Observational evidence for interhemispheric hydroxyl-radical parity.

The hydroxyl radical (OH) is a key oxidant involved in the removal of air pollutants and greenhouse gases from the atmosphere. The ratio of Northern Hemispheric to Southern Hemispheric (NH/SH) OH concentration is important for our understanding of emission estimates of atmospheric species such as nitrogen oxides and methane. It remains poorly constrained, however, with a range of estimates from 0.85 to 1.4 (refs 4, 7-10). Here we determine the NH/SH ratio of OH with the help of methyl chloroform data (a proxy for OH concentrations) and an atmospheric transport model that accurately describes interhemispheric transport and modelled emissions. We find that for the years 2004-2011 the model predicts an annual mean NH-SH gradient of methyl chloroform that is a tight linear function of the modelled NH/SH ratio in annual mean OH. We estimate a NH/SH OH ratio of 0.97 ± 0.12 during this time period by optimizing global total emissions and mean OH abundance to fit methyl chloroform data from two surface-measurement networks and aircraft campaigns. Our findings suggest that top-down emission estimates of reactive species such as nitrogen oxides in key emitting countries in the NH that are based on a NH/SH OH ratio larger than 1 may be overestimated.

Global-scale seasonally resolved black carbon vertical profiles over the Pacific.

[1] Black carbon (BC) aerosol loadings were measured during the High-performance Instrumented Airborne Platform for Environmental Research Pole-to-Pole Observations (HIPPO) campaign above the remote Pacific from 85°N to 67°S. Over 700 vertical profiles extending from near the surface to max ∼14 km altitude were obtained with a single-particle soot photometer between early 2009 and mid-2011. The data provides a climatology of BC in the remote regions that reveals gradients of BC concentration reflecting global-scale transport and removal of pollution. BC is identified as a sensitive tracer of extratropical mixing into the lower tropical tropopause layer and trends toward surprisingly uniform loadings in the lower stratosphere of ∼1 ng/kg. The climatology is compared to predictions from the AeroCom global model intercomparison initiative. The AeroCom model suite overestimates loads in the upper troposphere/lower stratosphere (∼10×) more severely than at lower altitudes (∼3×), with bias roughly independent of season or geographic location; these results indicate that it overestimates BC lifetime.

A corticosteroid receptor in neuronal membranes.

Steroids may rapidly alter neuronal function and behavior through poorly characterized, direct actions on neuronal membranes. The membrane-bound receptors mediating these behavioral responses have not been identified. [3H]Corticosterone labels a population of specific, high-affinity recognition sites (dissociation constant = 0.51 nanomolar) in synaptic membranes from an amphibian brain. These binding sites were localized by receptor autoradiography in the neuropil, outside the regions of perikarya. The affinities of corticoids for this [3H]corticosterone binding site were linearly related to their potencies in rapidly suppressing male reproductive behavior. Thus, it appears that brain membranes contain a corticosteroid receptor that could participate in the regulation of behavior.

A subset of kappa opioid ligands bind to the membrane glucocorticoid receptor in an amphibian brain.

Previous studies demonstrated that a membrane receptor for glucocorticoids (mGR) exists in neuronal membranes from the roughskin newt (Taricha granulosa) and that this receptor appears to be a G protein-coupled receptor (GPCR). The present study investigated the question of whether this mGR recognizes nonsteroid ligands that bind to cognate receptors in the GPCR superfamily. To address this question, ligand-binding competition studies evaluated the potencies of various ligands to displace [3H]corticosterone (CORT) binding to neuronal membranes. Initial screening studies tested 21 different competitors and found that [3H]CORT binding was displaced only by dynorphin 1-13 amide (an endogenous kappa-selective opioid peptide), U50,488 (a synthetic kappa-specific agonist) and naloxone (a nonselective opioid antagonist). Follow-up studies revealed that the kappa agonists bremazocine (BRE) and ethylketocyclazocine (EKC) also displaced [3H]CORT binding to neuronal membranes, but that U69,593 (a kappa specific agonist) and nor-BNI (a kappa specific antagonist) were ineffective. The Ki values measured for the opioid competitors were in the subnanomolar to low micromolar range and had the following rank-order: dynorphin > U50,488 > naloxone > BRE > EKC. Because these ligands displaced, at most, only 70% of [3H]CORT specific binding, it appears that some [3H]CORT binding sites are opioid insensitive. Kinetic analysis of [3H]CORT off-rates in the presence of U50,488 and/or CORT revealed no differences in dissociation rate constants, suggesting that there is a direct, rather than allosteric, interaction with the [3H]CORT binding site. In summary, these results are consistent with the hypothesis that the high-affinity membrane binding site for [3H] CORT is located on a kappa opioid-like receptor.

