Bird rookery nutrient over-enrichment as a potential accelerant of mangrove cay decline in Belize.

Coastal eutrophication is an issue of serious global concern and although nutrient subsidies can enhance primary productivity of coastal wetlands, they can be detrimental to their long-term maintenance. By supplying nutrients to coastal ecosystems at levels comparable to intensive agriculture practices, roosting colonial waterbirds provide a natural experimental design to examine the impacts of anthropogenic nutrient enrichment in these systems. We tested the hypothesis that long-term nutrient enrichment from bird guano deposition is linked to declines in island size, which may subsequently decrease the stability and resilience of mangrove cays in Belize. We combined remote sensing analysis with field- and lab-based measurements of forest structure, sediment nutrients, and porewater nutrients on three pairs of rookery and control cays in northern, central, and southern Belize. Our results indicate that rookery cays are disappearing approximately 13 times faster than cays without seasonal or resident seabird populations. Rookery cays were associated with a significantly higher concentration of nitrogen (N) in mangrove leaves and greater aboveground biomass, suggesting that eutrophication from bird guano contributes to increased aboveground productivity. Sediments of rookery cays also had lower percentages of soil organic matter and total N and carbon (C) than control islands, which suggests that eutrophication accelerates organic matter decomposition resulting in lower total C stocks on rookery cays. Our results indicate that coastal eutrophication can reduce ecosystem stability by contributing to accelerated cay loss, with potential consequences for mangrove resilience to environmental variability under contemporary and future climatic scenarios.

Five-century record of climate and groundwater recharge variability in southern California.

Modifications to the rates of water flowing from the surface to groundwater (groundwater recharge) due to climate variability are the most difficult to assess because of the lack of direct long-term observations. Here, we analyze the chloride salt distribution below the surface soil on a plateau near Los Angeles to reconstruct the amount of recharge that occurred in the last five centuries. Over this time interval, periods of major high and low recharge with different duration follow each other and this cyclicity is consistent with long-term atmospheric forcing patterns, such as the Pacific Decadal Oscillation. This study determines the range and the natural variability of recharge to groundwater, which sustains local freshwater flow system, and helps forecast future availability of groundwater resource in southern California, where water scarcity is critical to both local and global populations.

Gonadal hormones, but not sex, affect the acquisition and maintenance of a Go/No-Go odor discrimination task in mice.

In mice, olfaction is crucial for identifying social odors (pheromones) that signal the presence of suitable mates. We used a custom-built olfactometer and a thirst-motivated olfactory discrimination Go/No-Go (GNG) task to ask whether discrimination of volatile odors is sexually dimorphic and modulated in mice by adult sex hormones. Males and females gonadectomized prior to training failed to learn even the initial phase of the task, which involved nose poking at a port in one location obtaining water at an adjacent port. Gonadally intact males and females readily learned to seek water when male urine (S+) was present but not when female urine (S-) was present; they also learned the task when non-social odorants (amyl acetate, S+; peppermint, S-) were used. When mice were gonadectomized after training the ability of both sexes to discriminate urinary as well as non-social odors was reduced; however, after receiving testosterone propionate (castrated males) or estradiol benzoate (ovariectomized females), task performance was restored to pre-gonadectomy levels. There were no overall sex differences in performance across gonadal conditions in tests with either set of odors; however, ovariectomized females performed more poorly than castrated males in tests with non-social odors. Our results show that circulating sex hormones enable mice of both sexes to learn a GNG task and that gonadectomy reduces, while hormone replacement restores, their ability to discriminate between odors irrespective of the saliency of the odors used. Thus, gonadal hormones were essential for both learning and maintenance of task performance across sex and odor type.

A sex comparison of the anatomy and function of the main olfactory bulb-medial amygdala projection in mice.

