Drosophila social clustering is disrupted by anesthetics and in narrow abdomen ion channel mutants.

Members of many species tend to congregate, a behavioral strategy known as local enhancement. Selective advantages of local enhancement range from efficient use of resources to defense from predators. While previous studies have examined many types of social behavior in fruit flies, few have specifically investigated local enhancement. Resource-independent local enhancement (RILE) has recently been described in the fruit fly using a measure called social space index (SSI), although the neural mechanisms remain unknown. Here, we analyze RILE of Drosophila under conditions that allow us to elucidate its neural mechanisms. We have investigated the effects of general volatile anesthetics, compounds that compromise higher order functioning of the type typically required for responding to social cues. We exposed Canton-S flies to non-immobilizing concentrations of halothane and found that flies had a significantly decreased SSI compared with flies tested in air. Narrow abdomen (na) mutants, which display altered responses to anesthetics in numerous behavioral assays, also have a significantly reduced SSI, an effect that was fully reversed by restoring expression of na by driving a UAS-NA rescue construct with NA-GAL4. We found that na expression in cholinergic neurons fully rescued the behavioral defect, whereas expression of na in glutamatergic neurons did so only partially. Our results also suggest a role for na expression in the mushroom bodies (MBs), as suppressing na expression in the MBs of NA-GAL4 rescue flies diminishes SSI. Our data indicate that RILE, a simple behavioral strategy, requires complex neural processing.

Volatile general anesthetics reveal a neurobiological role for the white and brown genes of Drosophila melanogaster.

Molecular and cellular evidence argues that a heterodimer between two ABC transporters, the White protein and the Brown protein, is responsible for pumping guanine into pigment-synthesizing cells of the fruit fly, Drosophila melanogaster. Previous studies have not detected White or Brown outside pigment-synthesizing cells nor have behavioral effects of null mutants been reported, other than those that are visually dependent. Nevertheless, we show here that exposure to the volatile general anesthetic (VGA) enflurane reveals a difference in neuromuscular performance between wild-type flies and those that carry a null allele in either the white or brown gene. Specifically, in a test of climbing ability, w1118 or bw1 flies are much less affected by enflurane than are congenic controls. Altered anesthetic sensitivity is still observed when visual cues are reduced or eliminated, arguing that white and brown contribute to neural function outside the eye. This hypothesis is supported by the detection of white message in heads of flies that are genetically altered so as to lack pigment-producing cells. The w1118 or bw1 mutations also alter the response to a second VGA, halothane, albeit somewhat differently. Under some conditions, the combination of w1118 with another mutation that affects anesthesia leads to a drastically altered phenotype. We consider several ways by which diminished transport of guanine could influence neural function and anesthetic sensitivity.

Pathways for repair of topoisomerase I covalent complexes in Saccharomyces cerevisiae.

BACKGROUND: The covalent linkage between DNA and the active site tyrosine of topoisomerase I can be stabilized by chemotherapeutic agents, adjacent DNA lesions, or mutational defects in the topoisomerase itself. Following collision with a replication fork, the covalent complex can be converted to a double-strand break. Tdp1, an enzyme that can hydrolyse the bond between topoisomerase I and DNA, is thought to be involved in the repair of these lesions, but little is known about how such repair is accomplished. RESULTS: Reaction kinetics with model substrates reveal that the catalytic efficiency of Saccharomyces cerevisiae Tdp1 is relatively poor when the scissile bond is located in the middle of a duplex, but much better when it is located at the end of a structure. Survival of yeast after induction of a toxic topoisomerase is substantially reduced by inactivation of the TDP1 gene. Comparison of survival of single and double mutants places TDP1 and RAD52 in the same epistasis group but TDP1 and RAD9 in different epistasis groups. In the absence of RAD9, inactivation of TDP1 has a significant effect on the survival of cells following exposure to camptothecin but is without consequence for the survival of agents that do not target topoisomerase I. CONCLUSIONS: Tdp1 acts as a specific repair enzyme for topoisomerase I lesions. Rather than working at their earliest occurrence, the enzyme acts after covalent complexes have been converted to DSBs. A second repair pathway also exists that functions independently of Tdp1 but requires RAD9 function to efficiently repair topoisomerase I-linked DSBs. The efficiency of these pathways differs for complexes induced with the chemotherapeutic agent camptothecin vs. those accumulated by mutant forms of topoisomerase I.

