Changes in Both Neuron Intrinsic Properties and Neurotransmission Are Needed to Drive the Increase in GnRH Neuron Firing Rate during Estradiol-Positive Feedback.

Central output of gonadotropin-releasing hormone (GnRH) neurons controls fertility and is sculpted by sex-steroid feedback. A switch of estradiol action from negative to positive feedback initiates a surge of GnRH release, culminating in ovulation. In ovariectomized mice bearing constant-release estradiol implants (OVX+E), GnRH neuron firing is suppressed in the morning (AM) by negative feedback and activated in the afternoon (PM) by positive feedback; no time-of-day-dependent changes occur in OVX mice. In this daily surge model, GnRH neuron intrinsic properties are shifted to favor increased firing during positive feedback. It is unclear whether this shift and the observed concomitant increase in GABAergic transmission, which typically excites GnRH neurons, are independently sufficient for increasing GnRH neuron firing rate during positive feedback or whether both are needed. To test this, we used dynamic clamp to inject selected previously recorded trains of GABAergic postsynaptic conductances (PSgs) collected during the different feedback states of the daily surge model into GnRH neurons from OVX, OVX+E AM, and OVX+E PM mice. PSg trains mimicking positive feedback initiated more action potentials in cells from OVX+E PM mice than negative feedback or OVX (open feedback loop) trains in all three animal models, but the positive-feedback train was most effective when applied to cells during positive feedback. In silico studies of model GnRH neurons in which >1000 PSg trains were tested exhibited the same results. These observations support the hypothesis that GnRH neurons integrate fast-synaptic and intrinsic changes to increase firing rates during positive feedback.SIGNIFICANCE STATEMENT Infertility affects 15%-20% of couples; failure to ovulate is a common cause. Understanding how the brain controls ovulation is critical for new developments in both infertility treatment and contraception. Ovarian estradiol alters both the intrinsic properties of gonadotropin-releasing hormone (GnRH) neurons and synaptic inputs to these cells coincident with production of sustained GnRH release that ultimately triggers ovulation. We demonstrate here using dynamic clamp and mathematical modeling that estradiol-induced shifts in synaptic transmission alone can increase firing output, but that the intrinsic properties of GnRH neurons during positive feedback further poise these cells for increased response to higher frequency synaptic transmission. These data suggest that GnRH neurons integrate fast-synaptic and intrinsic changes to increase firing rates during the preovulatory GnRH surge.

Estradiol Enhances the Depolarizing Response to GABA and AMPA Synaptic Conductances in Arcuate Kisspeptin Neurons by Diminishing Voltage-Gated Potassium Currents.

Synaptic and intrinsic properties interact to sculpt neuronal output. Kisspeptin neurons in the hypothalamic arcuate nucleus help convey homeostatic estradiol feedback to central systems controlling fertility. Estradiol increases membrane depolarization induced by GABAA receptor activation in these neurons. We hypothesized that the mechanisms underlying estradiol-induced alterations in postsynaptic response to GABA, and also AMPA, receptor activation include regulation of voltage-gated potassium currents. Whole-cell recordings of arcuate kisspeptin neurons in brain slices from ovariectomized (OVX) and OVX+estradiol (OVX+E) female mice during estradiol negative feedback revealed that estradiol reduced capacitance, reduced transient and sustained potassium currents, and altered voltage dependence and kinetics of transient currents. Consistent with these observations, estradiol reduced rheobase and action potential latency. To study more directly interactions between synaptic and active intrinsic estradiol feedback targets, dynamic clamp was used to simulate GABA and AMPA conductances. Both GABA and AMPA dynamic clamp-induced postsynaptic potentials (PSPs) were smaller in neurons from OVX than OVX+E mice; blocking transient potassium currents eliminated this difference. To interrogate the role of the estradiol-induced changes in passive intrinsic properties, different Markov model structures based on the properties of the transient potassium current in cells from OVX or OVX+E mice were combined in silico with passive properties reflecting these two endocrine conditions. Some of tested models reproduced the effect on PSPs in silico, revealing that AMPA PSPs were more sensitive to changes in capacitance. These observations support the hypothesis that PSPs in arcuate kisspeptin neurons are regulated by estradiol-sensitive mechanisms including potassium conductances and membrane properties.SIGNIFICANCE STATEMENT Kisspeptin neurons relay estradiol feedback to gonadotropin-releasing hormone neurons, which regulate the reproductive system. The fast synaptic neurotransmitters GABA and glutamate rapidly depolarize arcuate kisspeptin neurons and estradiol increases this depolarization. Estradiol reduced both potassium current in the membrane potential range typically achieved during response to fast synaptic inputs and membrane capacitance. Using simulated GABA and glutamate synaptic inputs, we showed changes in both the passive and active intrinsic properties induced by in vivo estradiol treatment affect the response to synaptic inputs, with capacitance having a greater effect on response to glutamate. The suppression of both passive and active intrinsic properties by estradiol feedback thus renders arcuate kisspeptin neurons more sensitive to fast synaptic inputs.

