Altered activity, social behavior, and spatial memory in mice lacking the NTAN1p amidase and the asparagine branch of the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. N-terminal asparagine and glutamine are tertiary destabilizing residues, in that they are enzymatically deamidated to yield secondary destabilizing residues aspartate and glutamate, which are conjugated to arginine, a primary destabilizing residue. N-terminal arginine of a substrate protein is bound by the Ubr1-encoded E3alpha, the E3 component of the ubiquitin-proteasome-dependent N-end rule pathway. We describe the construction and analysis of mouse strains lacking the asparagine-specific N-terminal amidase (Nt(N)-amidase), encoded by the Ntan1 gene. In wild-type embryos, Ntan1 was strongly expressed in the branchial arches and in the tail and limb buds. The Ntan1(-/-) mouse strains lacked the Nt(N)-amidase activity but retained glutamine-specific Nt(Q)-amidase, indicating that the two enzymes are encoded by different genes. Among the normally short-lived N-end rule substrates, only those bearing N-terminal asparagine became long-lived in Ntan1(-/-) fibroblasts. The Ntan1(-/-) mice were fertile and outwardly normal but differed from their congenic wild-type counterparts in spontaneous activity, spatial memory, and a socially conditioned exploratory phenotype that has not been previously described with other mouse strains.

Behavioral characterization of mice lacking the ubiquitin ligase UBR1 of the N-end rule pathway.

The N-end rule pathway, a subset of the ubiquitin pathway, relates the in vivo half-life of a protein to the identity of its N-terminal residue. Mice lacking NTAN1, a component of the N-end rule pathway, showed altered learning and memory, and socially conditioned behavioral alteration (Balogh, Kwon, & Denenberg 1999, 2000; Kwon, Balogh et al. 2000; Balogh et al. 2001). Mice lacking UBR1 (E3alpha), one of at least three recognition components of the N-end rule pathway, are viable and outwardly normal. Here we describe behavioral characterizations of UBR1 knockout (UBR1-/-) mice. Compared to congenic littermates, UBR1-/- mice exhibited less spontaneous activity in an open field and took longer to locate the hidden platform during eight-week Morris water maze retention. In contrast, they performed better in horizontal-vertical discrimination and Lashley III maze testing. No statistically significant differences in inhibitory learning were observed. With the exception of enhanced Lashley III maze performance, these data parallel findings with NTAN1-/- mice lacking the upstream component of UBR1. These results suggest that, like NTAN1, UBR1 is involved in learning and memory.

Learning deficits in forebrain-restricted brain-derived neurotrophic factor mutant mice.

Brain-derived neurotrophic factor (BDNF) participates in synaptic plasticity and the adaptive changes in the strength of communication between neurons thought to underlie aspects of behavioral adaptation. By selectively deleting BDNF from the forebrain of mice using the Cre site-specific DNA recombinase, we were able to study the requirements for BDNF in behaviors such as learning and anxiety. Early-onset forebrain-restricted BDNF mutant mice (Emx-BDNF(KO)) that develop in the absence of BDNF in the dorsal cortex, hippocampus, and parts of the ventral cortex and amygdala failed to learn the Morris Water Maze task, a hippocampal-dependent visuo-spatial learning task. Freezing during all phases of cued-contextual fear conditioning, a behavioral task designed to study hippocampal-dependent associative learning, was enhanced. These mice learned a brightness discrimination task well but were impaired in a more difficult pattern discrimination task. Emx-BDNF(KO) mice did not exhibit altered sensory processing and gating, as measured by the acoustic startle response or prepulse inhibition of the startle response. Although they were less active in an open-field arena, they did not show alterations in anxiety, as measured in the elevated-plus maze, black-white chamber or mirrored chamber tasks. Combined, these data indicate that although an absence of forebrain BDNF does not disrupt acoustic sensory processing or alter baseline anxiety, specific forms of learning are severely impaired.

Role of neuronal nicotinic receptors in the effects of nicotine and ethanol on contextual fear conditioning.