The development and regression of deciduosarcomas and other lesions caused by estrogens and progestins in rabbits.

A series of experiments were conducted to study the histopathological effects of a combination of exogenous estrogens and progestins in mature rabbits. Estradiol (14-45 microg/day) and levonorgestrel (30-233 microg/day) were administered by intravaginal or subdermal Silastic devices for various time intervals to study the development of lesions with time and to determine if lesions regressed following withdrawal of the steroids. The origin of splenic decidual tumors (primary or metastasis from the uterus) was determined by administering the same steroid combination to castrated male rabbits. It was determined that uterine decidualization is present after 7 days of steroid treatment and that neoplasms of decidual cells may appear in the uterus after only 30 days of steroid administration. Decidual changes were observed frequently in uterine arteries, often concurrent with infarct-like areas of necrosis of the uterine wall. Withdrawal of contraceptive steroids for 14-120 days after 60 days' administration resulted in atrophy and disappearance of decidual cells and decidual tumors. Decidual neoplasms developed in the spleen of all castrated male rabbits given subdermal steroids, demonstrating that these tumors can arise as primary neoplasms of the spleen. The foregoing lesions appear to be peculiar to the rabbit and, together with previous data, suggest the rabbit to be a poor model for evaluating the effects of contraceptive steroids in other species.

The roles of estrogen and progestin in producing deciduosarcoma and other lesions in the rabbit.

The interactions of estrogens and progestins in producing decidualization, deciduosarcoma. and other lesions in the rabbit were explored. Steroids were delivered by silicone elastomer implants placed subdermally except for oral dosing in 1 experiment. Varying doses of levonorgestrel (LNG) were given with and without estradiol (E2) and varying doses of E2 with and without LNG. LNG alone delivered at an estimated mean dose of 233 microg/day did not result in endometrial decidualization or deciduosarcoma. Both conditions occurred when E2 was added to the regimen and increased as the dose of E2 was increased. Sixty microg of E2 per day produced endometrial decidualization in all test animals in a 2-month exposure, but deciduosarcoma occurred only when LNG was also supplied and increased as the LNG dose was increased. Progesterone given with E2 resulted in deciduosarcoma in most rabbits. Ethynylestradiol alone at 30 microg/day delivered by implants produced splenic and ovarian deciduosarcomas in 1 of 5 test animals. Adding LNG resulted in more numerous and widespread deciduosarcomas. These experiments indicate that exogenous estrogen is necessary for decidualization of the endometrium and to production of deciduosarcoma in the nonpregnant rabbit. Exogenous progestin promotes the process. Necrosis of the uterine wall tended to increase with increasing dose of estrogens.

Blockage of one class of potassium channel alters the effectiveness of halothane in a brain circuit of Drosophila.

At concentrations comparable to those used in the clinic, halothane has profound effects on a neuronal pathway devoted to the escape reflex of the fruit fly, Drosophila melanogaster. We studied the influence of the potassium channel that is encoded by the Shaker gene on the halothane sensitivity of this circuit. Shaker channels were specifically inactivated either by genetic means, using strains with two different severe Shaker mutations, or by pharmacologic means, using ingestion of millimolar concentrations of 4-aminopyridine. In all cases, halothane potency decreased substantially. To ensure that the genetic alteration was specific, both mutations were studied as stocks that had been repeatedly backcrossed to a control strain. The specificity of the pharmacologic inhibition was demonstrated by the fact that 4-aminopyridine had no effect on halothane potency in a Shaker mutant. Quantitative differences in the effects of channel inhibition between males and females suggested a sexual dimorphism in the functional brain anatomy of the reflex circuit.