Antibodies against different forms of GnRH distinguish different populations of cells and axonal pathways in a urodele amphibian, Taricha granulosa.

Neurons immunoreactive to the peptide hormone gonadotropin-releasing hormone (GnRH) have been identified in the posterior diencephalon or anterior midbrain of diverse vertebrates. These cells are located caudal to the more well-characterized GnRH neurons in the nervus terminalis and septo-preoptic area, and are believed to express one or more of the nonmammalian forms of the GnRH. The present study utilized immunocytochemical techniques to determine whether the posterior GnRH group is present in a urodele amphibian, the newt Taricha granulosa. Antibodies directed against different molecular forms of GnRH were used to evaluate the immunological properties of GnRH-containing neurons in amphibians. An antibody selective for mammalian GnRH labeled perikarya in the nervus terminalis (terminal nerve) and septo-preoptic region, as described previously. Thick fibers that arise from terminal nerve and septo-preoptic neurons project mainly to the median eminence, medial pallium and habenula. An antibody selective for chicken GnRH II labeled cell bodies in the paraventricular organ and posterior tubercle of the caudal diencephalon, and thin fibers that project widely throughout the central nervous system. Region-specific staining with different GnRH antibodies supports the interpretation that different molecular forms of GnRH are expressed by neuroanatomically distinguishable systems.

Neuroanatomical distribution of androgen and estrogen receptor-immunoreactive cells in the brain of the male roughskin newt.

Immunohistochemistry was used to investigate the neuroanatomical distribution of androgen and estrogen receptors in brains of adult male roughskin newts, Taricha granulosa, collected during the breeding season. Immunoreactive cells were found to be widely distributed in specific brain areas of this urodele amphibian. Androgen receptor-immunoreactive (AR-ir) cells were observed in the olfactory bulbs, habenula, pineal body, preoptic area, hypothalamus, interpeduncular nucleus, area acusticolateralis, cerebellum, and motor nuclei of the medulla oblongata. Estrogen receptor-immunoreactive (ER-ir) cells were found in the lateral septum, amygdala pars lateralis, pallium, preoptic area, hypothalamus, and dorsal mesencephalic tegmentum. This immunocytochemical study of the newt brain reveals AR-ir and ER-ir cells in several regions that have not been previously reported to contain androgen and estrogen receptors in non-mammalian vertebrates. Additionally, the distribution of AR-ir and ER-ir cells in the newt brain, in general, is consistent with previous studies, suggesting that the distribution of sex steroid receptor-containing neurons in some brain regions is relatively conserved among vertebrates.

Neuroanatomical distribution of vasotocin in a urodele amphibian (Taricha granulosa) revealed by immunohistochemical and in situ hybridization techniques.

