Draft Genome Sequence of the Nonmotile Tremellomycetes Yeast Naganishia albida, Isolated from Aircraft.

The Basidiomycota yeast Naganishia albida strain 5307AI was isolated from an aircraft polymer-coated surface. The genome size is 20,642,279 bp, with a G+C content of 53.99%. The genome contains fatty acid transporters, cutinases, hydroxylases, and lipases that are likely used for survival on polymer coatings on aircraft.

Comparison of two diphenyl polyenes as acid-sensitive additives during the biodegradation of a thermoset polyester polyurethane coating.

AIMS: Biochemical hydrolysis and chemical catalysis are involved in the successful biodegradation of polymers. In order to evaluate the potential separation between biochemical and chemical catalysis during the biodegradation process, we report the use of two diphenylpolyenes (DPPs), all trans-1,4-diphenylbutadiene (DPB) and all trans-1,6-diphenylhexatriene (DPH), as potential acid-sensitive indicators in polymers. METHODS AND RESULTS: 1,4-Diphenylbutadiene and DPH (0.1% w/w) were melt-cast successfully with poly(ethylene succinate) hexamethylene (PES-HM) polyurethane (thermoset polyester polyurethane) coatings above 80℃. When these two DPP/PES-HM coatings were exposed to a concentrated supernatant with significant esterase activity resulting from the growth of a recently isolated and identified strain of Tremellomycetes yeast (Naganishia albida 5307AI), the DPB coatings exhibited a measurable and reproducible localized decrease in the blue fluorescence emission in regions below where hydrolytic biodegradation was initiated in contrast with DPH blended coatings. The fluorescence changes observed in the biodegraded DPB coating were similar to exposing them to concentrated acids and not bases. CONCLUSIONS: Our experiments resulted in (1) a method to blend DPP additives into thermoset coatings, (2) the first report of the biodegradation of polyester polyurethane coating by N. albida, and (3) demonstration that hydrolytic supernatants from this strain generate acidic region within degrading polyester coatings using DPB as the indicator. SIGNIFICANCE AND IMPACT OF THE STUDY: Our experiments confirm that N. albida is an active polyester degrader and that DPB is a promising acid sensitive polymer coating additive.

In situ Linkage of Fungal and Bacterial Proliferation to Microbiologically Influenced Corrosion in B20 Biodiesel Storage Tanks.

Renewable fuels hold great promise for the future yet their susceptibility to biodegradation and subsequent corrosion represents a challenge that needs to be directly assessed. Biodiesel is a renewable fuel that is widely used as a substitute or extender for petroleum diesel and is composed of a mixture of fatty acid methyl esters derived from plant or animal fats. Biodiesel can be blended up to 20% v/v with ultra-low sulfur diesel (i.e., B20) and used interchangeably with diesel engines and infrastructure. The addition of biodiesel, however, has been linked to increased susceptibility to biodegradation. Microorganisms proliferating via degradation of biodiesel blends have been linked to microbiologically influenced corrosion in the laboratory, but not measured directly in storage tanks (i.e., in situ). To measure in situ microbial proliferation, fuel degradation and microbially influenced corrosion, we conducted a yearlong study of B20 storage tanks in operation at two locations, identified the microorganisms associated with fuel fouling, and measured in situ corrosion. The bacterial populations were more diverse than the fungal populations, and largely unique to each location. The bacterial populations included members of the Acetobacteraceae, Clostridiaceae, and Proteobacteria. The abundant Eukaryotes at both locations consisted of the same taxa, including a filamentous fungus within the family Trichocomaceae, not yet widely recognized as a contaminant of petroleum fuels, and the Saccharomycetaceae family of yeasts. Increases in the absolute and relative abundances of the Trichocomaceae were correlated with significant, visible fouling and pitting corrosion. This study identified the relationship between fouling of B20 with increased rates of corrosion and the microorganisms responsible, largely at the bottom of the sampled storage tanks. To our knowledge this is the first in situ study of this scale incorporating community and corrosion measurements in an active biodiesel storage environment.

