Robert Brenner - US grants

The goal of this study is to develop an antiprogestin-releasing intrauterine device (IUD), which might be applied as a contraceptive, or to inhibit endometrial bleeding. In collaboration with Leiras OY, Finland, a subsidiary of Schering AG, we tested two types of IUDs that release the new antiprogestin ZK 230 211 (ZK211). One released a high dose (26-30.2 ?g ZK 211/day) and the other a low dose (3.3-4.5 ?g ZK 211/day). Stumptail macaques were originally considered as the most suitable species for this project as they have a cervix that is straight compared to the S-shaped cervix of other common laboratory macaques. However, we had great difficulty in cannulating the cervix of the stumptails and discovered that while the external os and the cervical canal are straight and easy to cannulate, the internal os is extremely small and is itself S-shaped. To provide information relevant to the project goals, high and low dose IUDs were inserted in stumptailed and pig tail ed macaques by hysterectomy. Our first study was designed to test whether antiprogestin IUDs could inhibit the endometrial effects of progesterone. Both the low and high dose IUDs induced menstruation within three days of inserting the IUDs. This indicated that the amount of antiprogestin produced locally by both the high and low dose IUDs was sufficient to prevent systemic progesterone from maintaining the endometrium in a progestational state. The monkeys were treated to induce artificial menstrual cycles and tissues were collected at the end of one cycle. The endometrium exposed to the blank IUD showed no major differences from a typical progestational endometrium, except that the amount of endometrial tissue was somewhat less than would be expected at the end of a normal cycle. The antiprogestin IUDs caused a severe compaction of the stroma and an inhibition of the effects of P on both glandular sacculation and spiral artery development. There was evidence of perivascular hy aline degeneration similar to that observed after long term systemic antiprogestin. These results suggest that acute administration of local antiprogestin by IUD can act to inhibit endometrial development and may represent an alternative to systemic treatment to control endometrial bleeding or as a uterine based contraceptive. FUNDING Lalor Foundation PUBLICATIONS None

In primates, treatment with antiprogestins including RU 486 and ZK 137 316 will both block progesterone (P) action, and also inhibit estrogen stimulated proliferation in the endometrium. This action of antiprogestins would be of benefit to women treated with estrogen replacement therapy as a method of blocking untoward effects of estrogen on the endometrium. ZK 230 211 (ZK211) is a potent new generation antiprogestin produced by Schering AG, that can be administered both orally and systemically. The goal of this research was to determine if ZK211 will inhibit the proliferative actions of estrogen delivered continuously for 5 months. Six groups of rhesus macaques (n = 5 each) were treated with estradiol (E2)-filled implants for 5 months. Three of the groups were also injected daily with 3 different doses of ZK211 (0. 01, 0.05 and 0.25 mg/kg). P is known to oppose the proliferative action of E2 in the endometrium. Therefore, for comparison, 1 group received E2 plus a low dose of P (2 cm P implants) and one group received E2 plus a high dose of P (6 cm P implants). Control animals (group 6) received estrogen alone. Treatment with ZK211 resulted in a severe reduction of endometrial mass, which was associated with a dose dependent inhibition of epithelial cell proliferation. At higher doses of ZK211, this effect resulted in the upper portions of the glands degenerating and trapping secretory material. These higher doses led to formation of moderate size endometrial cysts, which appeared to contain trapped secretory material. There was no evidence of endometrial hyperplasia or metaplasia. As anticipated, long-term E2 + P treatment resulted in a dose-dependent decidualization response that was marked by extensive spiral artery hypertrophy. This effect on the spiral arteries was opposite to the reaction to that seen with ZK 211 which inhibited vascular development. We conclude that inhibition of vascular development by ZK 211 may be the key factor underlying the degenerative inhibition and blockade of estrogen-dependent endometrial proliferation induced by this antiprogestin. FUNDING Contract with Schering AG, Berlin, Germany PUBLICATIONS None