Immunohistochemical and in situ hybridization techniques were used to investigate the neuroanatomical distribution of arginine vasotocin-like systems in the roughskin newt (Taricha granulosa). Vasotocin-like-immunoreactive neuronal cell bodies were identified that, based on topographical position, most likely, are homologous to groups of vasopressin-immunoreactive neuronal cell bodies described in mammals, including those in the bed nucleus of the stria terminalis, medial amygdala, basal septal region, magnocellular basal forebrain-including the horizontal limb of the diagonal band of Broca, paraventricular and supraoptic nuclei, suprachiasmatic nucleus, and dorsomedial hypothalamic nucleus. Several additional vasotocin-like-immunoreactive cell groups were observed in the forebrain and brainstem regions; these observations are compared with previous studies of vasotocin- and vasopressin-like systems in vertebrates. Arginine vasotocin-like-immunoreactive fibers and presumed terminals also were widely distributed with high densities in the basal limbic forebrain, the ventral preoptic and hypothalamic regions, and the brainstem ventromedial tegmentum. Based on in situ hybridization studies with synthetic oligonucleotide probes for vasotocin and the related neuropeptide mesotocin, as well as double-labeling studies with combined immunohistochemistry and in situ hybridization, we conclude that the vasotocin immunohistochemical procedures used identify vasotocin-like, but not mesotocin-like, elements in the brain of T. granulosa. The distribution of arginine vasotocin-like systems in T. granulosa is greater than the distribution previously reported for any other single vertebrate species; however, it is consistent with an emerging pattern of distribution of vasotocin- and vasopressin-like peptides in vertebrates. Complexity in the vasotocinergic system adds further support to the conclusion that this peptide regulates multiple neurophysiological and neuroendocrinological functions.

N-ethylmaleimide (NEM) can significantly improve in situ hybridization results using 35S-labeled oligodeoxynucleotide or complementary RNA probes.

We predicted that a significant source of background labeling after in situ hybridization (ISH) using 35S-labeled probes is attributable to a chemical reaction between the phosphorothioate moiety of the probe [O3P = S] and disulfides in tissue. These covalent bonds would immobilize probe in the tissue, thereby increasing background labeling. On the basis of this view, we have explored the use of N-ethylmaleimide (NEM) to irreversibly alkylate the phosphorothioate moiety of the probe and/or to alkylate free sulfhydryls in tissue to block the formation of disulfides as a method of reducing background labeling. We report that NEM can significantly decrease background labeling of 35S-labeled oligodeoxynucleotide or cRNA probes but does not affect specific labeling. We conclude that the use of NEM in ISH protocols, as outlined here, may be an additional element researchers may consider to improve the signal-to-noise ratio.

Steroid-neuropeptide interactions that control reproductive behaviors in an amphibian.

Investigations into the neuroendocrine regulation of reproductive behaviors in an amphibian (Taricha granulosa) reveal the same basic repertoire of chemical messengers as regulators of male behaviors in other vertebrates. These studies have identified seasonal neural interactions between gonadal steroids and neuropeptides that facilitate male courtship behavior. In addition, this species has served to elucidate how stress-induced suppression of courtship is mediated by corticosterone action through a neuronal membrane receptor and subsequent, rapid neurophysiological effects. These findings indicate that a principal mechanism by which steroids and neuropeptides control male reproductive behavior is the modulation of neural processing of specific sensory stimuli.

Partial purification and biochemical characterization of a membrane glucocorticoid receptor from an amphibian brain.

A membrane receptor for corticosterone (mGR) in the brain of the roughskin newt (Taricha granulosa) has been previously identified. This manuscript reports the evaluation of several chromatographic resins for enrichment of the newt mGR solubilized from neuronal membranes. A protein with an apparent molecular weight of 63 kDa was purified to near homogeneity following sequential purification using ammonium sulfate fractionation, wheat germ agglutinin (WGA)-agarose chromatography, hydroxylapatite chromatography, and an immobilized ligand affinity resin (Corticosterone-Sepharose). Other studies employed a novel protein differential display strategy and a photoaffinity labeling strategy to visualize candidate receptor proteins following SDS-PAGE. Both of these techniques also identified a 63 kDa protein, agreeing with the estimation of molecular weight from the purification data. Furthermore, the use of 2D SDS-PAGE following the photolabeling procedure showed the candidate 63 kDa protein to have a pI of approximately 5.0. Taken together these data suggest that the newt mGR is an acidic glycoprotein with an apparent molecular weight of 63 kDa. Because these characteristics of newt mGR are inconsistent with the characteristics of intracellular glucocorticoid receptors, these two receptor proteins are apparently distinct.

Solubilization and pharmacological characterization of a glucocorticoid membrane receptor from an amphibian brain.