Organism Engineering for the Bioproduction of the Triaminotrinitrobenzene (TATB) Precursor Phloroglucinol (PG).

Organism engineering requires the selection of an appropriate chassis, editing its genome, combining traits from different source species, and controlling genes with synthetic circuits. When a strain is needed for a new target objective, for example, to produce a chemical-of-need, the best strains, genes, techniques, software, and expertise may be distributed across laboratories. Here, we report a project where we were assigned phloroglucinol (PG) as a target, and then combined unique capabilities across the United States Army, Navy, and Air Force service laboratories with the shared goal of designing an organism to produce this molecule. In addition to the laboratory strain Escherichia coli, organisms were screened from soil and seawater. Putative PG-producing enzymes were mined from a strain bank of bacteria isolated from aircraft and fuel depots. The best enzyme was introduced into the ocean strain Marinobacter atlanticus CP1 with its genome edited to redirect carbon flux from natural fatty acid ester (FAE) production. PG production was also attempted in Bacillus subtilis and Clostridium acetobutylicum. A genetic circuit was constructed in E. coli that responds to PG accumulation, which was then ported to an in vitro paper-based system that could serve as a platform for future low-cost strain screening or for in-field sensing. Collectively, these efforts show how distributed biotechnology laboratories with domain-specific expertise can be marshalled to quickly provide a solution for a targeted organism engineering project, and highlights data and material sharing protocols needed to accelerate future efforts.

Draft Genome Sequence of Filamentous Fungus Phialemoniopsis curvata, Isolated from Diesel Fuel.

Phialemoniopsis curvata D216 is a filamentous fungus isolated from contaminated diesel fuel. The genome size is 40.3 Mbp with a G+C content of 54.81%. Its genome encodes enzymes and pathways likely involved in the degradation of and survival in fuel, including lipases, fatty acid transporters, and beta oxidation.

Finished Genome Sequence of a Polyurethane-Degrading Pseudomonas Isolate.

Pseudomonas sp. strain WP001 is a laboratory isolate capable of polyurethane polymer degradation and harbors a predicted lipase precursor gene. The genome of strain WP001 is 6.15 Mb in size and is composed of seven scaffolds with a G+C content of 60.54%. Strain WP001 is closely related to Pseudomonas fluorescens based on ribosomal DNA comparisons.

Carbon Catabolite Repression and Impranil Polyurethane Degradation in Pseudomonas protegens Strain Pf-5.

Polyester polyurethane (PU) coatings are widely used to help protect underlying structural surfaces but are susceptible to biological degradation. PUs are susceptible to degradation by Pseudomonas species, due in part to the degradative activity of secreted hydrolytic enzymes. Microorganisms often respond to environmental cues by secreting enzymes or secondary metabolites to benefit their survival. This study investigated the impact of exposing several Pseudomonas strains to select carbon sources on the degradation of the colloidal polyester polyurethane Impranil DLN (Impranil). The prototypic Pseudomonas protegens strain Pf-5 exhibited Impranil-degrading activities when grown in sodium citrate but not in glucose-containing medium. Glucose also inhibited the induction of Impranil-degrading activity by citrate-fed Pf-5 in a dose-dependent manner. Biochemical and mutational analyses identified two extracellular lipases present in the Pf-5 culture supernatant (PueA and PueB) that were involved in degradation of Impranil. Deletion of the pueA gene reduced Impranil-clearing activities, while pueB deletion exhibited little effect. Removal of both genes was necessary to stop degradation of the polyurethane. Bioinformatic analysis showed that putative Cbr/Hfq/Crc-mediated regulatory elements were present in the intergenic sequences upstream of both pueA and pueB genes. Our results confirmed that both PueA and PueB extracellular enzymes act in concert to degrade Impranil. Furthermore, our data showed that carbon sources in the growth medium directly affected the levels of Impranil-degrading activity but that carbon source effects varied among Pseudomonas strains. This study uncovered an intricate and complicated regulation of P. protegens PU degradation activity controlled by carbon catabolite repression. IMPORTANCE: Polyurethane (PU) coatings are commonly used to protect metals from corrosion. Microbiologically induced PU degradation might pose a substantial problem for the integrity of these coatings. Microorganisms from diverse genera, including pseudomonads, possess the ability to degrade PUs via various means. This work identified two extracellular lipases, PueA and PueB, secreted by P. protegens strain Pf-5, to be responsible for the degradation of a colloidal polyester PU, Impranil. This study also revealed that the expression of the degradative activity by strain Pf-5 is controlled by glucose carbon catabolite repression. Furthermore, this study showed that the Impranil-degrading activity of many other Pseudomonas strains could be influenced by different carbon sources. This work shed light on the carbon source regulation of PU degradation activity among pseudomonads and identified the polyurethane lipases in P. protegens.