The activation of the hypothalamic-pituitary-adrenal axis in fetal sheep is the primary initiator of parturition; however, this same mechanism has not been proven to be important in primate parturition. To determine if adrenocorticotropic hormone (ACTH) can induce premature parturition in primates, we infused ACTH (0.1 U in 30 min, every 2 hrs; n=7) beginning on day 135 of pregnancy (term=167 days) until delivery occurred. A control group of animals (n=3) received saline vehicle infusions. All animals were surgically instrumented with fetal and maternal vascular and amniotic fluid catheters, fetal ECG and myometrial EMG electrodes. Uterine activity (UA) and fetal ECG were recorded continuously. The results of ACTH and vehicle infusion are listed in the following table (values are mean + SEM) * = P<0.05 (t-test) ACTH vs Vehicle Begin Infusion (day of preg) Onset Nocturnal UA (days) Days to Delivery Delivery (Day of Pregnancy) ACTH (n=6) 135.3 + 0.6 6.3 + 0.8 15.2 + 1.3 149.7 + 1.3 Vehicle (n=3) 135.7 + 1.8 24.7 + 2.7* 27.6 + 3.2* 163.3 + 1.5* One ACTH-treated animal was delivered by cesarean section at 165 days of pregnancy after prolonged labor and dystocia and was excluded from the data set. Mean (+ SEM) maternal and fetal steroid changes in ACTH-treated animals *=P<0.005 (Rank Sum Test) (n=6) Fetal Cortisol (ng/ml) Fetal DHEA-Sulfate (?g/ml) Fetal Androstene-dione (ng/ml) Fetal Estrone (pg/ml) Maternal Estradiol (pg/ml) Baseline 79 + 11 0.4 + .08 0.8 + 0.2 97 + 27 360 + 45 Labor 276 + 48* 6.6 + 3.1* 7.4 + 1.7* 519 + 170* 877 + 187* We conclude that infusion of ACTH into the fetal circulation activates fetal adrenal steroid biosynthesis causing increased secretion of glucocorticoids and androgens leading to increased estrogen and cortisol production and pre-term delivery in rhesus monkeys. FUNDING NIH HD06159, HD18185 PUBLICATIONS None

The overall goal of this project is to define the factors underlying the blunted growth hormone (GH) response to pharmacological stimulation that is characteristic of children and adults with depression and to utilize this information to identify neural systems which may underlie the development of affective disorders. Blunted stimulation of the anterior pituitary hormone, GH, after a number of pharmacological stimuli, occurs in patients with depression throughout the lifespan, and the blunted GH response is one of the most consistent physiological measures documented in children and adults with depression. Children with depression tend to exhibit less exploratory behavior and more inhibited behavior in a variety of psychological tests. We are performing the current study in the monkey to determine (1) if inhibited affect and blunted GH responsiveness are linked traits, and (2) if inhibited affect and blunted GH responsiveness predispose an individual to the development of anxious and /or depressive behaviors. Therefore, this project is quantifying the behavioral characteristics of young monkeys with regard to exploratory versus inhibitory behavior, and quantifying GH responsiveness to stimulation, and then assessing the degree of correlation between these parameters. Over the past 22 years, we developed 4 behavioral testing paradigms, based directly on paradigms used in children to assess these characteristics. In 1999, we tested 110 monkeys and we found a strong correlation between blunted GH responsiveness and Alow reactivity@ in the playroom and stranger approach test s. Thus, we have evidence that at least in some individuals, these traits are linked. FUNDING NIMH MH41712 PUBLICATIONS Coleman K, Dahl RE, Ryan N, Cameron JL. Behavioral inhibition A new look at a familiar trait. Soc Neurosci Abstr 24:525, 1998 (abstract #211.1). Cameron JL, Coleman K, Bench LM, Sabatini M, Owenby T, Kupfer DJ. Differential development of anxious and depressive behaviors in rhesus monkeys dependent on the timing of maternal separation. Soc Neurosci Abstr 24:526, 1998 (abstract #211.7).

