON and PVN neuronal perikarya and regenerating axons by 2 weeks. NOS-positive neurites were observed to regrow into the adjacent median eminence and insinuate into the lumen of the third cerebral ventricle. By 4 weeks posthypophysectomy, NOS staining of SON and PVN neurons and their regrown neurites had returned to normal control levels. Despite this fact, large complexes of apparent magnocellular neurites remained upon the floor of the third cerebral ventricle as observed with SEM. These observations support the hypothesis that NO may play a fundamental role in the process of regeneration, plasticity, and retargeting of SON and PVN axons following injury.

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.

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.

Fine structural correlates and functional parameters were measured in pinealectomized rats following grafting of the pineal gland into the third cerebral ventricle. Pinealectomy caused a significant decrease in serum melatonin concentration of animals compared to that in normal controls. No significant difference was observed in the serum melatonin concentration between pinealectomized rats and those receiving sham transplantation with fragments of occipital cortex. By 6 weeks nearly 50% of pinealectomized rats receiving pineal transplants demonstrated a significant increase in the serum melatonin concentration in contrast to that of pinealectomized rats and pinealectomized animals receiving sham transplants. Pinealocytes survived and flourished following transplantation from the epithalamic region to the third cerebral ventricle of the hypothalamus in host rats. These cells were found to be arranged individually or in clusters surrounding fenestrated capillaries of the graft. Moreover, these pinealocytes demonstrated ultrastructural features indicative of an active secretory process, including densecore and clear vesicles as well as vacuoles containing flocculent material. Additional characteristics distinctive of normal control pinealocytes were observed in surviving cells of grafts, such as synaptic ribbons, synaptic ribbon fields, and myeloid bodies. Bundles of unmyelinated axons and apparent adrenergic nerve endings were observed with transmission electron microscopy and immunocystochemistry using antisera against tyrosine hydroxylase (TH). Nerve fibers and terminals were found within perivascular spaces surrounding fenestrated capillaries of viable grafts. These reported observations suggest that a significant population of transplanted pinealocytes recover functional activity (e.g., heightened melatonin secretion) following stereotaxic grafting into the third cerebral ventricles of pinealectomized animals. This apparent recovery of function may be linked directly to reinnervation of the gland by nerve fibers that appear to arise from the underlying median eminence.

The aim of the present study is to compare c-fos expression in identified hypothalamic vasopressin (AVP) and oxytocin (OT) neurons in developing (PN7 and PN14) and adult rats following hypophysectomy using dual-labeled immunostaining. Our results showed that hypophysectomy induced c-fos expression in supraoptic (SON) and paraventricular (PVN) nuclei in both the developing and adult rats. Few or no positive cells were observed in the same nuclei in sham-operated animals. Quantitative analysis for c-fos and either of the above named neuropeptides revealed that almost all AVP and OT neurons in the adult and PN14 groups expressed c-fos in response to hypophysectomy. In PN7, hypophysectomy also induced all AVP neurons to express c-fos in SON and PVN. However, few OT neurons in the SON and PVN produced c-fos after hypophysectomy. In addition, the time course of c-fos expression was different in the developing and adult rats after hypophysectomy. The c-fos expression in the developing rats exhibited a more prolonged induction in which staining for c-fos persisted for at least 3 days after hypophysectomy compared with that in the adult in which c-fos immunoreactivity disappeared within 24 hr post-lesion. This study demonstrates that c-fos expression after hypophysectomy is regulated differently during development. Anat Rec, 290:1050–1056, 2007. 2007 Wiley-Liss, Inc.