Nicotine can enhance contextual learning while ethanol impairs some forms of learning. Nicotine can overcome some of the impairing effects of ethanol when the two drugs are co-administered. The specific brain nicotinic acetylcholine receptors (nAChRs) that mediate nicotine's effects on contextual learning and whether any of ethanol's actions are mediated by nAChRs are unknown. The potential roles of nAChRs in contextual and cued fear conditioning as well as the effects of nicotine, ethanol, or co-administration of nicotine and ethanol were examined in wild type and homozygous null mutant mice from alpha7, beta2, beta3, and beta4 mouse lines at 24 h after training. Nicotine was given prior to training and testing, whereas ethanol was given only before training. Nicotine enhanced contextual learning in both alpha7 wild types and mutants when mice were trained at 0.17 mA, but not 0.35 mA. Mutants lacking the alpha7 subunit were less sensitive to the memory impairing effects of ethanol trained at 0.35 mA. beta2 Null mutants receiving saline showed a small, but significant, impairment in contextual learning compared with wild type littermates when the shock stimulus was 0.35 mA. Beta2 Null mutant mice also did not respond to the cognitive enhancing effects of nicotine alone, or after ethanol administration. beta3 and beta4 null mutants did not differ from wild types either after saline or any of drug treatments. These results show that beta2-containing nAChRs, but not beta3- or beta4-containing receptors, mediate the enhancing effects of nicotine on contextual learning and confirm previous studies implicating beta2 in other forms of learning. A new role for alpha7 nAChRs in regulating sensitivity to the cognitive disrupting effects of ethanol is proposed.

Spatial ability of XY sex-reversed female mice.

Perinatal gonadal hormones significantly affect subsequent sex differences in reproductive and non-reproductive behaviors in rodents. However, the influence of the sex chromosomes on these behaviors has been largely ignored. To assess the influence of the non-pseudoautosomal region of the Y chromosome, C57BL/JEi male and female mice and mice from the C57BL/6JEi-Y(POS) consomic strain were given behavioral tests known to distinguish males from females. The C57BL/6JEi-Y(POS) strain contains sex-reversed XY-females which, when compared to their XX-female siblings, allow assessment of the influence of the Y chromosome in a female phenotype. XX-females and XY-females did not differ on open-field activity, the Lashley maze, or active avoidance learning, but XY-females were significantly better than XX-females on the Morris hidden platform spatial maze. These findings suggest that males may have both a genetic and a hormonal mechanism to ensure visuospatial superiority.

Facilitated stimulus-response associative learning and long-term memory in mice lacking the NTAN1 amidase of the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Inactivation of the NTAN1 gene encoding the asparagine-specific N-terminal amidase in mice results in impaired spatial memory [26]. The studies described here were designed to further characterize the effects upon learning and memory of inactivating the NTAN1 gene. NTAN1-deficient mice were found to be better than wild-type mice on black-white and horizontal-vertical discrimination learning. They were also better at 8-week Morris maze retention testing when a reversal trial was not included in the testing procedures. In all three tasks NTAN1-deficient mice appeared to use a strong win-stay strategy. It is concluded that inactivating the asparagine-specific branch of the N-end rule pathway in mice results in impaired spatial learning with concomitant compensatory restructuring of the nervous system in favor of non-spatial (stimulus-response) learning.

A behavioral and neuroanatomical assessment of an inbred substrain of 129 mice with behavioral comparisons to C57BL/6J mice.

The inbred 129 substrains have been characterized as poor learners that display hypoplasia of the corpus callosum. However, they are used extensively as a source of embryonic stem (ES) cells for creating mice carrying altered copies of a targeted gene ('knockout mice'). The present research investigated callosal agenesis and behavior in the 129/SvEvTac substrain and compared their behavior to that of C57BL/6J mice. In addition, the degree to which callosal agenesis affected behavior was assessed. Nearly 80% of 129/SvEvTac mice in the current sample exhibited callosal hypoplasia, although this was not subsequently found to be associated with any measure of cognition. They learned the Morris maze and a non-spatial pattern discrimination task, though at a level inferior to C57BL/6J mice. They were unable to learn shuttlebox avoidance or the Lashley III maze. The only measure on which they performed better than C57BL/6J mice was a simple water escape task. Thus, 129/SvEvTac mice, in addition to displaying aberrant neuroanatomy, perform poorly on many behavioral tasks, resulting in potential interpretational difficulties.