The overall goal of this grant is to experimentally test the hypothesis that the pubertal period represents a Awindow of heightened sensitivity@ to external perturbations that may have long-term health consequences. Specifically, this project is determining whether monkeys going through the pubertal transition are more sensitive to the various health consequences of social stress and moderately vigorous exercise training than monkeys in the early post-pubertal period. The health consequences that are being monitored are (1) reproductive function, including menstrual cyclicity and reproductive hormone secretion, (2) bone structure and growth, (3) body fat distribution, (4) serum lipid content, (5) glucose tolerance, (6) immune function, (7) susceptibility to viral infection, and (8) cardiovascular function, including resting heart rate, heart rate response to stress, and echocardiographic measurements. These parameters are being studied because they are known to be i nfluenced by social stress and exercise and they are all thought to be affected by reproductive steroid hormone levels. Thus, they are likely to be affected by the pubertal transition as well as by the external perturbations that are being studied. FUNDING The John D. and Catherine T. MacArthur Foundation PUBLICATIONS Rogers CJ, Brissette-Storkus CS, Hayes LA, Cameron JL, Chambers WH. Selective reduction in CD2 expression on CD2bright/CD8+ lymphocytes from cynomolgus monkeys (Macaca fascicularis) in response to acute stress. J Neuroimmunol 86:61-72, 1998. Rogers CJ., Brissette-Storkus CS, Chambers WH, Cameron JL. Acute stress-induced increase in NK cell granule content in monkeys. Soc Neurosci Abstr 24:1862,1998 (abstract 739.2). Rogers CJ, Brissette-Storkus CS, Cameron JL, Chambers WH. Acute stress alters both NK cell conjugation and lytic granule content in monkeys. Society for Natural Immunity Abstract, 1998.

The mid Cretaceous (Albian) is recognized as a greenhouse world under which atmospheric carbon dioxide concentrations, mean annual global temperature, and global precipitation rates were all much higher than present day. Geologic proxy data as well as general circulation models (GCM's) have been utilized in attempts to quantify climate variables (e.g. temperature and precipitation rates). We have utilized the oxygen isotopic composition (18-O) of siderite in spherical nodules (sphaerosiderites found in wetland paleosols) and an isotope mass balance model to quantify changes in precipitation rates along the coastal plains of the Cretaceous Western Interior Basin (KWIB) of North America (Kansas to Alaska). Our calculations suggest that at mid-high latitude precipitation rates greatly exceeded modern rates (290-390% greater in mid latitudes and 450% greater at high latitudes), that the subtropical dry-belt region had a greater precipitation deficit (i.e. was drier), and that the precipitation rates in the tropical-equatorial regions did not differ significantly from the modern. While our precipitation rates at mid latitudes and the sub-tropical dry belts precipitation deficit estimates are much greater than those estimated by GCM's, our tropical-equatorial region precipitation rates are much lower than GCM's estimates. However, empirical data from the tropics and sub-tropics that are needed to further constrain our model are lacking. This project will expand the northern hemisphere paleolatitudinal 18-O proxy coverage for the mid-Cretaceous (Albian-Cenomanian) to cover the mid-Cretaceous Americas of the northern hemisphere. The expanded latitudinal coverage will allow 1) refinements of isotope mass-balance models and constrain precipitation-evaporation fluxes for the sub-tropical to equatorial regions; 2) minimize uncertainties on modeling the hydrologic cycle in the northern hemisphere; and 3) better quantification of vapor sources and moisture recycling for the KWIB during the Albian. Paleoprecipitation 18-O compositions in the dry-belt region (15 to 30 N paleolatitude) will be determined by sampling and analyzing calcretes in siliciclastic and carbonate coastal plain deposits, and limestones from isolated carbonate platforms in Texas and Mexico, and freshwater lacustrine carbonates in central and southern Mexico. Equatorial (~10 S to 10 N paleolatitude) paleoprecipitation 18-O values will be determined by sampling and analyzing sphaerosiderites from nonmarine and coastal plain deposits in Colombia.