Wide variation in reproductive success is common among amphibians that breed in seasonal ponds, but persistence of adults can buffer against these fluctuations, particularly for long-lived species. We hypothesized that the frequent episodes of catastrophic failure of the marbled salamander (Ambystoma opacum) enhance the importance of high terrestrial survival. At Rainbow Bay in South Carolina reproductive success was poor (< 1 metamorph/breeding female) in nearly half of the 22 years that the species bred. Complete failure occurred in 6 of 22 years. To study catastrophic failure, we adapted an age-structured, individual-based model with density-dependent growth and survival of larvae. The model was based on extensive data from local field studies and experiments. With consistently good survival in the pond stages, the simulated population required survival probabilities in the upland stages (juveniles and adults) near 0.5/year to persist and near 0.8/year to achieve the increases observed. Catastrophic failure, occurring randomly with probability 0.5/year created additional fluctuations in the population, raised the thresholds of survival required for persistence, and caused extinction under conditions that were otherwise favorable. The marbled salamander at Rainbow Bay is not at great risk of extinction because of catastrophic failure, but the risk increases dramatically if life span is decreased or frequency of failure is increased. Any reduction in terrestrial survival will have deleterious consequences by reducing the breeding populations at equilibrium, even if it does not jeopardize persistence. Our model provides assessments of risk that can be applied to poorly studied species with similar life histories, such as the endangered flatwoods salamander (A. cingulatum).

We measured plasma levels of testosterone, dihydrotestosterone (DHT), and corticosterone for male marbled salamanders (Ambystoma opacum) collected during the breeding season. Our goal was to ascertain whether steroid levels changed in response to particular reproductive behaviors or laboratory confinement. Six groups of salamanders were examined: (a) MIGRATING, males migrating toward the pond basin during the breeding season; (b) LABORATORY, males kept under confined conditions in the laboratory for 10 days; (c) LAB-FIELD, laboratory males that were later released into seminatural enclosures in the field; (d) COURTING, males from male-female pairs in which the male actively courted the female (and deposited at least one spermatophore); (e) SOLO, males that were individually isolated from conspecifics; and (f) MALE-MALE, males that were placed together in pairs, and in which one male actively courted the other male. In three groups (COURTING, SOLO, and MALE-MALE), salamanders were placed in containers for observation and each male was observed for at least 2 hr prior to a plasma sample being taken. Circulating levels of testosterone, DHT, and corticosterone did not differ significantly for males in these groups. The similarity of androgen levels among the three groups indicated a lack of behaviorally evoked change under experimental conditions designed to reveal a behavior-androgen response. Male A. opacum differ taxonomically from other amphibians showing a behavior-androgen response (three species of toads in the genus Bufo) and also lack amplexus and male-male combat during competition for mates. The effects of confinement were indicated by levels of testosterone and DHT in LABORATORY males that were significantly lower than average levels of males in the following groups: MIGRATING, LAB-FIELD, and MALE-MALE. We inferred that LAB-FIELD males, following their release to seminatural enclosures, were able to regain plasma androgen levels typical of migrating males. This increase is one of very few demonstrations for amphibians of an increase in androgen levels upon release from laboratory confinement. Levels of corticosterone did not differ significantly between males that were active in the field and males that were kept in the laboratory. The similarity of corticosterone levels among these groups differs from the typical pattern of elevated corticosterone and depressed androgen levels in captive amphibians. Maximal corticosterone levels in breeding male A. opacum may act differently than in other species in which chronic elevations inhibit the pituitary-gonadal axis.