A new assay for the genetic study of general anesthesia in Drosophila melanogaster: use in analysis of mutations in the X-chromosomal 12E region.

We describe a new measure of the influence of general anesthetics on Drosophila that uses the robust tendency of fruit flies to briskly walk upwards after being tapped down. We expose flies to a fixed concentration of anesthetic gas in a 50 ml tube for a period of up to 1 h and then test the distribution of flies in the tube shortly after tapping them to its bottom. By measuring the effect of a series of anesthetic concentrations on the fraction of flies that fail to climb, we derive quantitative descriptors of the potency of the drug. This "distribution test" is superior to previous assays of anesthetic potency in terms of ease and reliability. We have used the assay to further the genetic analysis of several mutations that cluster on the X chromosome and are known to influence both neural function and anesthesia sensitivity. The results establish complementation patterns between the mutations, refine their genetic map positions, and open the way for the molecular identification of the relevant gene(s).

Mushroom body defect, a gene involved in the control of neuroblast proliferation in Drosophila, encodes a coiled-coil protein.

Neurogenesis relies on the establishment of the proper number and precisely controlled proliferation of neuroblasts, the neuronal precursor cells. A role for the mushroom body defect (mud) gene in both of these aspects of neuroblast behavior, as well as possible roles in other aspects of fruit fly biology, is implied by phenotypes associated with mud mutations. We have localized mud by determining the sequence change in one point mutant, identifying a predicted ORF affected by the mutation, and showing that an appropriate segment of the genome rescues mud mutant phenotypes. An analysis of mud cDNAs and a survey of mud transcripts by Northern blotting indicate that the gene is subject to differential splicing and is expressed primarily during embryogenesis but also, at lower levels, during subsequent developmental stages in a sexually dimorphic manner. The gene is predicted to encode a polypeptide without obvious homologs but with two prominent structural features, a long coiled coil that constitutes the central core of the protein and a carboxyl-terminal transmembrane domain.

Vaginal epithelial surface appearances in women using vaginal rings for contraception.

Vaginal inspections using colposcopy before insertion of contraceptive vaginal rings and at 2-month intervals during ring use were conducted on 169 users of four different formulations. The rings studied released Nestorone alone (50, 75, 100 g daily over 6 months); ethinyl estradiol: Nestorone (30:100 and 15:150 g daily over 6 months); ethinylestradiol:norethindrone acetate (20:1000 and 15:1000 g daily over 4 months); and ethinyl estradiol:norethindrone acetate (20:1000 g daily over 12 months). A total of 88 altered or atypical conditions of the vaginal surface appearance were recorded in 507 inspections (17.4% of inspections). Many of these atypical appearances were quite subtle. The incidence was significantly higher (p <0.01) than in the single pretreatment examinations (11 in 158 inspections; 7.0%), but closely matched that of a "control group" of sexually active women who were the subject of an earlier study by the same investigators. In that study, the incidence was 18% (57 atypical conditions in 317 inspections). In all, 83% of atypical conditions identified in the vagina during ring use had disappeared by the next scheduled colposcopy despite continued ring use. Findings of potential significance were conservatively defined as all ulcerations, those abrasions and ecchymoses that were >0.5 cm in any direction, and fields of five or more petechiae. Findings fitting those criteria comprised 30% of atypical conditions in ring users, 33% in the control group, and 27% pretreatment. The corresponding incidence as a percentage of inspections were 5.3%, 6. 0%, and 2.5% in the ring users, control groups, and pretreatment groups, respectively. These differences were not statistically significant. The findings suggest that the vaginal rings included in the studies contributed little, if at all, to clinically significant lesions or to total lesion incidence. Further definition would require a larger and longer-term study with matched controls.

Genetic effects on an anesthetic-sensitive pathway in the brain of Drosophila.