DESCRIPTION (provided by applicant): The Mirena(c) (Leiras, OY, Finland) is an intrauterine system (IUS) that releases levonorgestrel (LNG) and provides women with highly effective contraception for 5 years. However, serious breakthrough bleeding (BTB) can occur that leads many patients to discontinue treatment. In women using subcutaneous Norplant contraception, BTB can be suppressed by intermittent antiprogestin therapy with mifepristone. NICHD has a new antiprogestin, CDB-2914, that is more potent than mifepristone. The goal of this proposal is to determine whether CDB-2914 can suppress the BTB associated with the LNG-IUS. Preclinical studies will be done first in rhesus macaques fitted with an LNG-IUS and the information gained will be transmitted to Professor Hilary Critchley, University of Edinburgh, who will select a test population of 150 women from a pool of over 400 who are being fitted annually with an LNG-IUS for contraception. The bleeding-suppressive dose and schedule of CDB-2914 that works well in macaques will be adapted for use in women. The work consists of two major aims: Aim 1: to establish an effective, bleeding-suppressive dose schedule of CDB 2914 in macaques, and to assess spatio-temporal expression of bleeding-associated factors in the endometrium. Aim 2: to conduct a randomized, placebo controlled trial to evaluate CDB-2914 suppression of BTB in women being fitted with the LNG-IUS; and for added value, to develop a questionnaire to assess acceptability of the proposed treatment to women. This proposal involves translation of the information gained from basic research with macaques into clinical practice in women within the time frame of the grant. It is essential to conduct the clinical work at the University of Edinburgh, Scotland, UK, because no clinic in the USA has such a large patient population of women being fitted with the LNG-IUS for contraception. The macaque model provides excellent predictability for human studies, and Drs. Brenner and Critchley have published collaboratively on data from macaques and women in several recent papers. The proposal brings basic scientists and clinicians together for a "bench to bedside" approach that will advance women's health and improve contraceptive technology.

DESCRIPTION (provided by applicant): Potassium channels have the role of reducing excitability and are pivotal in controlling seizures. The large conductance calcium-activated potassium channels (BK-type channels) have the specialized role of regulating membrane excitability that is coincident with calcium influx. Previously, heterologous co- expression of the pore-forming a subunit with the novel [unreadable]4 accessory subunit was found to produce BK currents with properties of so called neuronal "type II BK channels". The [unreadable]4 subunit confers resistance to iberiotoxin block, slow gating kinetics, and a reduced open probability. In addition, the [unreadable]4 subunit may confer sensitivity to pKA dependent phosphorylation in neurons. We have generated [unreadable]4 gene knockout mice. Our preliminary data provides direct evidence that BK channel assembly with the [unreadable]4 subunit underlies type II BK channels. In the dentate gyrus (DG), knockout cells are more excitable and support high frequency firing. Finally, EEG recording demonstrate that the [unreadable]4 knockout mice exhibit non-convulsive partial seizures. The [unreadable]4 knockout mice may be the first genetic model for non-convulsive temporal lobe epilepsy. Investigation of these mice will allow us the unique opportunity to have a biophysical understanding of the changes in ion channel properties that may underlie this class of epilepsy. Our interest is to understand how a/[unreadable]4 subunit BK channels regulate the input/output properties of DG cells, and thereby contribute to resistance of synchronized epileptiform activity that is a property of the DG. Our working hypothesis is that beta4 subunits down-regulates BK channels, resulting in increased calcium influx and reduced excitability by recruitment of SK channels. We have 3 aims. 1) Utilizing patch clamp/slice recordings from DG cells, understand the change in membrane properties that result in increased AP firing in the knockout mice. 2) Determine how the ([unreadable]4 subunit modulates sensitivity of BK channels to pKA dependent phosphorylation in the DG, and thereby regulates excitability by metabotropic glutamate receptors 3) Utilize immunohistochemistry and electrophysiological recording techniques to determine the subcellular localization of the [unreadable]4 subunit in nerve terminals and its'contribution to neurotransmission in the mossy fiber terminals. These studies will provide the first understanding of a relatively uncharacterized BK channel subtype and further our understanding of this channels'function in the context of a novel genetic model for epilepsy.

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to understand the mechanism of action of calcium signaling pathways in airway smooth muscle in order to identify novel bronchodilators for the clinic. The immediate goals for this proposal are twofold. The research will provide an understanding (at the cellular level) of how a novel bronchodilator, UCL 1684, modulates SK channels and causes airway smooth muscle relaxation. The research will determine the mechanism by which SR SK potassium channels affect sarcoplasmic reticulum and cytosolic calcium load, excitability and contractility of ASM cells using calcium imaging and electrophysiology of airway smooth muscle cells. Second, the research will investigate UCL 1684 efficacy as a bronchodilator in vivo in animal models of asthma and models of ¿2 agonist tachyphylaxis. The approach will be to use whole animal plethysmography, and the forced oscillation techniques on asthmatic mice to evaluate UCL 1684 effects on airway severity and airway resistance, respectively. In addition, the experiments will examine UCL1684 effects on remodeling during asthma with a particular emphasis on its effects on airway muscle remodeling.

