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.

The effects of sex steroids and vasotocin on behavioral responses to visual and olfactory sexual stimuli in ovariectomized female roughskin newts.

Previous studies have found that vasotocin (AVT) administration to male roughskin newts (Taricha granulosa) enhances courtship clasping as well as appetitive responses to specific sexual stimuli and that treating female newts with androgens plus AVT induces the expression of male-typical courtship clasping (the selective clasping of females). However, the unique and/or interactive effects of sex steroids and AVT on appetitive responses to specific sexual stimuli have not yet been determined. To first identify male-typical, sexually dimorphic appetitive responses to female sexual stimuli, we tested intact newts during the breeding season and found that males, but not females, are attracted to female visual and pheromonal sexual stimuli. We then used ovariectomized (ovx) females implanted with empty silastic capsules (Blk) or with capsules containing testosterone (T), dihydrotestosterone (DHT), or estradiol (E2) and then injected with either saline or AVT to determine the effects of steroids and AVT, alone or in combination with each other, on male-typical behavioral responses to those stimuli. E2 treatment depressed responses toward female visual stimuli independently of AVT. On the other hand, only T-implanted, AVT-injected females displayed male-typical behavioral responses toward female olfactory stimuli, preferring to spend more time in proximity to female-scented than unscented newt models and selectively clasping the female-scented models. Together, these results support the conclusion that sex steroids and AVT influence behavioral responses to sexual stimuli via sensory-specific mechanisms. Furthermore, they suggest that T and AVT interact within the brain to influence sensorimotor processing in the pathways that integrate olfactory sexual stimuli into male-typical courtship behaviors.

A gene family required for human germ cell development evolved from an ancient meiotic gene conserved in metazoans.

The Deleted in AZoospermia (DAZ) genes encode potential RNA-binding proteins that are expressed exclusively in prenatal and postnatal germ cells and are strong candidates for human fertility factors. Here we report the identification of an additional member of the DAZ gene family, which we have called BOULE. With the identification of this gene, it is clear that the human DAZ gene family contains at least three members: DAZ, a Y-chromosome gene cluster that arose 30-40 million years ago and whose deletion is linked to infertility in men; DAZL, the "father" of DAZ, a gene that maps to human chromosome 3 and has homologs required for both female and male germ cell development in other organisms; and BOULE, a gene that we propose is the "grandfather" of DAZ and maps to human chromosome 2. Human and mouse BOULE resemble the invertebrate meiotic regulator Boule, the proposed ortholog of DAZ, in sequence and expression pattern and hence likely perform a similar meiotic function. In contrast, the previously identified human DAZ and DAZL are expressed much earlier than BOULE in prenatal germ stem cells and spermatogonia; DAZL also is expressed in female germ cells. These data suggest that homologs of the DAZ gene family can be grouped into two subfamilies (BOULE and DAZL) and that members of the DAZ family evolved from an ancestral meiotic regulator, Boule, to assume distinct, yet overlapping, functions in germ cell development.

Rapid changes in monoamine levels following administration of corticotropin-releasing factor or corticosterone are localized in the dorsomedial hypothalamus.

Monoaminergic systems are important modulators of the neuroendocrine, autonomic, and behavioral responses to stress-related stimuli. The male roughskin newt (Taricha granulosa) was used as a model system to investigate the effects of corticotropin-releasing factor (CRF) or corticosterone administration on tissue concentrations of norepinephrine, epinephrine, dopamine, 3,4-dihydroxyphenylacetic acid, serotonin, and 5-hydroxyindoleacetic acid (5-HIAA) in microdissected brain areas. Intracerebroventricular infusion of 25 or 50 ng of CRF increased locomotor activity and site-specifically increased dopamine concentrations within the dorsomedial hypothalamus 30 min after treatment when compared to vehicle-treated controls. In further studies, male newts were treated as follows: (1) no injection, no handling, (2) saline injection, or (3) 10 microg corticosterone and then placed in a novel environment. Monoamine and monoamine metabolite concentrations were similar in the unhandled and saline-injected controls 20 min after treatment. In contrast, corticosterone-injected newts had elevated concentrations of dopamine, serotonin, and 5-HIAA in the dorsomedial hypothalamus (a region that contains dopamine- and serotonin-accumulating neuronal cell bodies in representatives of all vertebrate classes) but not in several other regions studied. These site-specific neurochemical effects parallel neurochemical changes observed in the dorsomedial hypothalamic nucleus of mammals following exposure to a variety of physical and psychological stress-related stimuli. Therefore, these changes may reflect highly conserved, site-specific neurochemical responses to stress and stress-related neurochemicals in vertebrates. Given the important role of the dorsomedial hypothalamus in neuroendocrine, autonomic, and behavioral responses to stress, and a proposed role for this region in fast-feedback effects of glucocorticoids on the hypothalamo-pituitary-adrenal axis, these stress-related monoaminergic changes are likely to have important physiological or behavioral consequences.