Central to this investigation are several basic hypotheses that are designed to test the role of nitric oxide (NO) in the complex process of central regeneration and plasticity in a well established model system of the mammalian brain. We have employed histochemical techniques at the light and ultrastructural level coupled with correlative scanning electron microscopy, ommunoelectron microscopy, and in situ hybridization in order to determine the functional significance of the increased expression of nitric oxide synthase (NOS) in neurons of the supraoptic (SON) and paraventricular (PVN) nuclei which accompanies regeneration of their axotomized neurites following hypophysectomy. The aim of this investigation was to determine the potential role and temporal up-regulation of NOS in this basic regenerative process and to establish the ultrastructural and neuroanatomical correlates during critical periods of regeneration and regrowth of SON and PVN axons following hypophysectomy in the endocrine hypothalamus of the rat. Our data support the hypothesis that NO may serve as a second messenger molecule that may act in some fashion to govern not only the process of central regeneration and regrowth of magnocellular (SON/PVN) axons into the median eminence, neural stem, and neural lobe (the neurohypophyseal system) but may also influence the regeneration of neurites into new neuroanatomical domains such as the adjacent lumen of the third cerebral ventricle. We have demonstrated a distinct temporal relationship between injury (axotomy) of SON/ PVN axons and the establishment of new neurovascular zones following hypophysectomy with the up-regulation of NOS. This up-regulation appears to correlate well with successful regeneration in the mammalian neurohypophyseal system. We have also successfully inhibited axonal regeneration with the use of nitroarginine, a competitive antagonist of NO. NOS up-regulation attendant to regeneration of SON and PVN axons may have inestimable clinical implications, particularly with respect to closed head injury and cerebral contusion that involves the mechanical shearing of the infundibular stalk. In addition, this investigation has reaffirmed that large numbers of bona fide neurons migrate and emerge upon the floor of the adjacent third cerebral ventricle shortly following hypophysectomy (within 2 weeks). The origin and mechanisms of neuronal migration and plasticity following hypohysectomy are the subject of interpretation and discussion in this investigation.

The survival, revascularization, reinnervation, and recovery of function of transplanted rat pineal glands were studied following grafting into four different locations in pinealectomized rats. Pineal grafts were well vascularized by fenestrated capillaries. Pinealocytes in the grafts maintained high-metabolic activity. More nerve fibers and terminals were observed in the grafts within the anterior chamber of the eye than in the third cerebral ventricle and the pineal region (in situ transplantation). No fibers or terminals were found in grafts placed beneath the renal capsule. Nighttime serum melatonin levels increased significantly in pinealectomized rats with transplants into either the third cerebral ventricle or the anterior eye chamber. This increase might reflect graft reinnervation. Yet day-night differences in serum melatonin were observed only in host rats receiving transplants in the anterior eye chamber. In conclusion, pinealocytes survived transplantation into different locations and exhibited ultrastructural features indicative of active secretory processes; however, day-night differences in serum melatonin are only restored following transplants into the anterior eye chamber. Reinnervation of the grafts by the host superior cervical ganglion is necessary for this restoration.

Thirty-two Sprague-Dawley rats were divided into four groups, eight rats per group. Animals were hypophysectomized with removal of both the pars distalis and the neural lobe of the neurohypophysis. Groups of eight rats were euthanized 1, 2, 4 and 8 weeks following hypophysectomy and prepared for routine scanning electron microscopy (SEM) and correlative immunoelectron microscopy employing antisera against arginine vasopressin (AVP). Eight normal rats served as controls. In experimental rats that survived one to eight weeks posthypophysectomy, remarkable neuroanatomical alterations were notable in the median eminence and adjacent third cerebral ventricular lumen. In contrast to normal control rats, large numbers of neuntes were observed with SEM to insinuate from the lateral recess into the cerebral ventricular lumen and as early as one week following hypophysectomy they overgrew the apical surfaces of ependymal cells that constitute the lining of the cerebral ventricle. Immunoelectron microscopy revealed that a significant proportion of these neurites were magnocellular in origin in that they harbored AVP-positive neurosecretory vesicles. In addition to large numbers of invading magnocellular neurites, neuronal perikayria with apparent axosomatic synapses were observed to emerge upon the thick feltwork of invading axons, the latter of which appeared to freely terminate within the ventricular lumen. AVP-positive axon profiles were, in addition, seen to terminate upon the basal lamina of portal perivascular spaces in the zona externa of the median eminence. These data are consistent with the idea that following hypophysectomy (to include high stalk section of the neurohypophyseal system), that there is rapid, and dynamic sprouting and regrowth of AVP-positive axons into the adjacent third cerebral ventricular lumen and to the contact zone of the median eminence as well. This phenomenon may represent a compensatory physiological response to injury of the neurohypophyseal system characterized by a highly plastic neuroanatomical reorganization of magnocellular elements which appear to utilize the CSF of the third cerebral ventricle as a functional terminus for the neurocisternal secretion of AVP which ultimately enters the systemic circulation.