We previously reported that some main olfactory bulb (MOB) mitral/tufted (M/T) cells send a direct projection to the "vomeronasal" amygdala in female mice and selectively respond to volatile male mouse urinary odors. We asked whether MOB M/T cells that project to the vomeronasal amygdala exist in male mice and whether there is a sexually dimorphic response of these neurons to volatile male urinary pheromones. Gonadectomized male and female mice received bilateral injections of the retrograde tracer, Cholera toxin-B (CTb) into the medial amygdala (Me), which is part of the vomeronasal amygdala. All subjects were then treated with estradiol benzoate and progesterone before being exposed to volatile male urinary odors whereupon they were sacrificed 90 min later. Sections of the MOB were immunostained for Fos protein and/or CTb. Male mice, like females, displayed a small population of MOB M/T cells that project to the Me. While the general localization of these cells was similar in the two sexes, there were statistically significant sex differences in the percentage of MOB M/T cells in the anterior and posterior medial segments of the MOB that were retrogradely labeled by CTb. Male urinary volatiles stimulated equivalent, significant increases in Fos expression by MOB M/T neurons projecting to the Me in the two sexes. By contrast, in the same mice exposure to male urinary volatiles stimulated a significant increase in Fos expression by mitral cells in the accessory olfactory bulb (AOB) only in female subjects. Thus any sexually dimorphic behavioral or neuroendocrine responses to male urinary volatiles likely depend on the differential processing of these odor inputs in the AOB and/or other downstream forebrain structures after their detection by the main olfactory system.

Improved resolution of ambient flow through fractured rock with temperature logs.

In contaminant hydrogeology, investigations at fractured rock sites are typically undertaken to improve understanding of the fracture networks and associated groundwater flow that govern past and/or future contaminant transport. Conventional hydrogeologic, geophysical, and hydrophysical techniques used to develop a conceptual model are often implemented in open boreholes under conditions of cross-connected flow. A new approach using high-resolution temperature (+/-0.001 degrees C) profiles measured within static water columns of boreholes sealed using continuous, water-inflated, flexible liners (FLUTe) identifies hydraulically active fractures under ambient (natural) groundwater flow conditions. The value of this approach is assessed by comparisons of temperature profiles from holes (100 to 200 m deep) with and without liners at four contaminated sites with distinctly different hydrogeologic conditions. The results from the lined holes consistently show many more hydraulically active fractures than the open-hole profiles, in which the influence of vertical flow through the borehole between a few fractures masks important intermediary flow zones. Temperature measurements in temporarily sealed boreholes not only improve the sensitivity and accuracy of identifying hydraulically active fractures under ambient conditions but also offer new insights regarding previously unresolvable flow distributions in fractured rock systems, while leaving the borehole available for other forms of testing and monitoring device installation.

Sex and gonadal steroid modulation of pheromone receptor gene expression in the mouse vomeronasal organ.

Non-volatile chemosignals in rodents are detected by unique receptors in the vomeronasal organ of the accessory olfactory system. Although the vomeronasal organ has been implicated in the regulation of sexually dimorphic behavioral and neuroendocrine functions, the underlying cellular mechanisms are undetermined. In previous studies we showed that exposure to soiled male bedding augmented immediate early gene immunoreactivity in neurons of the basal zone of the vomeronasal organ, an effect that depended on gender and sex steroid expression. To determine whether this effect could be due to differences in vomeronasal organ receptor expression, we examined two representatives (VR1 and VR4) from different subfamilies of the V2R family of receptors that are expressed in the basal zone of the vomeronasal organ. Adult Swiss-Webster male and female mice were gonadectomized and implanted with capsules containing 17beta-estradiol, testosterone or neither steroid (control). Two weeks later vomeronasal organs were processed for in situ hybridization using probes from the N-terminal extracellular domains of VR1 and VR4. Expression of both VR1 and VR4 was significantly higher in males than in females. Estradiol, but not testosterone-treated, males had significantly lower levels of VR1 expression in the caudal vomeronasal organ compared with untreated gonadectomized males. In contrast, testosterone enhanced VR4 expression in males relative to similarly treated females. Despite these effects, we found no evidence that vomeronasal organ neurons express either androgen or estrogen receptors. These data show that expression of vomeronasal organ receptors in mice is sexually dimorphic and regulated by sex steroids. Thus, gonadal hormones may affect the response of vomeronasal organ neurons to chemosignals by altering levels of the receptors to which they bind.