Physiological functions of steroid hormones involve activation of intracellular receptors as well as poorly understood membrane receptors. We report the pharmacological characterization of a solubilized corticosterone receptor from neuronal membranes. This receptor previously was shown to localize with plasma membrane subcellular fractions and to be involved in the modulation of courtship behaviors in the roughskin newt (Taricha granulosa). We describe procedures with non-ionic detergents that solubilize the receptor and maintain high affinity [3H]corticosterone binding. The pharmacology of the solubilized corticosterone receptor resembles that of the membrane receptor with high affinity for [3H]corticosterone and an identical rank-order potency for other steroid ligands (corticosterone>cortisol>aldosterone>dexamethasone). Unlike binding in membrane preparations, [3H]corticosterone binding to the solubilized receptor is insensitive to negative modulation by guanyl nucleotides and only modestly sensitive to the presence of Mg2+. We also identified two ligands that exhibit high affinity binding to the solubilized receptor and have the potential to be used in an affinity purification scheme. They are corticosterone-3-carboxymethyloxime (CORT-3-CMO), which may be covalently attached to a Sepharose resin, and a derivitized azide form of CORT-3-CMO which can be covalently coupled to the solubilized receptor itself. The stability of the solubilized [3H]corticosterone receptor in the detergent system will facilitate further purification and molecular characterization.

Evolutionary precedents for behavioral actions of oxytocin and vasopressin.

It is clear that the behavioral actions of oxytocin and vasopressin in mammals are not newly acquired, but have evolutionary antecedents. Injection studies with fish, amphibians, reptiles, and birds indicate that AVT can activate certain reproductive behaviors. The strongest evidence that AVT acts centrally to control reproductive behaviors comes from research on T. granulosa. In this amphibian, injections of AVT agonists activate courtship behaviors (amplectic clasping) in males and egg-laying behaviors in females, whereas injections of AVT antagonists inhibit the behaviors. Also, in Taricha males, AVT concentrations in specific brain areas are associated with the expression of courtship behaviors. Several conclusions about steroid-peptide interactions can be drawn, based on research with this amphibian. First, gonadal steroid hormones act to maintain the behavioral actions of AVT in both males and females. In Taricha, gonadectomy abolishes and steroid implants restore AVT-induced courtship in males and egg-laying in females. Second, gonadal steroids maintain the behavioral actions of AVT, in part, by modulating AVT receptor numbers on target neurons. In Taricha males and females, gonadectomy reduces AVT receptor concentrations (but not binding affinity) in certain brain areas (amygdala pars lateralis) and not others. Third, the type of gonadal steroid determines whether AVT elicits male-like or female-like reproductive behaviors. Ovariectomized Taricha females respond to AVT injections with egg-laying behaviors when implanted with estradiol and with male-like amplectic clasping when implanted with dihydrotestosterone. Fourth, the masculinization of AVT-induced behaviors in females most likely reflects site-specific actions of androgens on AVT-synthesizing neurons. In Taricha, AVTir concentrations in the optic tectum are sexually dimorphic (higher in males than females) and reach peak levels in males during the breeding season. Fifth, AVT content in specific brain areas increase as a function of performing the behaviors. In Taricha, AVTir concentrations in DPOA, CSF, and ventral infundibulum are higher in males that exhibit courtship behaviors than in males that do not. These conclusions illustrate how steroid-peptide interactions in the control of behaviors entail multiple neuroanatomical sites and neurochemical actions.

Intracranial administration of corticotropin-like peptides increases incidence of amphibian reproductive behavior.

Male rough-skinned newts (Taricha granulosa) exhibit an increase in sexual behavior (amplectic clasping) following intracerebroventricular (ICV) injection of adrenocorticotropin (ACTH 1-39), ACTH 4-10, or melanocyte-stimulating hormone (alpha MSH). In contrast, intraperitoneal (IP) administration of ACTH 1-39 or corticosterone significantly decreases the incidence of sexual behavior in male newts. These results suggest that a corticotropin-like peptide acts centrally to enhance sexual behavior and that systemic ACTH acts on the interrenal tissue to inhibit sexual behavior by stimulating the release of corticosterone.

Arginine vasotocin induces sexual behavior of newts by acting on cells in the brain.