Vasotocin stimulates appetitive responses to the visual and pheromonal stimuli used by male roughskin newts during courtship.

It is now well established that vasotocin (AVT) and its mammalian homologue vasopressin influence various social behaviors in vertebrates, but less is known about the mechanisms through which these peptides modulate behavior. In male roughskin newts, Taricha granulosa, AVT stimulates a courtship behavior, amplectic clasping. Three general explanations for how AVT affects male courtship behavior have been considered: by enhancing a central state of sexual motivation, by affecting sensorimotor integration mechanisms in individual sensory modalities, or by influencing a nonspecific state of attention, arousal, or anxiety. AVT administration enhanced appetitive responses to visual and olfactory sexual stimuli, as would be expected if AVT affects a state of sexual motivation that affects behavioral responses to sexual stimuli regardless of the sensory modality in which they are processed. However, AVT selectively enhanced responses to female olfactory stimuli (sex pheromones), but similarly enhanced responses to female and food-related visual stimuli (worms), thus questioning the utility of such a motivational mechanism, as responses to female stimuli were not selectively enhanced in all sensory modalities. We therefore propose that exogenous AVT independently influences olfactory processes associated with orientation/attraction toward a female sex pheromone and visual processes associated with orientation/attraction toward a visual feature common to females and worms. In further experiments AVT administration failed to stimulate feeding behavior but did decrease locomotor activity. Thus, AVT does not stimulate courtship behavior in this species by enhancing the animals' general state of attention or by decreasing general anxiety, as responses to nonsexual, attractive stimuli were not uniformly enhanced, nor by stimulating general arousal, as activity levels did not increase. Rather, the data support the conclusion that AVT affects courtship by influencing specific sensorimotor processes associated with behavioral responses to individual releasing stimuli, which suggests a mechanistic framework for understanding socially motivated behavior is this species.

Behaviroal, pharmacological, and molecular characterization of an amphibian cannabinoid receptor.

Investigation of cannabinoid pharmacology in a vertebrate with a phylogenetic history distinct from that of mammals may allow better understanding of the physiological significance of cannabinoid neurochemistry. Taricha granulosa, the roughskin newt, was used here to characterize an amphibian cannabinoid receptor. Behavioral experiments demonstrated that the cannabinoid agonist levonantradol inhibits both newt spontaneous locomotor activity and courtship clasping behavior. Inhibition of clasping was dose-dependent and potent (IC(50) = 1.2 microgram per animal). Radioligand binding studies using [(3)H]CP-55940 allowed identification of a specific binding site (K(D) = 6.5 nM, B(max) = 1,853 fmol/mg of protein) in brain membranes. Rank order of affinity of several ligands was consistent with that reported for mammalian species (K(D), nM) : CP-55940 (3.8) > levonantradol (13.0) > WIN55212-2 (25.7) >> anandamide (1,665) approximately anandamide 100 microM phenylmethylsulfonyl fluoride (2,398). The cDNA encoding the newt CB1 cannabinoid receptor was cloned, and the corresponding mRNA of 5.9 kb was found to be highly expressed in brain. A nonclonal Chinese hamster ovary cell line stably expressing the newt CB1 cannabinoid receptor was prepared that allowed demonstration of cannabinoid-mediated inhibition of adenylate cyclase (EC 4.6.1.1) activity. This inhibition was dose-dependent and occurred at concentrations consistent with affinities determined through radioligand binding experiments. The behavioral, pharmacological, and molecular cloning results demonstrate that a CB1 cannabinoid receptor is expressed in the CNS of the roughskin newt. This amphibian CB1 is very similar in density, ligand binding affinity, ligand binding specificity, and amino acid sequence to mammalian CB1. The high degree of evolutionary conservation of cannabinoid signaling systems implies an important physiological role in vertebrate brain function.