Naris occlusion alters transductory protein immunoreactivity in olfactory epithelium.

We have recently shown that unilateral naris occlusion (UNO) causes an increase in olfactory marker protein (OMP) immunoreactivity (IR) in mouse olfactory sensory neurons (OSN) from the occluded side of the nasal cavity and a decrease in OMP-IR on the non-occluded side, relative to controls. Given OMP's demonstrated role in olfactory modulation, these OMP-IR changes have been interpreted as a compensatory response by OSNs to odor deprivation on the occluded side and to supernormal exposure to odor on the non-occluded side of the nasal cavity. In the current study, we examined the developmental timing and the regional distribution of this process throughout the nasal cavity using immunocytochemistry. Results demonstrate that OMP-IR diverges in OSNs from the occluded side relative to the non-occluded side of the nasal cavity within eleven days after UNO, with statistically significant differences measurable after 17 days (n=16). We also measured relative levels of the Type 4 phosphodiesterase (PDE4A), another potential olfactory modulator, in nasal cavity tissue from UNO (n=8) and untreated mice (n=9) using western blots and immunocytochemistry. Like OMP, PDE4A-IR increased on the occluded side of the nasal cavity after UNO. Finally, we used immunocytochemistry to assess relative levels of olfactory-specific adenylyl cyclase (ACIII, n=4) and G-protein (Golf, n=2) in OSNs from the occluded and non-occluded sides of the nasal cavity of UNO mice. Following UNO, ACIII but not Golf -IR levels diverged comparing the occluded to the non-occluded sides of the nasal cavity. Taken together, our findings provide support for the previously unknown phenomenon of compensatory responses by OSNs to odor environment.

Vertical cross contamination of trichloroethylene in a borehole in fractured sandstone.

Boreholes drilled through contaminated zones in fractured rock create the potential for vertical movement of contaminated ground water between fractures. The usual assumption is that purging eliminates cross contamination; however, the results of a field study conducted in a trichloroethylene (TCE) plume in fractured sandstone with a mean matrix porosity of 13% demonstrates that matrix-diffusion effects can be strong and persistent. A deep borehole was drilled to 110 m below ground surface (mbgs) near a shallow bedrock well containing high TCE concentrations. The borehole was cored continuously to collect closely spaced samples of rock for analysis of TCE concentrations. Geophysical logging and flowmetering were conducted in the open borehole, and a removable multilevel monitoring system was installed to provide hydraulic-head and ground water samples from discrete fracture zones. The borehole was later reamed to complete a well screened from 89 to 100 mbgs; persistent TCE concentrations at this depth ranged from 2100 to 33,000 microg/L. Rock-core analyses, combined with the other types of borehole information, show that nearly all of this deep contamination was due to the lingering effects of the downward flow of dissolved TCE from shallower depths during the few days of open-hole conditions that existed prior to installation of the multilevel system. This study demonstrates that transfer of contaminant mass to the matrix by diffusion can cause severe cross contamination effects in sedimentary rocks, but these effects generally are not identified from information normally obtained in fractured-rock investigations, resulting in potential misinterpretation of site conditions.

Effect of source variability and transport processes on carbon isotope ratios of TCE and PCE in two sandy aquifers.