Brain function is dependent upon proteins that allow ions to pass across neuron cell membranes (called ion channels) to create currents that mediate the electrical activity of neurons. Protein channels that permit potassium ions to pass through neuron membranes are generally thought to quiet excessive neuron activity. However, there is a growing list of examples indicating that these "BK-type" potassium ion channels may also increase neuron excitability. The fact that BK channels are expressed throughout the nervous system suggests that understanding such paradoxical effects is important for understanding brain function in general. The investigators will record the activity of neurons in mice to learn the conditions that cause BK channels to quiet or excite neurons. The results will be used to make computational models that predict the anti-excitatory or pro-excitatory behaviors of BK channels. The project will train graduate students and a postdoctoral fellow to use state-of-art methods for studying brain function and anatomy and to make computational models, and will support the development of an integrated online virtual laboratory, "The Ion Channel laboratory," to teach users about ion channels and the electrical excitability of neurons.

The mechanisms underlying how slow- and fast-gating BK channel types either depress or paradoxically enhance a neuron's likelihood for firing an action potential (AP) will be studied in dentate gyrus neurons of the hippocampus. Using wild type neurons that express slow-gating BK channels, and transgenic neurons (BK beta4 knockout) that express fast-gating channels, the investigators will directly measure the cause-and-effect relationship between BK-regulated AP shape, activation of spike-triggered bulk and local calcium, recruitment of interspike conductances, and AP frequency. These data will be used to generate a computational model of dentate gyrus neurons that predicts the context in which BK channels reduce or enhance neuronal excitability. The model will then be experimentally tested in hippocampus CA1 pyramidal cells and cerebellum purkinje neurons to determine if pro-excitatory effects uncovered in dentate gyrus neurons are features observed in other neuron types that express BK channels. The findings of this study will create new understanding and new neuronal computational models of spike-influenced conductance and its effect on intrinsic excitability.

Project Summary Although epilepsy is diagnosed as unprovoked seizures, it is generally accepted that seizures can be precipitated in epileptic individuals by endogenous and exogenous stimulants. Stress and anxiety are among the most frequently reported precipitants for seizures in epileptic individuals. Understanding the pathophysiology of anxiety-induced seizures can dramatically benefit management of epileptic patients. Here, we present preliminary data for a novel mouse model for anxiety-induced seizures. The model is regulated by cre-induced expression of a human BK-type potassium channel gene containing an epilepsy mutation (D369G transgene). Preliminary studies indicate the D369G mice have increased anxiety, and handling-induced epileptic seizures. Our overarching hypothesis is that these transgenic mice will serve as a model for anxiety-induced seizures in epilepsy. As a first step to understanding anxiety-induced seizures, we propose an R03 pilot study to identify the regions of the brain where expression of the D369G causes seizures. We propose two aims. For aim 1, we will more thoroughly characterize the mouse phenotypes and screen the remaining founder lines for handling- induced seizures. By also crossing D369G lines to the BK channel knockout mice, we will eliminate endogenous expression and determine if D369G transgene expression reflects the normal BK channel expression pattern. For aim 2, we will use tissue and developmental-specific cre driver lines to determine the brain region/s responsible for the D369G phenotype. We hypothesize that handling-induced seizures are mediated by D369G BK channel post-development expression in excitatory cells of the hippocampus. Using temporal and tissue- specific cre driver lines we will examine 1) if prenatal or postnatal expression is required for the phenotype, 2) if excitatory or inhibitory neurons confer the phenotype, or 3) if expression in the hippocampus or amygdala confer increased anxiety and handling induced seizures. Success of this proposal will create a foothold for future in- depth electrophysiology studies regarding how activity in the identified brain region intersects with anxiety to precipitate seizures, and potentially provide drug targets to prevent or limit stress-induced seizure onset.