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.

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.

Sexual dimorphism in numbers of vasotocin-immunoreactive neurons in brain areas associated with reproductive behaviors in the roughskin newt.

Vasotocin (VT) and vasopressin control many endocrine and neuroendocrine functions, including the regulation of reproductive behaviors. In the roughskin newt (Taricha granulosa), VT administration can enhance courtship behaviors in males and egg-laying behaviors in females. This study used immunohistochemistry to investigate whether there are sex differences in VT in specific brain areas, and whether these differences persist in nonbreeding animals. Numbers of VT immunoreactive (ir) cell bodies were counted in males and females collected in February, April, June, and August. Radioimmunoassay of plasma samples confirmed that testosterone and 5alpha-dihydrotestosterone concentrations were higher in males than females, and that 17beta-estradiol concentrations were higher in females than males. In 11 brain areas, no sexual or seasonal differences in the number of VTir cells were found. But in 3 brain regions-the bed nucleus of the stria terminalis (BNST), the nucleus amygdalae dorsolateralis (AMYG), and the anterior preoptic area (aPOA)-there were significantly greater numbers of VTir cells in males than in females, and these differences did not change seasonally. In the aPOA, an area important to male sex behaviors, the sexual dimorphism in VTir was particularly pronounced. In four brain regions, there were significantly greater numbers of VTir cells in females than males, but only in specific seasons. In April-collected (breeding) animals, more VTir cells were found in females than in males in the populations of VT cells within the pars dorsalis hypothalami and ventromedial hypothalamus, brain regions frequently associated with stress responses and female mating behaviors. In August-collected (nonbreeding) animals, more VTir cells were found in females than in males, in the region of the bed nucleus of the decussation of the fasciculus lateralis telencephali and in the nucleus visceralis superior, nucleus isthmi region. Significantly greater numbers of VTir cells were observed in the magnocellular preoptic area of males and females collected in February. These results indicate that the functional interactions between gonadal steroid hormones and VT are complex and appear to involve site-, sex-, and season-specific regulatory mechanisms. Furthermore, it seems likely that populations of VT neurons in the BNST, AMYG, and aPOA are involved in regulating male-specific behaviors, and that the VT neurons in the pars dorsalis hypothalami/ventromedial hypothalamus may be involved in female-specific behaviors.

Steroid hormones use non-genomic mechanisms to control brain functions and behaviors: a review of evidence.

Progestins, estrogens, androgens, and corticosteroids are capable of modifying brain functions and behaviors by mechanisms that involve the classic genomic model for steroid action. However, experimental evidence indicates that some responses to steroid hormones use non-classical, non-genomic mechanisms. This paper reviews the evidence that steroids can bind to receptors in the plasma membrane, activate cell signaling pathways, and regulate responses on a time scale of seconds or a few minutes. The existence of these alternative regulatory pathways for steroid hormones should make endocrinologists and neurobiologists change how they think about steroid hormones. It is no longer valid to assume that minute-to-minute changes in steroid concentrations are not regulating biologically important, short-term responses, or that the only steroids with biological functions are the ones that bind with high affinity to intracellular steroid receptors.

A neurobehavioral model for rapid actions of corticosterone on sensorimotor integration.