Chlorinated ethenes often migrate over extended distances in aquifers and may originate from different sources. The aim of this study was to determine whether stable carbon isotope ratios remain constant during dissolution and transport of chlorinated ethenes and whether the ratios can be used to link plumes to their sources. Detailed depth-discrete delineation of the carbon isotope ratio in a tetrachloroethene (PCE) plume and in a trichloroethene (TCE) plume was done along cross-sections orthogonal to groundwater flow in two sandy aquifers in the Province of Ontario, Canada. At the TCE site, TCE concentrations up to solubility were measured in one high concentration zone close to the bottom of the aquifer from where dense non-aqueous phase liquid (DNAPL) was collected. A laboratory experiment using the DNAPL indicated that only very small carbon isotope fractionation occurs during dissolution of TCE (0.26 per thousand), which is consistent with field observations. At most sampling points, the delta(13)C of dissolved TCE was similar to that of the DNAPL except for a few sampling points at the bottom of the aquifer close to the underlying aquitard. At these points, a (13)C enrichment of up to 2.4 per thousand was observed, which was likely due to biodegradation and possibly preferential diffusion of TCE with (12)C into the aquitard. In contrast to the TCE site, several distinct zones of high concentration were observed at the PCE site and from zones to zone, the delta(13)C values varied substantially from -24.3 per thousand to -33.6 per thousand. Comparison of the delta(13)C values in the high concentration zones made it possible to divide the plume in the three different domains, each probably representing a different episode and location of DNAPL release. The three different zones could still be distinguished 220 m from the DNAPL sources. This demonstrates that carbon isotope ratios can be used to differentiate between different zones in chlorinated ethene plumes and to link plume zones to their sources. In addition, subtle variations in delta(13)C at plume fringes provided insight into mechanisms of plume spreading in transverse vertical direction. These variations were identified because of the high-resolution provided by the monitoring network.

Interactive effects of testosterone and superior cervical ganglionectomy on attraction thresholds to volatile urinary odors in gonadectomized mice.

Volatile urinary odors contribute to mate recognition in mice after their detection by the main olfactory epithelium (MOE). We used a habituation/dishabitution task to ask whether the capacity of gonadectomized mice of both sexes to detect and investigate decreasing concentrations of volatile urinary odors from either breeding males or estrous females is modulated by administering androgen or estrogen and if so, whether any effects of these sex steroids are altered by disrupting the sympathetic innervation of the MOE via bilateral superior cervical ganglionectomy (SCGx). In tests given, beginning 51 days after gonadectomy without steroid treatment both male and female subjects detected even the lowest concentrations (1:120 and 1:160 dilutions by volume) of male urinary odors, provided they were SCGx as opposed to sham operated. In subsequent tests given after estradiol benzoate (EB) followed later by 5alpha-dihydrotestosterone (DHT) treatments, neither male nor female subjects detected low concentrations of male urinary odors regardless of whether or not their SCG's were intact. Administration of testosterone (T) prior to a final series of tests restored the ability of gonadectomized subjects of both sexes to detect low concentrations of male urinary odors regardless of their SCG status. This suggests that T, but not its neural metabolites estradiol, or DHT, facilitates responsiveness to low concentrations of male odors in mice of both sexes. In tests given 51 days after gonadectomy without steroid treatment most male and female subjects readily detected the three highest concentrations of estrous female urinary odors whereas SCGx males and females failed to detect the lowest concentrations of these odors. After treatment with EB and then with DHT, gonadectomized mice of both sexes generally failed to detect the three lowest concentrations of estrous female urinary odors regardless of their SCG status. After T treatment; however, subjects of both sexes again detected most dilutions of estrous female urine, provided their SCG's were intact. Again, these results suggest that T, but not estradiol or DHT, facilitates responsiveness to estrous female urinary odors. Provided such an activational effect of T is present, sympathetic, noradrenergic inputs to the MOE may enhance odorant contrast, as previously suggested [Nat. Neurosci. 2 (1999) 106], by reducing the responsiveness of olfactory neurons to low (1:120 and 1:160 dilutions) concentrations of some biologically significant odorants (e.g. male urinary odors) while facilitating their responsiveness to low to moderate (1:80 dilution) concentrations of others (e.g. estrous female urinary odors).

Removal of the superior cervical ganglia fails to block Fos induction in the accessory olfactory system of male mice after exposure to female odors.