Ovarian steroids alter mu opioid receptor trafficking in hippocampal parvalbumin GABAergic interneurons.

The endogenous hippocampal opioid systems are implicated in learning associated with drug use. Recently, we showed that ovarian hormones regulate enkephalin levels in the mossy fiber pathway. This pathway overlaps with parvalbumin (PARV)-basket interneurons that contain the enkephalin-activated mu opioid receptors (MORs) and are important for controlling the "temporal timing" of granule cells. Here, we evaluated the influence of ovarian steroids on the trafficking of MORs in PARV interneurons. Two groups of female rats were analyzed: cycling rats in proestrus (relatively high estrogens) or diestrus; and ovariectomized rats euthanized 6, 24 or 72 h after estradiol benzoate (10 microg, s.c.) administration. Dorsal hippocampal sections were dually immunolabeled for MOR and PARV and examined by light and electron microscopy. As in males, in females MOR-immunoreactivity (-ir) was in numerous PARV-labeled perikarya, dendrites and terminals in the dentate hilar region. Variation in ovarian steroid levels altered the subcellular distribution of MORs in PARV-labeled dendrites but not terminals. In normal cycling rats, MOR-gold particles on the plasma membrane of small PARV-labeled dendrites (area <1 microm2) had higher density in proestrus rats than in diestrus rats. Likewise, in ovariectomized rats MORs showed higher density on the plasma membrane of small PARV-labeled dendrites 72 h after estradiol exposure. The number of PARV-labeled cells was not affected by estrous cycle phase or estrogen levels. These results demonstrate that estrogen levels positively regulate the availability of MORs on GABAergic interneurons in the dentate gyrus, suggesting cooperative interaction between opioids and estrogens in modulating principal cell excitability.

Ovarian steroids modulate leu-enkephalin levels and target leu-enkephalinergic profiles in the female hippocampal mossy fiber pathway.

In the hippocampal formation (HF), the enkephalin opioids and estrogen are each known to modulate learning and cognitive performance relevant to drug abuse. Within the HF, leu-enkephalin (LENK) is most prominent in the mossy fiber (MF) pathway formed by the axons of dentate gyrus (DG) granule cells. To examine the influence of ovarian steroids on MF pathway LENK levels, we used quantitative light microscopic immunocytochemistry to evaluate LENK levels in normal cycling rats and in estrogen-treated ovariectomized rats. Rats in estrus had increased levels of LENK-immunoreactivity (ir) in the DG hilus compared to rats in diestrus or proestrus. Rats in estrus and proestrus had higher levels of LENK-ir in CA3a-c compared to rats in diestrus. Ovariectomized (OVX) rats 24 h (but not 6 or 72 h) after estradiol benzoate (EB; 10 microg) administration had increased LENK-ir in the DG hilus and CA3c. Electron microscopy showed a larger proportion of LENK-labeled small terminals and axons in the DG hilus compared to CA3 which may have contributed to region-specific changes in LENK-ir densities. Next we evaluated the subcellular relationships of estrogen receptor (ER) alpha, ERbeta and progestin receptor (PR) with LENK-labeled MF pathway profiles using dual-labeling electron microscopy. ERbeta-ir colocalized in some LENK-labeled MF terminals and smaller terminals while PR-ir was mostly in CA3 axons, some of which also showed colocalization with LENK. ERalpha-ir was in dendritic spines, but no colocalization with LENK-labeled profiles was observed. The present studies indicate that estrogen can modulate LENK in subregions of the MF pathway in a dose-and time-dependent manner. These effects might be triggered by direct activation of ERbeta or PR in LENK-containing terminals.