General anesthetics are known to inhibit the electrically induced escape response of the fruitfly through action within the brain. We examined this response and its sensitivity to anesthetics in several mutants that cause significant disruption of the mushroom body and other structures of the central brain in adult flies. Because we show here that anesthesia sensitivity is influenced by genetic background, we have used a set of congenic mutant lines. Sensitivity to halothane is normal in most of these lines, indicating that the anesthetic target is unaffected by the gross status of the central brain. Thus, for the escape response, anesthetic sensitivity is not a global feature but reflects action at a localized target. Only the mushroom body defect (mud) line showed an increased sensitivity of the escape response to halothane. Sensitivity to two other anesthetics is also perturbed in this line, albeit less dramatically so. The behavior of mud/+ heterozygotes and the comparison of brain anatomy among all the mutant lines imply that the effect of the mud mutation on anesthesia is not via gross alteration of central brain structures. The possibility that an adventitious mutation in the mud line is responsible for the effects on anesthesia is disfavored by the behavior of a heterozygote between two mud alleles. Although we do not yet know whether the mud gene encodes an anesthetic target or influences the functioning of an anesthetic-sensitive neuron in this pathway, our work indicates that this gene regulates the effects of halothane on a circumscribed pathway.

Estradiol-delivering vaginal rings for hormone replacement therapy.

OBJECTIVES: This study was undertaken to determine the relief of climacteric symptoms by vaginal rings delivering estradiol and to monitor estrogen levels. STUDY DESIGN: Rings releasing in vitro either 60 or 140 microg/d estradiol were used by 35 women who had undergone hysterectomy for each dose level. Hot flash and night sweat incidences, vaginal conditions, and complaints were recorded at clinic visits pretreatment and at 1 week, 2 weeks, 1 month, and monthly thereafter through 6 months. Serum samples were assayed for estradiol, estrone, and estrone sulfate. RESULTS: Hot flash incidence was reduced by about 80% with either ring. Vaginal conditions and mood were improved. Fourteen of 70 women discontinued ring use during the trial, 5 because of ring expulsions. Mean (+/-SD) estradiol levels were 123 +/- 48 and 307 +/- 93 pmol/L for the low and high dosage levels, respectively. Mean estrone levels exceeded estradiol levels by 1.7-fold for the higher dosage ring and 2.6-fold for the lower dosage ring. Increases in estrone sulfate concentrations were many times greater than those of estradiol or estrone. CONCLUSIONS: Vaginal rings are an acceptable method of delivery for periods of >/=6 months of doses of estradiol that reduce vasomotor symptoms and improve vaginal conditions. There was little difference in these responses between the 2 dosage levels.

Yeast gene for a Tyr-DNA phosphodiesterase that repairs topoisomerase I complexes.

Covalent intermediates between topoisomerase I and DNA can become dead-end complexes that lead to cell death. Here, the isolation of the gene for an enzyme that can hydrolyze the bond between this protein and DNA is described. Enzyme-defective mutants of yeast are hypersensitive to treatments that increase the amount of covalent complexes, indicative of enzyme involvement in repair. The gene is conserved in eukaryotes and identifies a family of enzymes that has not been previously recognized. The presence of this gene in humans may have implications for the effectiveness of topoisomerase I poisons, such as the camptothecins, in chemotherapy.

Efficacy, bleeding patterns, and side effects of a 1-year contraceptive vaginal ring.