Avoidance learning in autoimmune mice.

Previous studies have shown that autoimmune mice perform very poorly on active avoidance learning tasks. In the current studies, mice with lupus-like systemic autoimmunity were able to learn active, as well as passive, avoidance protocols with shock as reinforcement. Therefore, the behavioral deficits seen in active avoidance tasks are not a consequence of the use of electric shock. Rather, the current findings suggest that the inability of autoimmune mice to learn shock motivated responding is due to multiple performance factors, including shock level and properties of the testing apparatus.

Effects of neocortical ectopias upon the acquisition and retention of a non-spatial reference memory task in BXSB mice.

BXSB mice have a 40-60% incidence of small, focal, neocortical dysplasias called ectopias. Ectopics and non-ectopics were compared on horizontal-vertical discrimination learning, a non-spatial cognitive task. No differences were found during acquisition. Ectopic mice, however, were superior when retested 8 weeks later. The results support prior findings that ectopic mice have better long-term spatial memory and extend this conclusion to the non-spatial domain.

Ketamine blocks a conditioned taste aversion (CTA) in neonatal rats.

These experiments explored the effects of glutamate, N-methyl-D-aspartate (NMDA) receptor blockade on the formation, retention, and expression of conditioned taste aversion (CTA) in young rats. Previous data from our laboratory suggested that ketamine administration potentiates a CTA in E18 rat fetuses. The current studies investigated this phenomenon in neonates. High-pressure liquid chromatography (HPLC) methods were used to determine the amount of ketamine that must be injected intraperitoneally (i.p.) to achieve brain ketamine levels in neonates comparable to those found in the fetuses from our previous experiments. Then, on their day of birth, Sprague-Dawley rat pups received injections of either 0.1, 10, or 70 mg/kg of ketamine HCI, i.p. or a Sal control injection. One-half hour later, pups were injected orally with either Saccharin (Sac; 10 microL of 0.3%) or water followed by an injection of either lithium chloride (LiCl; 81 mg/kg) or Sal (i.p.). The CTA was evaluated in two different tests. Two weeks after conditioning, the dam was anesthetized and the frequency with which pups attached to Sac-painted nipples versus nipples painted with water was measured (i.e., the nipple taste test, NTT). Controls for state-dependent learning were run in which 10 mg/kg of ketamine or saline (Sal) was administered before both taste aversion conditioning and the NTT. After weaning, the CTA was also evaluated by measuring the amount of Sac (0.3%) or water consumed during a two-bottle test. Neonates that received Sal control injections before the Sac + LiCl pairing acquired CTAs and avoided Sac-painted nipples. However, the pups injected with ketamine on the conditioning day only (P0) did not avoid Sac-painted nipples (as compared to controls). Pups that had ketamine both at the time of CTA training and testing, or just before the NTT, also failed to avoid Sac-painted nipples. Ketamine's acute effects apparently influenced the outcome of the NTT of state-dependent control subjects. Rat pups that received the highest doses of ketamine (10 or 70 mg/kg) and tasted Sac on P0 later failed to show a neophobia for Sac-painted nipples. Whereas, rat pups that received the high dose of ketamine and water on P0, later exhibited a neophobic response. These data suggest that ketamine did not impair the animal's ability to taste Sac. These data reflecting a ketamine-induced blockade of neonatal CTAs may be contrasted with our previous findings in which ketamine potentiated fetal CTAs. However, they are in consonance with data from adult rats suggesting that ketamine can cause an amnesia for CTAs. NMDA receptor blockade may shape memory formation in a manner that is dependent on the stage of brain development.

Vacuum aspiration with the IPAS Modified Gynecologic Syringe.