Adjacent tissue sections through the rat median eminence were examined for the distribution of gonadotropin-releasing hormone (GnRH) and catecholamines (CA). A simultaneous visualization technique was employed for this correlative neuroanatomical analysis. At rostral and mid-central levels of the median eminence the majority of GnRH terminals do not appear in coexistence with CA terminals; the latter were confined to the outer-most 10 micrometers of the median eminence while the densest concentration of GnRH terminals was located internal to this layer. However, individual GnRH fibers appeared to penetrate the outer CA zone wherein they were found in juxtaposition to portal capillaries. At caudal levels of the median eminence, there was an extensive overlap of CA and GnRH varicosities adjacent to the tubero-infundibular sulcus. In addition, numerous GnRH terminals were seen adjacent to portal vessels. The differences in the positions of CA and GnRH terminals between rostral and caudal median eminence may provide a morphological basis for the hypothesis of separate regulatory mechanisms for CA upon GnRH secretion at these two levels of the median eminence.

Primary cultures of dispersed cells were prepared from 3–5 mg pieces of basal hypothalami of 10–12-day-old rats. The tissue included median eminence, arcuate nucleus and variable amounts of adjacent hypothalamus and preoptic area. The dispersion procedure consisted basically of tissue trypsinization and mechanical dissociation of cells. They were cultured in a modified L-15 medium in an air atmosphere. Neurons survived approximately 3 months. On the basis of morphological characteristics, two basic cell types could be distinguished. One was a larger (50 μm diameter) multipolar cell; microspectrofluorometric analysis revealed that a small percentage of these neurons contained a catecholamine. A second type was smaller, fusiform or ovoid and generally bipolar; a significant number of these were immunoreactive for the releasing hormone LHRH.

This investigation has confronted some very basic questions of neurobiology and specifically deals with the neurovascular and neuroanatomical interactions that occur between graft and host following neural transplantation. Host Long-Evans rats with chronic autosomal diabetes insipidus (DI) received stereotaxic implants of normal 17 day post-coitus fetal hypothalamic fragments from the rostral (anterior) hypothalamus of normal Long-Evans pups. Following stereotaxic surgery DI hosts were killed 60 or 90 days later and their brain prepared for correlative microangiography-immunocytochemistry coupled with transmission electron microscopy. Explants were rapidly invaded by host vessels from several routes. 1. (1) Vessels appeared to arise from portal capillaries in the underlying median eminence and 2. (2) from adjacent vessels from the paraventricular nucleus and surrounding endocrine hypothalamus and 3. (3) possibly from intrinsic vessels of the graft. The former remained fenestrated and established bonafide neurovascular zones in the ventral regions and in actively growing explants. Small clusters of arginine vasopressin-positive fibers and neurophysin positive neurons were noted throughout the parenchyma of explants. Despite the presence of neurosecretory neurons and neurovascular (neurohemal) zones, none of the host rats exhibited a physiological return to normal parameters of water balance. However the active growth and development of explants in the third cerebral ventricle of DI host rats coupled with emergence of neurovascular zones lends support to a potential model for analyzing the development of anatomical substrates for the central delivery of neuropeptide hormones.