When exposed to female odors, testosterone-primed male mice show a robust expression of immediate early genes in the vomeronasal organ (VNO) and associated accessory olfactory structures. We asked whether the superior cervical ganglia (SCG), which provide autonomic inputs to the VNO, are required for odor induction of Fos. Gonadally intact male mice received sham, unilateral, or bilateral SCG lesions and were exposed to odors from estrous females. In comparison to clean bedding, female odors significantly increased neuronal Fos immunoreactivity in sites throughout the VNO projection pathway, but these responses were not reliably modified by SCG removal. Thus, noradrenergic inputs to the VNO, which regulate a pumping mechanism thought to facilitate entrance of chemosignals into the VNO lumen, are not required for odors to induce Fos in the mouse accessory olfactory system.

Induction of Fos in the accessory olfactory system by male odors persists in female mice with a null mutation of the aromatase (cyp19) gene.

The ability of odors from soiled male bedding to induce neuronal Fos-immunoreactivity (IR) in sensory neurons located in both the apical and basal zones of the vomeronasal organ (VNO) and in two segments of the VNO-projection pathway, the anterior nucleus of the medial amygdala and the bed nucleus of the stria terminalis (BNST), was significantly reduced in adult, ovariectomized, estrogen-treated female mice with a homozygous null mutation of the cyp19 gene (ArKO) which encodes the estrogen biosynthetic P450 enzyme, aromatase. However, a significant odor-induced activation of Fos-IR was seen in other segments of the VNO-projection pathway of ArKO females, including the accessory olfactory bulb (AOB) granule cell layer, the posterior-dorsal medial amygdala (MePD), and the medial preoptic area (MPA). These results suggest that the VNO/accessory olfactory pathway to the hypothalamus was functional in ArKO females even though they had presumably been exposed to less estrogenic stimulation than wild-type (WT) control females throughout development and until the time that estrogen treatment was begun in adulthood. Thus, the hypothesis of Toran-Allerand [Prog. Brain Res. 61 (1984) 63] that female-typical features of neuroendocrine and behavioral function require perinatal exposure to estrogen was not supported, at least for the VNO/accessory olfactory system.

Monitoring oxidation of chlorinated ethenes by permanganate in groundwater using stable isotopes: laboratory and field studies.

Permanganate injection is increasingly applied for in situ destruction of chlorinated ethenes in groundwater. This laboratory and field study demonstrates the roles that carbon isotope analysis can play in the assessment of oxidation of trichloroethene (TCE) by permanganate. In laboratory experiments a strong carbon isotope fractionation was observed during oxidation of TCE with similar isotopic enrichment factors (-25.1 to -26.8 per thousand) for initial KMnO4 concentrations between 67 and 1,250 mg/L. At the field site, a single permanganate injection episode was conducted in a sandy aquifer contaminated with TCE as dense nonaqueous liquid (DNAPL). After injection, enriched delta13C values of up to +204% and elevated Cl- concentrations were observed at distances of up to 4 m from the injection point. Farther away, the Cl- increased without any change in delta13C of TCE suggesting that Cl- was not produced locally but migrated to the sampling point Except for the closest sampling location to the injection point, the delta13C rebounded to the initial 613C again, likely due to dissolution of DNAPL Isotope mass balance calculations made it possible to identify zones where TCE oxidation continued to occur during the rebound phase. The study indicates that delta13C values can be used to assess the dynamics between TCE oxidation and dissolution and to locate zones of oxidation of chlorinated ethenes that cannot be identified from the Cl- distribution alone.

A controlled field experiment on groundwater contamination by a multicomponent DNAPL: creation of the emplaced-source and overview of dissolved plume development.