The IPAS Modified Gynecologic Syringe was compared with the electric pump and standard hand pump in a clinical trial of 1227 women undergoing vacuum aspiration. This modified double-valve syringe was designed to fit larger 8 mm, 10 mm and 12 mm cannulae, which are more suitable for late first trimester procedures. The study was conducted at four centers in which the double-valve syringe was used alternately with the vacuum source normally used at each clinic. Although there were notable inter-center differences with respect to certain outcome variables, results were similar for procedures performed at the same center. The only significant finding was the lower proportion of women requiring D & C to complete uterine evacuation among double-valve syringe patients than among hand pump cases at one center. The data indicate that the double-valve syringe is safe and effective for uterine aspiration in women up to 14 weeks' gestation and may be preferable for use in clinics with limited facilities.

Bleeding patterns and acceptability among Norplant users in two Asian countries.

Changes in menstrual bleeding patterns are the most frequent reason for discontinuing NORPLANT during the first year of use. Although NORPLANT is used by more women in Asia than in any other region, the changes that occur among these women are not well documented. In pre-introductory clinical trials of NORPLANT in the Philippines and Sri Lanka, 175 women were enrolled with greater than 95% continuing the method after one year. Daily menstrual bleeding calendars were kept by 94% of subjects for nine months and by 66% for one year. In contrast to findings in previous studies which showed that approximately 60% of NORPLANT users had bleeding disruptions in the first year of use, almost all of these women experienced bleeding disruptions. In a one-year study of menstrual pattern changes, increased bleeding was found to occur with the same frequency as reduced bleeding. However, the changes experienced by the Asian women tended to be decreases, rather than increases, in menstrual bleeding.

The effect of hydrogel on the reduction of bleeding associated with IUD use: a comparative study of the plain and Hydron-coated Spring Coil.

PIP: Alterations in the size, shape, and material composition or surface texture of IUDs, as well as the addition of medicaments, have been made to reduce bleeding, the most common side effect of intrauterine contraception. To determine if coating with a biocompatible hydrogel could diminish bleeding associated with the use of Spring Coils, these devices were randomly assigned to 464 women following an induced abortion; 241 women received the plain Spring Coil, 223 the Hydron-coated Spring Coil. The age/parity distribution and previous abortion experience of the 2 groups of women were almost identical. 1- and 2-year rates of pregnancy, expulsion, and removal for bleeding/pain were compared using life table analysis; differences between the 2 user groups were not significant. Menstrual pattern changes were also examined in terms of the women's perceptions of changes in their cycle length, amount of flow and duration, as well as episodes of intermenstrual bleeding. Only the increase in menstrual flow duration at 1 year follow-up was significantly lower among the Hydron-coated Spring Coil users than among women using the plain device. From the results of this study, it is unlikely that a modification to the surface material such as coating with hydrogel will significantly reduce bleeding associated with the use of the Spring Coil IUD.^ieng

Evaluation of safety, efficacy, and acceptability of NORPLANT implants in Sri Lanka.

This paper presents findings based on a one-year pre-introductory clinical investigation concerning safety, efficacy, and acceptability of the new contraceptive NORPLANT implants in Sri Lanka. The study is based on 400 acceptors of the implants at two clinics in Sri Lanka. Acceptors' mean age was 27 years, mean number of children was about two, and average educational attainment was eight years. Nearly half of the acceptors did not want to have another child. Follow-up was conducted at one, three, six, and twelve months. The one-year termination rate due to medical problems was very low, and the one-year life-table pregnancy rate was 0.5. Menstrual pattern disruption was the most frequently reported complaint with the implants. Amenorrhea increased in the first three months of use, and intermenstrual bleeding was common among those menstruating. In spite of these changes and some significant variations between the acceptors in the two study center, the overall degree of satisfaction with the implants was very high. The features most important to the acceptability of the implants were low risk of pregnancy, the long duration of action, and the convenience of use. The results of the study suggest that NORPLANT implants are safe, effective, and highly acceptable to Sri Lankan women.