Adult male Brattleboro rats with chronic diabetes insipidus underwent stereotaxic surgery wherein minced fragments of anterior hypothalamus from fetal rats, 17 days post-coitus, were stereotaxically positioned into the lumen of the host third cerebral ventricle. Host rats with fetal donor tissue were killed at various times following surgery and were prepared for correlative scanning-transmission electron microscopy. Examination with this technique revealed the presence of large neurografts which grew to occupy the entire lumen of the host third ventricle. Grafts were well vascularized and in addition exhibited remarkable numbers of supraependymal, cerebrospinal fluid-contacting neurons. The physical emergence of this cell line in proximity to viable grafts is discussed with respect to the biochemical influences that a neuropeptide producing fetal transplant has upon a peptide-deficient host.

A new method is described for producing plastic casts of murine Ventriculosubarachnoid space. Photographs of a representative cast demonstrate the highly detailed tridimensional anatomy obtainable with this method.

This report concerns a light and electron microscopic investigation of the median eminence and dorsal infundibular stem of the rat following surgical isolation (deafferentation). Using a modification of the Halasz technique, the basal hypothalamus, including the arcuate nucleus and median eminence were surgically isolated from surrounding structures. Special attention was directed to the contact (external) zone of the median eminence and rostral infundibulum where tuberohypophyseal axons as well as ependymal cell processes abut upon the abluminal basement membrane of the portal perivascular space. The results of this study to date suggest that 9, 20, and 40 days following surgical isolation, there is a distinct increase in the population of tuberohypophyseal dense core vesicles. It is suggested that deafferentation abolishes inhibitory and excitatory input that serves to modify the cellular dynamics of tuberohypophyseal neurosecretory elements. Comments are also made on the presence of cistern-like structures in the lateral median eminence; the presence of vesicle-like inclusions in terminal ependymal processes is discussed in relationship to the role that ependyma may play in linking the third ventricle with the adenohypophysis.

A light and electron microscopic autoradiographic analysis revealed that H3-valine infused into the lateral ventricle of normal and acutely dehydrated cats is preferentially taken up by the supraoptic (SON) and paraventricular nucleus (PVN) of the hypothalamus. Grain counts for these magnocellular neurons in normal unstressed cats were highest at one hour post infusion with a significant fall off by three hours. Uptake by the SON and PVN at one hour exceeded neighboring nuclear groups by a factor of 7 and 4 fold respectively. Electron microscopic autoradiographs from acutely dehydrated cats revealed the presence of emission grains in association with rough endoplasmic reticulum and large osmiophilic neurosecretory vesicles. In view of statistically significant uptake values and rapid turnover of H3-valine by SON and PVN in normal animals, coupled with emission tracks in direct association with underlying neurosecretory product in acutely dehydrated ones, it is speculated that valine may be an amino acid component of one or both of the neurophysins to which neurohypophyseal hormones are non-covalently linked.

An in vitro assay for thyrotrophin releasing factor (TRF) is described which is based upon release of TSH from hemi-pituitary glands of normal rats. Pre-incubation of the pituitary gland in rat plasma results in considerable increase in sensitivity to approximately 35 pg of TRF. The content of TRF in median eminence of the hypothalamus of normal rats is estimated to be in the range of 102–140 pg. When median eminence is incubated in vitro with 3H-TRF or when radiolabelled hormone is infused intraventricularly, autoradiographic evidence indicates that the hormone is present in specialized ependyma (tanycytes) of the median eminence.

Ultrastructural changes in the neural lobe related to lactation and release of oxytocin in response to suckling were observed. Neural lobes of nonlactating controls showed abundant stores of osmiophilic neurosecretion contained in vesicles in the axons. In contrast, the glands of lactating rats sacrificed during suckling by the young displayed extensive depletion as evidenced by decreased numbers of vesicles containing osmiophilic neurosecretion and increased numbers of optically clear neurosecretory vesicles. When a lapse of 15 minutes was allowed between removal of the suckling young and sacrifice of the mother, a high degree of restoration of neurosecretion had taken place, with approximately 50% of the axons filled with neurosecretion. After 1 hour the restoration of neurosecretion was virtually complete. Observations on synaptic vesicles were also made. Relations of these changes to hormonal events and mechanisms of release and restoration of neurosecretion are discussed.