A unique field experiment has been undertaken at the CFB Borden research site to investigate the development of dissolved chlorinated solvent plumes from a residual dense non-aqueous phase liquid (DNAPL) source. The "emplaced-source" tracer test methodology involved a controlled emplacement of a block-shaped source of sand containing chlorinated solvents below the water table. The gradual dissolution of this residual DNAPL solvent source under natural aquifer conditions caused dissolved solvent plumes of trichloromethane (TCM), trichloroethene (TCE) and perchloroethene (PCE) to continuously develop down gradient. Source dissolution and 3-D plume development were successfully monitored via 173 multilevel samplers over a 475-day tracer test period prior to site remediation research being initiated. Detailed groundwater level and hydraulic conductivity data were collected. Development of plumes with concentrations spanning 1-700,000 micrograms/1 is described and key processes controlling their migration identified. Plumes were observed to be narrow due to the weakness of transverse dispersion processes and long due to advection and significant longitudinal dispersion, very limited sorptive retardation and negligible, if any, attenuation due to biodegradation or abiotic reaction. TCM was shown to be essentially conservative, TCE very nearly conservative and PCE, consistent with its greater hydrophobicity, more retarded yet having a greater mobility than observed in previous Borden field tests. The absence of biodegradation was ascribed to the prevailing aerobic conditions and lack of any additional biodegradable carbon substrates. The transient groundwater flow regime caused significant transverse lateral plume movement, plume asymmetry and was likely responsible for most of the, albeit limited, transverse horizontal plume spreading. In agreement with the widespread incidence of extensive TCE and PCE plumes throughout the industrialized world, the experiment indicates such solvent plumes are likely to be highly mobile and persistent, at least in aquifers that are aerobic and have low sorption potential (low foc content).

Geochemical reactions resulting from in situ oxidation of PCE-DNAPL by KMnO4 in a sandy aquifer.

Although the potential for KMnO4 to destroy chlorinated ethenes in situ was first recognized more than a decade ago, the geochemical processes that accompany the oxidation have not previously been examined. In this study, aqueous KMnO4 solutions (10-30 g/L) were injected into an unconfined sand aquifer contaminated by the dense non-aqueous-phase liquid (DNAPL) tetrachloroethylene (PCE). The effects of the injections were monitored using depth-specific, multilevel groundwater samplers, and continuous cores. Two distinct geochemical zones evolved within several days after injection. In one zone where DNAPL is present, reactions between KMnO4 and dissolved PCE resulted in the release of abundant chloride and hydrogen ions to the water. Calcite and dolomite dissolved, buffering the pH in the range of 5.8-6.5, releasing Ca, Mg, and CO2 to the pore water. In this zone, the aqueous Ca/Cl concentration ratio is close to 5:12, consistent with the following reaction for the oxidation of PCE in a carbonate-rich aquifer: 3C2Cl4 + 5CaCO3(s) + 4KMnO4 + 2H+ --> 11CO2 + 4MnO2(s) + H2O + 12Cl- + 5Ca2+ + 4K+. In addition to Mg from dolomite dissolution, increases in the concentration of Mg as well as Na may result from exchange with K at cation-exchange sites. In the second zone, where lesser amounts of PCE were present, KMnO4 persisted in the aquifer for more than 14 months, and the porewater pH increased graduallyto between 9 and 10 as a resultof reaction between KMnO4 and H2O. A small increase in SO4 concentrations in the zones invaded by KMnO4 suggests that KMnO4 injections caused oxidation of sulfide minerals. There are important benefits of carbonate mineral buffering during DNAPL remediation by in situ oxidation. In a carbonate-buffered system, Mn(VII) is reduced to Mn(IV) and is immobilized in the groundwater by precipitating as insoluble manganese oxide. Energy-dispersive X-ray spectroscopy analyses of the manganese oxide coatings on aquifer mineral grains have detected the impurities Al, Ca, Cl, Cu, Pb, P, K, Si, S, Ti, U, and Zn indicating that, similar to natural systems, precipitation of manganese oxide is accompanied by coprecipitation of other elements. In addition, the consumption of excess KMnO4 by reaction with reduced minerals such as magnetite will be minimized because the rates of these reactions increase with decreasing pH. Aquifer cores collected after the KMnO4 injections exhibit dark brown to black bands of manganese oxide reaction products in sand layers where DNAPL was originally present. Mineralogical investigations indicate that the manganese oxide coatings are uniformly distributed over the mineral grains. Observations of the coatings using transmission electron microscopy indicate that they are on the order of 1 microm thick, and consequently, the decrease in porosity through the formation of the coatings is negligible.