Angiotensin II-induced hypertension differentially affects estrogen and progestin receptors in central autonomic regulatory areas of female rats.

Estrogen receptor (ER) activation in central autonomic nuclei modulates arterial blood pressure (ABP) and counteracts the deleterious effect of hypertension. We tested the hypothesis that hypertension, in turn, influences the expression and trafficking of gonadal steroid receptors in central cardiovascular circuits. Thus, we examined whether ER- and progestin receptor (PR)-immunoreactivity (ir) are altered in medullary and hypothalamic autonomic areas of cycling rats following chronic infusion of the hypertensive agent, angiotensin II (AngII). After 1 week AngII-infusion, systolic ABP was elevated from 103+/-4 to 172+/-8 mmHg (p<0.05; N=8/group) and all rats were in diestrus (low estrogen). In AngII-infused rats the number of PR-immunoreactive nuclei was reduced (-72%) compared to saline-infused controls also in diestrus (p<0.05). Furthermore, the intensity of ERalpha-ir increased selectively in nuclei (16%) and cytoplasm (21%) of cells in the commissural nucleus of the solitary tract (cNTS; p<0.05) while neither the number nor intensity of ERbeta-labeled cells changed (p>0.05). Following chronic AngII-infusion, electron microscopy showed a higher cytoplasmic-to-nuclear ratio of ERalpha-labeling selectively in tyrosine hydroxylase (TH)-labeled neurons in the cNTS. Furthermore, AngII-infusion increased ERalpha-ir in the cytosol of TH- and non-TH neuronal perikarya and increased the amount of ERalpha-ir associated with endoplasmic reticulum only in TH-containing perikarya. The data suggest that hypertension modulates the expression and subcellular distribution of ERalpha and PR in central autonomic regions involved in blood pressure control. Considering that ERalpha counteracts the central and peripheral effects of AngII, these receptor changes may underlie adaptive responses that protect females from the deleterious effects of hypertension.

Microsatellite loci for the invasive colonial hydrozoan Cordylophora caspia.

Cordylophora caspia, a colonial hydrozoan native to the Ponto-Caspian region, has become a common invader of both fresh and brackish water ecosystems of North America and Europe. We describe 11 polymorphic microsatellite loci for this species. Preliminary analyses indicate that population substructure may contribute to departures from Hardy-Weinberg equilibrium. In addition, new loci failed to consistently amplify Cordylophora samples known to be genetically distant from those utilized in this study, indicating the presence of cryptic diversity within the taxon.

Opioid systems in the dentate gyrus.

Opiate drugs alter cognitive performance and influence hippocampal excitability, including long-term potentiation (LTP) and seizure activity. The dentate gyrus (DG) contains two major opioid peptides, enkephalins and dynorphins, which have opposing effects on excitability. Enkephalins preferentially bind to delta- and mu-opioid receptors (DORs and MORs) while dynorphins preferentially bind to kappa-opioid receptors (KORs). Opioid receptors can also be activated by exogenous opiate drugs such as the MOR agonist morphine. Enkephalins are contained in the mossy fiber pathway, in the lateral perforant path (PP) and in scattered GABAergic interneurons. MORs and DORs are predominantly in distinct subpopulations of GABAergic interneurons known to inhibit granule cells, and are present at low levels within granule cells. MOR and DOR agonists increase excitability and facilitate LTP in the molecular layer. Anatomical and physiological evidence is consistent with somatodendritic and axon terminal targeting of both MORs and DORs. Dynorphins are in the granule cells, most abundantly in mossy fibers but also in dendrites. KORs have been localized to granule cell mossy fibers, supramammillary afferents to granule cells, and PP terminals. KOR agonists, including endogenous dynorphins, diminish the induction of LTP. Recent evidence indicates that opiates and opioids also modulate other processes in the hippocampal formation, including adult neurogenesis, the actions of gonadal hormones, and development of neonatal transmitter systems.