A combined contraceptive vaginal ring designed to last 12 months was tested at three clinic sites. This ring released approximately 1 mg of norethindrone acetate (NET-Ac) and 20 micrograms of ethinyl estradiol (EE) daily. A total of 60 women were enrolled to use the ring in a schedule of 3 weeks in/1 week out. Serum norethindrone (NET) and ethinyl estradiol (EE) levels were assayed twice weekly in cycles 6, 9, and 13. Mean NET concentrations between cycles 6 and 9 were relatively stable between 13 and 19 nmol/L but showed a 10%-21% decrease in all centers between cycles 9 and 13. Mean EE concentrations ranged from 75 to 103 pmol/L, but did not have the same decrease as NET between cycles 9 and 13. Cycles with progesterone peaks (> 9.6 nmol/L) compatible with some luteal activity occurred in 4% of cycles sampled in Sydney, 3% in Santo Domingo, and 26% in Los Angeles. Half of these cycles exhibited at least one progesterone value > 32 nmol/L with three of 18 occurring in noncompliant cycles. Heavier body weight was associated with increased probability of luteal activity. Based on serum estradiol peaks > 400 pmol/L, eight of 81 cycles appeared to have marked follicular activity with no luteal activity. No pregnancies occurred. Nausea was reported by about half the subjects in approximately 10% of the visits (mainly in the first 1-2 days in the first cycle immediately after ring insertion). Vomiting was reported by 20% of subjects early in the first cycle only. Headache was reported on occasion by nearly 50% of the women, but the relationship to ring use was uncertain. Vaginal discharge was reported by 17 women (82% of these were from one clinic). Of 60 women, 14 discontinued before completing the study, but only two discontinuations were for medical reasons. Small but statistically significant changes occurred in lipid levels in two of the three centers. All changes remained within normal limits and were similar to those seen with many oral contraceptives. It appears that this ring may perform slightly differently in different populations, but is a highly satisfactory method of contraception for many women. Minor modifications in design could provide higher levels of steroid release and in the later months of the ring life span would assure continuing high levels of contraceptive protection for heavier women.

PIP: This study determines the ovarian effects, contraceptive efficacy, and effects on serum levels of norethindrone acetate (NET-Ac) and ethinyl estradiol (EE) among women using a single contraceptive vaginal ring (CVR) cyclically over a period of 1 year. A total of 60 women were enrolled and used the ring according to a schedule of 3 weeks "in" and 1 week "out." Assays of serum norethindrone acetate (NET-Ac) ethinyl estradiol (EE) levels were taken twice weekly in cycles 6, 9, and 13. Despite luteal activity in some cycles, no pregnancies were noted within the 12-month study period. Heavier body weight was associated with increased probability of luteal activity. Mean serum levels decreased over the last 3 months of CVR use, accounting for the increase in luteal activity and possible ovulations in cycle 13. Among women in Sydney, by contrast with women in the other centers, a difference in the effect on lipids was seen. However, the changes in lipid levels were very small. The side effects were a little different from those experienced by women using a combined pill. Nausea and vomiting were largely confined to early cycles and most common in the first days of the first cycle. Weight gain was also not a problem, although there was a small mean increase in body weight over the 12-month treatment period. This study indicates that use of a single CVR releasing EE and NET-Ac over a period of 12 months constitutes an acceptable, safe and effective contraceptive method.

Variations in vaginal epithelial surface appearance determined by colposcopic inspection in healthy, sexually active women.

A total of 107 sexually active women, aged 18-35 years, was recruited through family planning clinics in four centres in different countries. Each woman underwent two or three gentle but thorough and systematic vaginal inspections using a consistent technique with colposcopic magnification over a 4-6 month period to look for changes in vaginal and cervical appearance which might be related to sexual intercourse, tampon use, contraceptive method used, cigarette smoking or other environmental factors. Obvious changes in appearance were photographed at x10 magnification. These 'appearances' or 'conditions' were classified according to a modification of the recommendations of a workshop sponsored by the World Health Organization, the Population Council and the Conrad Program. Most of these alterations in the appearance of the vaginal epithelium were judged to be of such minor clinical importance that they have been termed 'conditions' or 'changes in appearance' rather than 'lesions'. In all, 56 'conditions' or 'appearances' were noted in 314 inspections, the commonest being petechiae (53.6%). Potentially significant conditions justifying the term 'lesions' (three microulcerations, two ecchymoses, five abrasions and one mucosal tear; 3.5% of inspections) usually healed spontaneously and disappeared rapidly. The incidence of these conditions was highest when the inspections followed intercourse in the previous 24 h (25.2 versus 14.2%; P < 0.0008), or tampon use (32.4 versus 15.9%; P < 0. 0001). These processes may be regarded as a reflection of regular minor trauma to the vaginal epithelium.