To investigate whether arginine vasotocin (AVT) acts on target cells in the brain of Taricha granulosa (a urodele amphibian), the behavioral effects of intracerebroventricular (ICV) and intraperitoneal (IP) injections of AVT were compared. Male newts exhibited the greatest sexual activity (amplectic clasping) following an ICV injection of 0.1 microgram AVT. Another study showed that nanogram quantities of AVT, administered ICV, stimulated the behavior. An ICV injection of an antagonist to arginine vasopressin, d(CH2)5Tyr(Me)AVP, or an anti-AVT immune serum significantly inhibited the sexual behavior. Intracranial implants of 17 beta-estradiol (E2) or 5 alpha-dihydrotestosterone (DHT) in castrated males maintained the behavioral response to an injection of AVT. Another study found that an IP injection of DHT or E2 did not increase the incidence of newt sexual behavior during the 8 hours following the injection.

Gonadotropin-releasing hormones in microdissected brain regions of an amphibian: concentration and anatomical distribution of immunoreactive mammalian GnRH and chicken GnRH II.

Mammalian and chicken II gonadotropin-releasing hormones (mGnRH, cGnRH II) were extracted from 350 microns diameter punches from brains of a urodele amphibian, Taricha granulosa, and measured by means of radioimmunoassay (RIA) with specific antisera. Measurable quantities of both peptides were found in the lateral pallium, the subpallium (along the course of the nervus terminalis), the preoptic area, habenula, optic tectum, infundibulum, paraventricular organ/posterior tubercle of the caudal diencephalon, medulla, and cerebrospinal fluid. Highest concentrations of both peptides were in the preoptic area and infundibulum, suggesting a role in gonadotropin release. In most extrahypothalamic regions, cGnRH II concentrations exceeded those of mGnRH, suggesting that cGnRH II may function as a neurotransmitter in many sites, perhaps to control reproductive behaviors. Results are largely consistent with immunocytochemical (ICC) analyses, except that RIA revealed small amounts of both peptides not found by ICC in some areas of the brain. Results from this microdissection/RIA study and prior ICC studies in amphibians support the conclusions that GnRH cell bodies in the terminal nerve and preoptic area, which project mainly to the median eminence and habenula, express mGnRH, and that GnRH cell bodies in the caudal diencephalon, which project widely throughout the CNS, express cGnRH II. Comparative data support the view that cGnRH II, and the neural systems in which it is expressed, evolved early in vertebrate phylogeny and have been highly conserved.

Gonadectomy reduces the concentrations of putative receptors for arginine vasotocin in the brain of an amphibian.

Putative receptors for arginine vasotocin (AVT) in the brain of the newt (Taricha granulosa) were measured using quantitative autoradiography with tritium-labelled vasopressin. Specific binding sites were observed in the olfactory bulb, medial (hippocampal) pallium, dorsal pallium, amygdala para lateralis and tegmental region of the medulla oblongata. In both male and female newts, concentrations of binding sites in the amygdala, but not in the other four areas, were significantly lower in gonadectomized animals than in sham-operated controls. The equilibrium dissociation constants (KdS) were not altered by gonadectomy. Since long-tem castration abolishes the effect of AVT injection on sexual behaviors, these results support the hypothesis that gonadal steroids maintain sexual behaviors in this amphibian by maintaining AVT receptors in the amygdala.

Sexually dimorphic concentrations of arginine vasotocin in sensory regions of the amphibian brain.

Arginine vasotocin (AVT) regulates reproductive behaviors in amphibians. We measured AVT in the brains of bullfrogs (Rana catesbeiana) and newts (Taricha granulosa) using radioimmunoassay. In bullfrogs, AVT concentrations were greater in males, compared to females, in the amygdala pars lateralis, optic tectum, and tegmentum. Concentrations in the dorsolateral nucleus were greater in females. In newts, AVT concentrations were also greater in the tectum and tegmentum of males. AVT may modulate dimorphic behaviors by acting at these sites.

Ontogeny of immunoreactive gonadotropin-releasing hormone neuronal systems in amphibians.