Microspectrofluorometry, fluorescence histochemistry and light and electron microscopic autoradiography have established the presence of sub-populations of neurons in the arcuate-periventricular region of the rat hypothalamus that sequester both radiolabeled dopamine and demonstrate formaldehyde-induced fluorescence. These characteristics are consistent with a catecholaminergic function. Selective sequestration of 3H-dopamine at the light and ultrastructural level is discussed in the context of an ultrashort loop autoregulatory mechanism for this neuronal population.

The present ultrastructural study proves the existence of nerve terminals closely apposed to the plasmalemmata of tanycytes in the rat median eminence. Several of these “axo-tanycytic” endings display remarkable accumulations of agranular endoplasmic reticulum in the form of pleomorphic vesicles which are closely apposed on either side of the plasma membrane of each cell compartment. Some of these vesicular profiles give the impression of structural continuity across both membrane systems. This phenomenon is discussed in the context of being a potential substratum for communication between both cell compartments.

Light and electron microscopic autoradiography has been employed to define the neuroanatomical patterns of uptake and binding of radiolabelled L-dopa in the endocrine hypothalamus of the rat. A dorsomedial continuum of arcuate and periventricular neurons selectively sequester 3H L-dopa 20 min following its intraventricular infusion. By 40 and 60 min following the infusion labelling of neurons is minimal and supports the notion of rapid degradation. Other cell compartments such as tanycytes demonstrate uptake of 3H L-dopa. The ultrastructural localization and distribution of radiolabelled L-dopa (or its metabolites) in the rodent hypothalamus is discussed with respect to mechanisms and cell compartments involved in neuroendocrine regulatory processes.

Blocks of anterior hypothalamus were transplanted from 19 day-old fetuses of Wistar/Lewis rats into the third ventricle of adult male Brattleboro rats. Physiological changes in graft recipients and in sham-operated animals were monitored daily. Twenty days after surgery, the graft recipients and sham-operated animals were killed and their brains examined by correlative scanning and transmission electron microscopy. Host animals that exhibited both decreased polydipsia and increased urine concentration were found to have viable grafts within the third ventricle. The observed physiological changes suggested that synthesis and release of vasopressin occurred in the transplanted neurons. Grafts were well vascularized by vessels arising from the host hypothalamus. Neurons, with perikarya ranging from 8 to 30 micron in diameter, glial cells, and neurites were located throughout the transplants. A neurohemal contact zone, similar to that normally seen in the median eminence, could not be demonstrated in the grafts. The absence of complete glial and ependymal barriers indicates a relatively close association between cells in the transplants and the cerebrospinal fluid. A large increase in supraependymal neurons and their processes, including an eruption of neurons through the floor of the third ventricle in one animal, was observed in graft recipients but not in sham-operated animals.

This investigation has combined microangiography, immunocytochemistry, coupled with transmission and scanning electron microscopy to discuss the neuroanatomical interactions that occur in the brains of Brattleboro rats with diabetes insipidus, following stereotaxic placement of normal fetal hypothalamic fragments into the third cerebral ventricle. Following surgical placement of 17 day post-coitus hypothalamic fragments, host rats with chronic autosomal homozygous diabetes insipidus were killed and their brains were prepared for analysis. A significant degree of explants (68%) flourished and grew in the lumen of the third cerebral ventricle of recipient hosts. Explants were rapidly invaded by host vessels from two routes. (1) Vessels arose from the underlying mantle plexus of portal capillaries which remained fenestrated in the lower one-third of the explants and developed neurovascular (neurohemal) zones. (2) The second source of vessels arose from bed capillaries of the adjacent paraventricular nucleus and adjacent hypothalamus. In contrast to vessels arising from the contact zone, these latter vessels remained unfenestrated. Small clusters of immunocytochemically positive neurons (neurophysin positive) were seen throughout the explants. Numerous healthy magnocellular neurons harboring numerous dense core vesicles and exhibiting multiple axosomatic and axodendritic synapses were seen throughout the neuropil of explants. Axon profiles were noted to terminate upon the abluminal basal lamina of perivascular spaces surrounding fenestrated capillaries in the lower one third of explants. None of the host animals exhibited physiological return to normal parameters of urine output, drinking behavior, and/or urine osmolarity. However the growth and development of explants in the third cerebral ventricle of DI hosts coupled with the emergence of bonafide neurovascular zones supports a potential anatomical substrate for the central delivery of neuropeptide hormones in this experimental model.