Surgically induced cryptorchidism-related degenerative changes in spermatogonia are associated with loss of cyclic adenosine monophosphate-dependent phosphodiesterases type 4 in abdominal testes of rats.

The present study was undertaken to investigate the role of phosphodiesterase type 4 (PDE4) enzymes in cryptorchidism-induced apoptosis of the germ cells. Regulation of expression of PDE4 enzymes was studied in the abdominal and scrotal testes of surgically induced cryptorchid rats for 10, 20, and 30 days. In some cases orchidopexy was performed after 30 days of cryptorchidism, and rats were allowed to recover for an additional 50 days. Upon histological examination, marked degenerative changes in the epithelial lining of the seminiferous tubules within abdominal testes were observed compared with contralateral control or age-matched sham-operated rats. These changes included degeneration of some spermatogonia, apoptosis of the secondary spermatocytes, incomplete spermatogenesis, and lack of spermatozoa in the lumen. In contrast, contralateral scrotal testes exhibited normal histology. Significant improvement in the regeneration of spermatogonia was observed in rats after 50 days of recovery following orchidopexy. Immunocytochemical examination suggested the presence of PDE4A in germ cells while PDE4B was predominantly expressed on somatic cells. Western blotting using PDE4 subtype-selective antibodies showed the presence of two PDE4A variants (a 109-kDa PDE4A8 and a previously uncharacterized 88-kDa PDE4A variant) and two PDE4B (78-kDa PDE4B2 and 66-kDa PDE4B variant) bands. In unilaterally cryptorchid animals, the abdominal testis showed a time-dependent decrease in both PDE4A8 and 88-kDa PDE4A variants. In contrast, the expression of 66-kDa PDE4B was markedly increased in a time-dependent fashion in abdominal testes of cryptorchid rats. Animals surgically corrected for cryptorchidism and allowed to recover for 50 days exhibited normal expression of both PDE4A and PDE4B variants compared with aged-matched, sham-operated controls. In conclusion, this study suggests that down-regulation of PDE4A variants in cryptorchid testes may play an important role in the degeneration of spermatogonia and increased apoptotic activity in the germ cells.

Sex difference and steroid modulation of pheromone-induced immediate early genes in the two zones of the mouse accessory olfactory system.

Two anatomically and neurochemically distinct zones within the vomeronasal organ (VNO) and accessory olfactory bulb (AOB) have been identified that are responsible for the detection of pheromones. Using markers to distinguish between apical and basal neurons of the VNO neuroepithelium and rostral versus caudal AOB glomeruli, we examined immediate early gene immunoreactivity (IEG-IR) in gonadectomized, steroid-treated mice in response to pheromones of male and female conspecifics. After exposure of estradiol-treated females to soiled male bedding, more VNO neurons in the basal than the apical layer exhibited IEG-IR compared with VNO neurons of estradiol-treated males. Conversely, whereas soiled female bedding failed to induce IEG-IR in VNO neurons of estradiol-treated males or females, both apical and basal neurons were activated in testosterone-treated males. Male and female pheromones also activated mitral and granule cells in the AOBs of all subjects, but responses to different pheromones were distributed across the boundary of the rostral and caudal regions. These data show that differences in the response of males and females to the same pheromonal stimulus are found in the sensory neurons of the VNO. We propose that centrifugal, noradrenergic inputs to VNO neurons, which may differ in the two sexes and respond differently to adult sex steroids, modulate sensitivity to pheromonal stimulation.

Diazepam and rolipram differentially inhibit cyclic AMP-specific phosphodiesterases PDE4A1 and PDE4B3 in the mouse.