Methylphenidate administration to juvenile rats alters brain areas involved in cognition, motivated behaviors, appetite, and stress.

Thousands of children receive methylphenidate (MPH; Ritalin) for attention deficit/hyperactivity disorder (ADHD), yet the long-term neurochemical consequences of MPH treatment are unknown. To mimic clinical Ritalin treatment in children, male rats were injected with MPH (5 mg/kg) or vehicle twice daily from postnatal day 7 (PND7)-PND35. At the end of administration (PND35) or in adulthood (PND135), brain sections from littermate pairs were immunocytochemically labeled for neurotransmitters and cytological markers in 16 regions implicated in MPH effects and/or ADHD etiology. At PND35, the medial prefrontal cortex (mPFC) of rats given MPH showed 55% greater immunoreactivity (-ir) for the catecholamine marker tyrosine hydroxylase (TH), 60% more Nissl-stained cells, and 40% less norepinephrine transporter (NET)-ir density. In hippocampal dentate gyrus, MPH-receiving rats showed a 51% decrease in NET-ir density and a 61% expanded distribution of the new-cell marker PSA-NCAM (polysialylated form of neural cell adhesion molecule). In medial striatum, TH-ir decreased by 21%, and in hypothalamus neuropeptide Y-ir increased by 10% in MPH-exposed rats. At PND135, MPH-exposed rats exhibited decreased anxiety in the elevated plus-maze and a trend for decreased TH-ir in the mPFC. Neither PND35 nor PND135 rats showed major structural differences with MPH exposure. These findings suggest that developmental exposure to high therapeutic doses of MPH has short-term effects on select neurotransmitters in brain regions involved in motivated behaviors, cognition, appetite, and stress. Although the observed neuroanatomical changes largely resolve with time, chronic modulation of young brains with MPH may exert effects on brain neurochemistry that modify some behaviors even in adulthood.

Cellular and subcellular localization of androgen receptor immunoreactivity relative to C1 adrenergic neurons in the rostral ventrolateral medulla of male and female rats.

In male and female rats, high androgen levels can increase blood pressure. The C1 area of the rostral ventrolateral medulla (RVLM), which is crucial for blood pressure regulation, contains estrogen receptors (ERs) in pre- and postsynaptic neuronal compartments and is modulated by estrogens (Wang et al. [2006] Brain Res 1094:163-178). In this study, the cellular and subcellular localization of androgen receptors (ARs) in the C1 area was examined in sections from male, proestrus (high estrogen) and diestrus (low estrogen) female rat brains that were immunocytochemically labeled for AR and tyrosine hydroxylase (TH). By light and electron microscopy, AR-labeled nuclei were scattered among TH-labeled somata in the RVLM; significantly more AR-labeled nuclei were seen males compared to females. Electron microscopy revealed that extranuclear AR-immunoreactivity (ir) was in similar profile types in male and female rats. AR-ir was almost exclusively in myelinated and unmyelinated axons and in glia. Rarely, AR-ir was in axon terminals that contacted TH-containing dendrites. AR-labeled axon terminals had large diameters and contained numerous dense-core vesicles, resembling peptide-containing hypothalamic or solitary tract inputs. No nuclear or extranuclear AR-ir was found in TH-labeled perikarya and dendrites although a few non-TH- labeled dendrites contained AR-ir. Qualitatively, more axonal profiles appeared to be present in males compared to females. These studies suggest that, unlike ERs, ARs in male and female rats are almost exclusively positioned on afferents and glia, suggesting that androgens modulate RVLM C1 neurons, and thus blood pressure, through presynaptic and glial signaling.