PIP: This paper presents findings based on a 1 year pre-introductory clinical investigation concerning safety, efficacy, and acceptability of the new contraceptive Norplant implants in Sri Lanka. The study is based on 400 acceptors of the implants at 2 clinics in Sri Lanka. Acceptors' mean age was 27 years, mean number of children was about 2, and average educational attainment was 8 years. Nearly 1/2 of the acceptors did not want to have another child. Follow-up was conducted at 1,3,6, and 12 months. The 1 year termination rate due to medical problems was very low, and the 1 year life-table pregnancy rate was 0.5. Menstrual pattern disruption was the most frequently reported complaint with the implants. Amenorrhea increased in the 1st 3 months of use, and intermenstrual bleeding was common among those menstruating. In spite of these changes and some significant variations between the acceptors in the 2 study centers, the overall degree of satisfaction with the implants was very high. The features most important to the acceptability of the implants were low risk of pregnancy, the long duration of action, and the convenience of use. The results of the study suggest that Norplant implants are safe, effective, and highly acceptable to Sri Lankan women when administered by health personnel who are well trained in counseling women about the features of the method and in the proper insertion and removal techniques. Despite the overall high level of acceptability, some important variations were found with respect to the sociodemographic characteristics, effectiveness, satisafction, and adverse experiences between the 2 groups of acceptors in the study centers. This variation underscores the importance of conducting the study in more than 1 clinic within a country.

Previous abdominal surgery as a risk factor in interval laparoscopic sterilization.

Previous abdominal surgery has been termed a relative contraindication for laparoscopic sterilization. This study quantifies the relative risks of surgical difficulties, immediate complications, and early follow-up complications/complaints of interval sterilization in women with versus women without previous abdominal surgery (PAS and NPAS groups, respectively). This multicenter study included women sterilized by electrocoagulation, the tubal ring, and the spring-loaded clip. Within each center the PAS and NPAS women were generally similar with respect to sociodemographic variables and preexisting medical conditions. Stratified analysis determined that the rate of surgical difficulties was significantly higher in PAS women (relative risk = 2.3, P less than 0.05), but most of these difficulties were overcome without resorting to another technique or approach. The rates of complications as well as the mean operation times and mean durations of hospitalization (indices of medical costs) did not differ significantly between the PAS and NPAS groups.

Varying intertrial interval reveals temporally defined memory deficits and enhancements in NTAN1-deficient mice.

The N-end rule is one ubiquitin-proteolytic pathway that relates the in vivo half-life of a protein to the identity of its N-terminal residue. NTAN1 deamidates N-terminal asparagine to aspartate, which is conjugated to arginine by ATE1. An N-terminal arginine-bearing substrate protein is recognized, ubiquitylated by UBR1/E3alpha, and subsequently degraded by 26S proteasomes. Previous research showed that NTAN1-deficient mice exhibited impaired long-term memory in the Lashley III maze. Therefore, a series of studies, designed to assess the role of NTAN1 in short- and intermediate-term memory processes, was undertaken. Two hundred sixty mice (126 -/-; 134 +/ +) received Lashley III maze training with intertrial intervals ranging from 2-180 min. Results indicated that inactivation of NTAN1 amidase differentially affects short-, intermediate-, and long-term memory.

Long-term use of intrauterine devices.