Cyclic AMP is hydrolyzed by members of at least eight classes of cyclic nucleotide phosphodiesterases (PDEs). Although it has been reported that cyclic AMP PDE activity in mammalian tissues can be inhibited by benzodiazepines, it has not been conclusively demonstrated that members of the class of cyclic AMP-specific, rolipram-inhibitable PDEs (PDE4s) are targets for these drugs. Moreover, no PDE4s expressed in mice have been characterized. To address these issues, we isolated two cDNAs representing homologues of PDE4A1 and PDE4B3 from a mouse brain library. After transient transfection in human embryonic kidney (HEK) 293 cells, the mouse PDEs hydrolyzed cyclic AMP with a low K(m) and were inhibited by rolipram; both are properties typical of other mammalian PDE4 enzymes. In addition, we found that diazepam inhibited cyclic AMP hydrolysis by the mouse PDE4 subtypes. Interestingly, PDE4B was significantly more sensitive to inhibition by both rolipram and diazepam than the PDE4A subtype. This is the first demonstration that recombinantly expressed PDE4s are inhibited by diazepam, and should facilitate future studies with mouse models of depression and anxiety.

Central forebrain Fos responses to familiar male odours are attenuated in recently mated female mice.

Exposure of recently mated female mice to the urinary odours of an unfamiliar male blocks pregnancy (the Bruce effect). The absence of a pregnancy block in response to the stud male's familiar odours depends on an olfactory memory that is formed in the accessory olfactory bulb (AOB) in response to vomeronasal organ (VNO) inputs during mating. Sexually naive Balb/c female mice in pro-oestrus/oestrus were either placed onto soiled bedding ('bedding-only' females) from, or allowed to mate with, a Balb/c male ('recently mated' females). After 42 h, females were placed for 90 min onto clean bedding (controls) or onto soiled bedding from either a C57BL/6 male (unfamiliar bedding) or a Balb/c male (familiar bedding). Significant increases in Fos-immunoreactivity (Fos-IR, a marker of neuronal activation) occurred in the medial amygdala and the medial preoptic area (MPA) of 'bedding only' females exposed to either unfamiliar or familiar bedding and in 'recently mated' females exposed to unfamiliar bedding but not to familiar bedding. This suggests that a mating-induced memory prevents the later activation by the familiar stud male's odours of neurons in forebrain regions that receive inputs from the VNO--AOB. 'Bedding-only' females later exposed to either familiar or unfamiliar bedding had increased Fos-IR in the G alpha(o) protein-expressing basal zone of the VNO whereas no such effect occurred in 'recently mated' females. Familiar, as well as unfamiliar, male odours augmented Fos-IR in significantly more rostral than caudal AOB granule cells in all groups, with the effect being strongest in 'recently mated' females exposed to familiar male bedding. This outcome is consistent with the absence of odour-induced Fos-IR in forebrain regions of these females and, presumably, the absence of a pregnancy block.

Cyclic AMP phosphodiesterases in the zebra finch: distribution, cloning and characterization of a PDE4B homolog.

Songbirds are important animal models for studying neural mechanisms underlying learning and memory. While evidence has emerged that cAMP plays a significant role in invertebrate and mammalian learning, little is known about the role of cAMP pathways in regulating neuronal function in birds. With the goal of identifying important components of this pathway, we report the first cloning of a cAMP-specific, Type IV phosphodiesterase (PDE4) in a non-mammalian vertebrate. A combination of PCR analysis and cDNA library screening was used to show that homologs of the four known mammalian PDE4 genes also exist in zebra finch. A full-length cDNA representing the zebra finch homolog of PDE4B1 was isolated from a telencephalic library. Expression of this cDNA in human embryonic kidney 293 (HEK) cells yielded an enzyme that hydrolyzed cAMP with a low K(m) and was inhibited by micromolar concentrations of rolipram; these properties are typical of all known mammalian PDE4s. In brain, northern blots revealed transcripts of 3.6 and 4.4 kb in adults, but only the 3.6 kb transcript in juveniles, suggesting that PDE4 expression is developmentally regulated. In situ hybridization of tissue sections demonstrated that PDE4 message was distributed widely throughout the adult zebra finch brain, including regions controlling the learning of songs and the acquisition of spatial memories. These data suggest that PDE4 enzymes may influence a variety of brain functions in these birds and play a role in learning.