Kappa opioid receptors in the rostral ventromedial medulla of male and female rats.

Kappa opioid receptor (KOR) ligands alter nociceptive responses when applied to the rostral ventromedial medulla (RVM). However, the effects of kappa opioid receptor ligands are distinct in males and females. The present study examined the distribution of kappa opioid receptor immunoreactivity in the RVM of male and female rats. KOR immunoreactivity was found at pre- and postsynaptic sites within the RVM of both sexes. The most common KOR-immunoreactive (KOR-ir) neuronal structures were unmyelinated axons, followed by axon terminals, dendrites, and somata. Different proportions of KOR-ir axon terminals and dendrites were found in females at different estrous stages. Specifically, dendrites containing KOR immunoreactivity were less abundant in proestrus females compared with estrus females and showed a trend toward being less abundant in males, suggesting that KOR ligands applied to the RVM may be less potent in proestrus females. These findings suggest that the distribution of KORs in the RVM may be influenced by reproductive hormone levels. We also found KOR immunoreactivity in many spinally projecting neurons within the RVM of female rats. These findings are consistent with the hypothesis that KOR ligands influence nociceptive behaviors by altering the activity of specific populations of neurons within the RVM. The abundance of KOR in axons and axon terminals in RVM indicates a substantial role for presynaptic effects of KOR ligands through pathways that have not been clearly delineated. Altering the balance between pre- and postsynaptic receptive sites may underlie differences in the effects of KOR agonists on nociceptive responses in males and females.

The role of neuronal signaling in controlling cerebral blood flow.

Well-regulated blood flow within the brain is vital to normal function. The brain's requirement for sufficient blood flow is ensured by a tight link between neural activity and blood flow. The link between regional synaptic activity and regional cerebral blood flow, termed functional hyperemia, is the basis for several modern imaging techniques that have revolutionized the study of human brain activity. Here, we review the mechanisms of functional hyperemia and their implications for interpreting the blood oxygen level-dependent (BOLD) contrast signal used in functional magnetic resonance imaging (fMRI).

Extranuclear estrogen receptor beta immunoreactivity is on doublecortin-containing cells in the adult and neonatal rat dentate gyrus.

In adult female rats, estrogen receptor (ER) activation, particularly of ERbeta, promotes hippocampal neurogenesis. We previously reported that extranuclear ERbeta immunoreactivity (ir) in adult rats is on cellular profiles in or near the granule cell layer, which is the location of newly generated cells. During development, cells in or near the granule cell layer transiently express high levels of estrogen binding and nuclear ERs. Thus, we sought to determine if extranuclear ERbeta is in newly generated cells in adult and neonatal rat dentate gyrus. Sections from the dentate gyrus of adult proestrus or postnatal day 7 and 14 female rats were dual-labeled for ERbeta and the new-cell marker doublecortin (DCX) and examined by electron microscopy. DCX-containing neurons were found in the subgranular hilus in adult rats and were more widespread throughout the granule cell layer and hilus of neonatal rats. In both adults and neonatal rats, ERbeta immunoreactivity was found in a subset of DCX-labeled neurons. Electron microscopic examination of the adult dentate gyrus revealed that most perikarya with DCX-ir had the morphological characteristics of granule cells, although a few resembled interneurons. Dendrites with DCX-ir also were observed. In both adults and neonates, DCX-labeled neuronal perikarya and dendrites contained ERbeta-ir; ERbeta-ir usually was aggregated near the plasma membrane, mitochondria or endoplasmic reticula. ERbeta-ir was in glial profiles that apposed DCX-labeled perikarya and dendrites. These findings are consistent with data showing that estrogens can exert non-genomic effects directly and indirectly on newly generated cells in neonatal and adult rat dentate gyrus.

Kappa opioid receptor (KOR) and GAD67 immunoreactivity are found in OFF and NEUTRAL cells in the rostral ventromedial medulla.