Stress-induced corticosterone (CORT) secretion that causes a rapid blockade of courtship clasping by male roughskin newts (Taricha granulosa) is mediated by a specific neuronal membrane receptor for CORT. Amplectic clasping, which can be triggered by pressure on the ventral body surface and cloaca, is controlled by the influence of medullary neurons on the spinal cord. Using clasping as a simple neurobehavioral model, we have focused our analysis of CORT effects on clasping by examining the steroid's effects on neurophysiological properties of medullary neurons, especially medullary reticulospinal neurons, the principal output cells from the brain to the spinal cord. Systemic CORT caused, within 3 min of injection, diverse reductions in reticulospinal neuron excitability. Another rapid CORT effect on medullary neurons was to depress responsiveness to pressure on the cloaca. Experiments with chronically implanted, freely moving newts revealed that the rapid CORT effects are quite specific to neural processes related to clasping. CORT injections rapidly blocked clasping in response to cloacal stimuli and concurrently depressed neuronal responses to cloacal pressure and firing associated with clasping. Activity of reticulospinal neurons was often associated with nonclasping movements and this activity was rarely altered by CORT. Thus, CORT mainly affected aspects of neuronal function related to clasping. In other neurophysiological experiments, we found that the neuropeptides vasotocin and corticotropin-releasing hormone modified the neural effects of CORT. Prior exposure of medullary neurons to either of these neuropeptides caused systemic CORT administration to rapidly potentiate neuronal responses to cloacal stimuli, indicating that the direction and potency of CORT effects depend critically on the prevailing neuroendocrine state of the brain.

Dynamical localization of ultracold sodium atoms.

We report a study of atomic motion in time-dependent optical potentials. We measure momentum transfer in parameter regimes for which the classical dynamics are chaotic, and observe the quantum suppression of chaos by dynamical localization. The high degree of control over the experimental parameters enables detailed comparisons with theoretical predictions, and opens new avenues for investigating quantum chaos.

Comparative neuroanatomy of vasotocin and vasopressin in amphibians and other vertebrates.

This review focuses on the neuroanatomical distribution of vasotocin (VT) and vasopressin (VP) and presents a comparative analysis of brain areas in which VT and VP cell bodies have been reported in fish, amphibians, reptiles, birds and mammals. A comparison of information from previous neuroanatomical studies of VT and VP with findings from a recent study of VT in an amphibian (Taricha granulosa) supports the conclusions that the VT/VP system can be subdivided into identifiable groups of cell bodies, based on neuroanatomical and cell morphology characteristics, and that these cell groups are not necessarily delimited by classical neuroanatomical boundaries. The comparative neuroanatomy of the distribution of VT and VP cell bodies also indicates that the neuroanatomy of the VT/VP system is fairly conserved among vertebrates. The review uses comparative data to present a series of tentative hypotheses about the homology of the VT cell groups and VP cell groups in the different vertebrate taxa.

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.

Rapid, corticosterone-induced disruption of medullary sensorimotor integration related to suppression of amplectic clasping in behaving roughskin newts (Taricha granulosa).

Endogenously secreted or injected corticosterone (CORT) rapidly suppresses courtship clasping in male roughskin newts (Taricha granulosa) by an action on a specific neuronal membrane receptor. Previous studies, using immobilized newts, showed that CORT administration rapidly depresses excitability of reticulospinal neurons and attenuates medullary neuronal responsiveness to clasp-triggering sensory stimuli. The present study used freely moving newts to examine clasping responses and concurrently record sensorimotor properties of 67 antidromically identified reticulospinal and other medullary reticular neurons before and after CORT injection. Before CORT, reticulospinal neurons fired in close association with onset and offset of clasps elicited by cloacal pressure. Reticulospinal neurons also showed firing correlates of nonclasping motor events, especially locomotion. Neuronal activity was typically reduced during clasping and elevated during locomotion. Medullary neurons that were not antidromically invaded (unidentified neurons) usually showed sensorimotor properties that resembled those of reticulospinal neurons. Intraperitoneal CORT (but not vehicle) reduced the probability and quality of hindlimb clasping in response to cloacal pressure, especially within 5-25 min of injection. Simultaneously, responses of reticulospinal and unidentified neurons to cloacal pressure and occurrence of clasping-related activity were attenuated or eliminated. CORT effects were relatively selective, altering clasping-related neuronal activity more strongly than activity associated with nonclasping motor events. The properties of CORT effects indicate that the hormone impairs clasping by depressing processing of clasp-triggering afferent activity and by disrupting the medullary control of clasping normally mediated by reticulospinal neurons. The rapid onset of these CORT effects implicates a neuronal membrane receptor rather than genomic action of the steroid.

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.