Norplant consensus statement and background review.

This review has highlighted the attributes of a very important new method of contraception. The signatories to this document agree that, with the provision of appropriate information and instruction for the user, Norplant is a good contraceptive choice to be made available worldwide in family planning programs that have the resources for appropriate training and counseling. The signatories to this document are acting in their own personal capacity and not as representatives of any particular organization.

PIP: The Norplant contraceptive implant has been registered in 60 countries and used by over 6 million women worldwide. Clinical studies have repeatedly confirmed this method's long-term efficacy and safety when used appropriately. Preliminary findings of the Post-Marketing Surveillance study of Norplant, a 5-year prospective study conducted in 32 family planning clinics in 8 developing countries in 1987-97, indicate an intrauterine pregnancy rate of 0.23/100 woman-years, an ectopic pregnancy rate of 0.03/100 woman-years, and a 67.3% continuation rate at 5 years. No significant excess of malignant neoplastic or cardiovascular disease has been observed. The major side effect is an irregular pattern of uterine bleeding, associated with about 25% of the discontinuations after 5 years of use. The quality of the family planning service is a major determinant of successful Norplant use and the degree of user satisfaction. Informed choice, the quality of follow-up care, easy access to removal services, and provider skills and attitudes are also critical. The signatories to this Consensus Statement (acting as individuals rather than representatives of their organizations) agree that, with the provision of appropriate information to users, Norplant is a good contraceptive choice that should be made available globally in all family planning programs with resources for appropriate training and counseling.

The visually-induced jump response of Drosophila melanogaster is sensitive to volatile anesthetics.

Fruit flies jump when startled by a sudden decrease in light intensity. We show that this visually-induced jump response in Drosophila melanogaster is sensitive to three general anesthetic agents: halothane, enflurane, and methoxyflurane. The concentration of anesthetic required to inhibit this response is similar to that needed for the inhibition of other assays in flies and other organisms. We believe the simplicity of this assay coupled with the insight gained from the more complex electrophysiological monitoring of the same neural pathway will be of value in the genetic identification of the molecular target of volatile anesthetics.

Estradiol delivery by vaginal rings: potential for hormone replacement therapy.

OBJECTIVES: To determine if delivery of estradiol from elastomeric vaginal rings gives estradiol blood levels in the range associated with effective estrogen replacement therapy and to determine the relation between in vitro estradiol release from the rings and blood levels in vivo. Secondary objectives related to changes in lipoprotein cholesterol, changes in climacteric symptoms, and evaluation of acceptability to users. METHODS: Three ring variants releasing approximately 100, 150 and 200 micrograms/day of estradiol in vitro were used through 22 days in 21 postmenopausal women, 7 on each dose levels. Blood samples for measurement of estradiol were taken at 3-4 day intervals. Lipoprotein cholesterol was measured before and at the end of treatment. Women were questioned about climacteric symptoms and about their satisfaction with the ring. RESULTS: Mean serum estradiol levels for the three groups of rings were 63 +/- 6, 94 +/- 5 and 136 +/- 13 pg/ml for the 100, 150 and 200 micrograms/day rings, respectively. FSH levels declined during ring use and the maturation values of cells collected on vaginal swabs markedly increased. Total and LDL cholesterol were significantly reduced and HDL cholesterol was not significantly changed. All women reported relief of postmenopausal symptoms. Vaginal discomfort during the first 3 days of use was reported by 12 women but overall satisfaction with the method was high. CONCLUSIONS: Women using the vaginal rings attained estradiol blood levels compatible with control of climacteric symptoms and bone loss. The relation between in vitro estradiol release and blood levels in vivo was essentially identical for all 3 doses. The use of vaginal rings to deliver estradiol for hormone replacement therapy is judged to merit further evaluation.