PIP: Analysis was performed of data relative to 10,001 IUD insertions (mostly Lippes Loop D and C) in 8091 women in Slovenia between 1965 and 1972. Data were gathered on sociodemographic characteristics of the users; cumulative life-table rates per 100 women for removals due to pregnancy, expulsion, bleeding/pain, other medical reasons, and personal reasons as well as continuation rates and the rate of follow-up; and the proportion of women with bleeding, spotting, vaginitis, and pelvic inflammatory disease (PID) at 1-, 5-, and 10-year follow-up. The most frequent reason for removal was bleeding. Increased duration of IUD use was associated with a smaller number of women with bleeding and PID episodes. Of those with first insertions, 608 were hospitalized for IUD-related complications (49% for bleeding requiring curettage and 20% with PID). IUD use for more than five years carried a relative risk of 3.9 for a severe episode when PID developed. Of the women who terminated use of their first IUD because of expulsions, pregnancy, or bleeding, 28% asked for reinsertion. Over the 15 years of IUD use, 853 pregnancies occurred with an IUD in situ. If the IUD was removed after conception, the pregnancy outcome did not differ from women without IUDs. If the IUD was not removed, there was an increased risk of spontaneous abortion, preterm labor, and intrauterine fetal infection. While the IUD users experienced the same risk of ectopic pregnancy as noncontraceptors, if the IUD user did become pregnant, their risk of ectopic pregnancy was twice as high as that faced by those who did not use IUDs. Duration of IUD use and type of IUD had no effect on time from removal to desired conception. This duration was increased, however, in women with a history of PID and in older women. Women over 40 tolerated the IUD better than younger women but as women approached 50, removals for bleeding became more frequent. In conclusion, the IUD is a very safe contraceptive agent and is well tolerated if users are selected carefully, are motivated, and receive help with side effects.^ieng

Sustained intrauterine release of AMCA to reduce menstrual blood loss.

PIP: Increased menstrual blood loss (MBL) is 1 of the side effects associated with IUDs. Attempts at reducing the increase in MBL have included the use of horomones, such as progesterone and levonorgestrel, and antifibrinolytic agents, such as tranexamic acid (AMCA) and aminiocaproic acid (EACA). This paper presents the results of a randomly allocated comparative study of menstrual pattern changes with a Lippes loop D (LLD) modfied to slowly release AMCA and a control LLD that released no AMCA. Quantitative MBL data were collected at a preinsertion cycle and at follow-up vistits scheduled 1, 2, 3, and 6months postinsertion for 11 control LLD cases and 10 cases assigned an AMCA-loaded LLD. The increase in MBL over preinsertion levels was significantly less with the AMCA device. The mean increase in MBL after the 1st cycle was 45.3 ml among control LLD acceptors compared to 16.0 ml among AMCA LLD users. These differences persisted through the 1st 3 postinsertion cycles, but disappeared by the 6th month. AMCA was effective in reducing the initial increase in MBL and other menstrual aside effects that occur with IUD insertion, but its effectiveness is limited by the amount of AMCA which can be loaded into the device.^ieng

A method for monitoring the fertility of workers. 1. Method and pilot studies.

A method has been developed for monitoring male and female industrial workers and other persons exposed to environmental agents which may impair fertility. The essential feature of the method is its ability to distinguish the potential effects of occupational exposure from a multitude of other factors known to affect fertility. Information is collected primarily through use of a questionnaire which take less than three minutes to complete and can be administered to persons of either sex. The advantages and limitations of the method are considered in the context of pilot studies at two small chemical plants and of a review of attempts to assess occupational effects of fertility from interviews of health records.

A method for monitoring the fertility of workers. 2. Validation of the method among workers exposed to dibromochloropropane.

A method has been developed for monitoring industrial workers and others exposed to environmental agents which may impair fertility. National birth probabilities specific for maternal birth cohort, age, parity, and race are used to derive expected fertility. Observed fertility is obtained by questionnaire. Standardized fertility ratios are computed for exposure and non-exposure periods and compared. The analytic techniques have been validated by applying the method to a group of 36 male factory employees working in an agricultural chemical division (ACD) where pesticides including the nematocide dibromochloropropane were formulated. Twelve of these employees in mid-1977 had been discovered to have severely depressed sperm counts related to occupational exposure. The standardized fertility ratio (SFR) computed from data available in mid-1977 for the period at risk from employment in the ACD (SFR = 0.75) was significantly lower than those derived for the entire not-at-risk period (SFR = 1.88) and the portion related to employment in other areas of the factory (SFR = 2.16). Similar differences also were evident from data available several years earlier, demonstrating that the surveillance technique would have been capable of detecting occupationally induced infertility among these workers in advance of the actual discovery date.