Light and electron microscopic autoradiographic analysis of the medial basal hypothalamus of rat brains incubated for 30 and 60 minutes in tritiated luteinizing releasing factor (3H-LRF) revealed selective uptake in the form of silver grains over specialized ependyma (tanycytes) and perivascular glial cells in the palisade-contact zone of the median eminence. The functional capacity of tanycytes and glia are discussed in light of these data.

Rats were given a standard scald burn on 60% of the body surface or only a sham burn and were sacrificed at intervals from 6 hr to 14 days later. Serum thyroxine (T 4 ), free thyroxine index (FT 4 I) and triiodothyronine (T 3 ) were depressed compared to values in respective shams as early as 6 hr post-burn. T 4 and FT 4 I were less depressed on post-burn days (PBD) 2–3 than on PBD 1 and then exhibited a further fall. T 3 , remained depressed through PBD 14. Pineal melatonin content was elevated at 6 hr and fell to the normal daytime range in subsequent samples. The ventral portion of the diencephalon was prepared for scanning electron microscopy. Only in the burned rats and beginning on PBD 2, large numbers of supraependymal neurons (SEN) appeared in the ventricular space attached to the inferior walls and floor of the third cerebral ventricle. Transmission electron microscopy was used to confirm the neuronal nature of the SEN. Viewed by scanning electron microscopy, these persisted through PBD 14. SEN were interconnected by cables of their neuntes exhibiting varicosities on individual neuntes as they passed over perikarya of other SEN. Some SEN were seen to be only partially emerged from the underlying tissue and others were seen to send a thick process into the hypothalamic tissue. These observations indicate that after peripheral injury there is marked plasticity of the brain in an area thought to control the endocrine systems that show abnormalities after such a peripheral injury. The timing, location and nature of these anatomic changes indicate the possibility that at least some aspects of central nervous orchestration of the endocrine metabolic response to injury may be related to the emergence of a neuronal system receiving or sending messages through the cerebrospinal fluid and/or through new neurite circuits along the surface of the third ventricular wall. These structures may appear in response to initial primary hormonal changes and/or may play a role in maintaining the post-injury hormonal milieu manifested in part by a subsequent second fall in serum T 4 .

The ultrastructure of the normal median eminence of the male rhesus monkey (Macaca mulatta) is described using high-voltage electron microscopy. Surface specializations of ependymal cells lining the infundibular recess included cilia, apical extrusions, and microvilli. Supraependymal cells were predominantly macrophage-like, but examples of lymphocytic types were also seen. Tanycytes had long, branching, basal processes filled with numerous microtubules, some lipid droplets, and granules. The zona interna was composed of large unmyelinated neurosecretory fibers. A few myelinated fibers were also seen, but their character as neurosecretory fibers could not be established. The zona externa was composed of densely-packed profiles of neuro secretory fibers of small diameter, was well-vascularized and contained the terminations of tanycytes. Perivascular glial cells, vesiculated elements, pituicytes, and cellular elements common to connective tissue were observed. The intricate relationships between both the cellular and fibrous elements of the median eminence can be appreciated with the capability of high-voltage electron microscopy to discern ultrastructure in sections 10 times thicker than those used for low-voltage electron microscopy. The median eminence of this primate species has an ultrastructural organization similar to that described for most other species.