The ontogeny of gonadotropin-releasing hormone (GnRH) systems was investigated in 3 anuran amphibians (genus Rana) by means of immunocytochemical (ICC) techniques and antibodies generated against 3 different forms of GnRH. Antisera that recognize primarily chicken II and mammalian GnRHs revealed two anatomically and developmentally distinct GnRH systems. One system, referred to here as the forebrain-spinal cord system, contained GnRH immunoreactive (ir) fibers extending from the rostral diencephalon through the ventromedial brainstem to the spinal cord. Intensity of labeling was robust in the youngest, premetamorphic tadpoles, but decreased with age. GnRH immunolabeling in the hypothalamic-pituitary tract was not detected until late prometamorphosis and increased with age. Development of GnRHir in the hypothalamic-pituitary tract coincided with first appearance of GnRHir in the terminal nerve in R. catesbeiana, but not in R. cascadae or R. aurora, suggesting species differences. Comparisons of results obtained with antisera to different forms of GnRH support the interpretation that the forebrain-spinal cord system, hitherto undescribed in amphibians, develops first and synthesizes a non-mammalian, chicken II-like GnRH, and that the hypothalamic-pituitary system develops later and synthesizes primarily mammalian GnRH. We speculate that the forebrain-spinal cord system may represent a GnRH innervation of frog sympathetic ganglia, and that the two GnRH systems are chemically and embryonically distinct.

Steroid modulation of GABAA receptors in an amphibian brain.

Steroids can modulate gamma-aminobutyric acid (GABAA) receptor function in rat brains, but the physiological relevance of this mechanism is still unclear. To determine whether this phenomenon is widespread among vertebrates, we investigated steroid modulation of GABAA receptors in amphibian brain tissue. Equilibrium binding parameters for t-butylbicyclophosphorothionate ([35S]TBPS) and [3H]flunitrazepam were similar in Taricha granulosa and mammalian brains, as was the allosteric regulation of [35S]TBPS and [3H]flunitrazepam binding by GABA. The rank order and absolute potencies of steroids to inhibit [35S]TBPS binding and enhance [3H]flunitrazepam binding were also similar in Taricha and rat brains. As in mammalian studies, physiological concentrations of corticosterone had no effect on ligand binding or GABA-stimulated Cl- uptake. In autoradiographic studies, 3 alpha-hydroxy-5 alpha-pregnan-20-one inhibited [35S]TBPS binding sites in all brain regions examined, whereas corticosterone had no effect on [35S]TBPS binding. These studies suggest that the steroid recognition sites on GABAA receptors have been highly conserved through vertebrate evolution and thus portend physiologically important functions. However, the pharmacological profiles for the GABAA receptor and the high-affinity corticosteroid receptor are apparently different, suggesting there are multiple types of steroid recognition sites on neuronal membranes.

Effects of corticotropin-releasing factor (CRF) and opiates on amphibian locomotion.

Male rough-skinned newts (Taricha granulosa) were used as a model for the study of the neuroendocrine regulation of locomotion. Intracerebroventricular (i.c.v.) injections of nanogram quantities of corticotropin-releasing factor (CRF) dose-dependently increased locomotion as measured in a circular open-field test arena. In other studies animals received intraperitoneal (i.p.) injections of saline or naloxone, a synthetic opioid antagonist, followed by i.c.v. injections of saline or CRF. With 1-min intervals between injections, neither i.p. saline nor naloxone injections modified the stimulatory effects of CRF injections on locomotor activity. In contrast, with 20-min intervals between injections, the naloxone-plus-CRF injected newts displayed more locomotor activity than the saline-plus-CRF injected newts, suggesting that the opioid system modulated the behavioral effects of CRF. An i.p. injection of bremazocine, an opiate kappa-receptor agonist, suppressed spontaneous locomotion but not CRF-induced locomotion. In contrast, an i.p. injection of morphine, an opiate mu-receptor agonist, did not affect spontaneous locomotion but reduced CRF-induced locomotion, indicating further that the opioid system may modulate the behavioral effects of CRF in this amphibian. The present study provides the first evidence that both CRF and opioids may be involved in the regulation of amphibian locomotor activity.