This study combines functional and anatomical characterization of neurons in the rostral ventromedial medulla (RVM) to show distinct neurochemical phenotypes between functional classes of neurons. The RVM contains three functional classes of neurons: off cells show a pause in spontaneous activity prior to a nociceptive withdrawal reflex; on cell activity increases prior to a nociceptive reflex; and neutral cell activity does not change significantly during the nociceptive reflex. We determined if serotonin, glutamate decarboxylase (GAD67), or the kappa opioid receptor (KOR) were differentially located within these cell types as predicted by previous studies. In this study, RVM neurons were recorded extracellularly, functionally characterized, and juxtacellularly labeled with biotinamide. Fixed sections were processed for detection of biotinamide and immunfluorescence either for serotonin or for KOR and GAD67. In the first study, serotonin was found exclusively in a subset of neutral cells (33%). These data substantiate previous findings that serotonin is found in some neutral cells whose role in nociception remains unclear. In the second study, we found KOR immunoreactivity in most off (86%) and neutral (80%) cells but rarely in on (13%) cells. We also found GAD67 immunoreactivity in most off (93%) and neutral cells (80%) but less frequently in on cells (63%). Most KOR-immunoreactive cells (16 of 17) also contained GAD67 immunoreactivity regardless of cell classification. These findings support the hypothesis that KOR agonists directly inhibit off and neutral cell activity. The majority of the off and neutral cells are GABAergic, and some on cells are also GABAergic.

Mu opioid receptors are extensively co-localized with parvalbumin, but not somatostatin, in the dentate gyrus.

In the rat dentate gyrus, mu opioid receptor (MOR) agonists disinhibit principal cells, promoting excitation, but whether MOR protein is differentially distributed to interneuron subtypes is unknown. Here, the distribution of MOR immunoreactivity was semi-quantitatively examined in neurochemically identified interneurons using fluorescence microscopy. We find that MOR- and parvalbumin-immunoreactivities are frequently co-localized, while MOR- and somatostatin-immunoreactivities are less commonly co-localized. This suggests that MORs are most frequently on interneurons specialized to inhibit granule cell output, and are on a limited number of interneurons that inhibit granule cell distal dendrites.

Evidence that estrogen directly and indirectly modulates C1 adrenergic bulbospinal neurons in the rostral ventrolateral medulla.

Blood pressure in women increases after menopause, and sympathetic tone in female rats decreases with estrogen injections in the rostral ventrolateral medulla (RVLM) region that contains bulbospinal C1 adrenergic neurons and is involved in blood pressure control. We investigated the anatomical and physiological basis for estrogen effects in the RVLM. Neurons with alpha- or beta-subtypes of estrogen receptor (ER) immunoreactivity (-ir) overlapped in distribution with tyrosine hydroxylase (TH)-containing C1 neurons. Immunoelectron microscopy revealed that ERalpha- and ERbeta-ir had distinct cellular and subcellular distributions. ERalpha-ir was most commonly in TH-lacking profiles, many of which were axons and peptide-containing afferents that contacted TH-containing dendrites. ERalpha-ir was also in some TH-containing dendrites. ERbeta-ir was most frequently in TH-containing somata and dendrites, particularly on endoplasmic reticula, mitochondria, and plasma membranes. In whole-cell patch clamp recordings from isolated bulbospinal RVLM neurons, 17beta-estradiol dose-dependently reduced voltage-gated Ca(++) currents, especially the long-lasting (L-type) component. This inhibition was reversed by washing or prevented by adding the non-subtype-selective ER antagonist ICI182780. An ERbeta-selective agonist, but not an ERalpha-selective agonist, reproduced the Ca(++) current inhibition. The data indicate that estrogens can modulate the function of RVLM C1 bulbospinal neurons either directly, through extranuclear ERbeta, or indirectly through extranuclear ERalpha in selected afferents. Moreover, Ca(++) current inhibition may underlie the decrease in sympathetic tone evoked by local 17beta-estradiol application. These findings provide a structural and functional basis for the effects of estrogens on blood pressure control and suggest a mechanism for the modulation of cardiovascular function by estrogen in women.