Lessons Learned From Delivering Imara, an HIV/STI Risk Reduction Intervention for African American Girls in Juvenile Detention.

A critical need exists for efficacious interventions to reduce sexual risk and sexually transmitted infections (STIs) among African American girls in juvenile detention. Adapting evidence-based interventions is one strategy for developing interventions that might protect detained African American girls from adverse sexual health outcomes. To support development and implementation of evidence-based HIV/STI prevention interventions for this population, this qualitative study describes lessons learned from delivering Imara, an adapted HIV/STI prevention intervention for detained African American girls. Program implementation includes one-on-one sessions in the detention facility that offer logistical advantages; provide intervention contact inside the facility, soon after release, and frequently thereafter; address STI treatment for girls and their sexual partners; tailor intervention content based on individual risk and learning needs; and identify and acknowledge girls' competing priorities. These lessons are discussed in the context of challenges encountered and solutions for addressing the challenges, and in terms of the structure and content of the intervention. The lessons learned from delivering Imara exemplify the continuous process of adapting an existing intervention for a new population and setting.

3D Data Mapping and Real-Time Experiment Control and Visualization in Brain Slices.

Here, we propose two basic concepts that can streamline electrophysiology and imaging experiments in brain slices and enhance data collection and analysis. The first idea is to interface the experiment with a software environment that provides a 3D scene viewer in which the experimental rig, the brain slice, and the recorded data are represented to scale. Within the 3D scene viewer, the user can visualize a live image of the sample and 3D renderings of the recording electrodes with real-time position feedback. Furthermore, the user can control the instruments and visualize their status in real time. The second idea is to integrate multiple types of experimental data into a spatial and temporal map of the brain slice. These data may include low-magnification maps of the entire brain slice, for spatial context, or any other type of high-resolution structural and functional image, together with time-resolved electrical and optical signals. The entire data collection can be visualized within the 3D scene viewer. These concepts can be applied to any other type of experiment in which high-resolution data are recorded within a larger sample at different spatial and temporal coordinates.

Slow plastic creep of 2D dusty plasma solids.

We report complex plasma experiments, assisted by numerical simulations, providing an alternative qualitative link between the macroscopic response of polycrystalline solid matter to small shearing forces and the possible underlying microscopic processes. In the stationary creep regime we have determined the exponents of the shear rate dependence of the shear stress and defect density, being α=1.15±0.1 and ß=2.4±0.4, respectively. We show that the formation and rapid glide motion of dislocation pairs in the lattice are dominant processes.

[Preoperative endorectal MRI in prostate cancer: a monocentric retrospective cohort]. / Place de l'IRM prostatique avant prostatectomie radicale. Cohorte monocentrique rétrospective.

INTRODUCTION: The aim of the study was to assess the added value of a prostatic MRI performed in a non-expert center before radical prostatectomy for prostate cancer. PATIENTS AND METHODS: All patients considered for a radical prostatectomy for a clinically localised prostate cancer in our institution between June 2006 and April 2011 were analysed. They underwent a systematic endorectal 1.5 T MRI [eMRI] (T2 and diffusion-weighted imaging). The procedure was performed at least 8 weeks after transrectal ultrasound guided prostate biopsies. They were analyzed by a single experimented genitourinary radiologist. The preoperative characteristics and biopsy data were collected, as the eMRI interpretation using a systematic scale. Correlation between eMRI and final histopathology has been analyzed (standardized pathological report using the Stanford procedure). We considered the following points: extracapsular extension, seminal vesicle invasion and cancer localisation. Cancer localization was analyzed both by halves (left/right) and on sextant-basis (base, middle, apex, left and right respectively). For each data, sensitivity, specificity, positive (PPV) and negative predictive value (NPV) were calculated. Finally, we compared the results with those from the Partin's table and the Kattan's nomogram for T stage. RESULTS: Two hundred and fifteen patients underwent a radical prostatectomy, having a mean age of 63 ± 6 years, a mean PSA: 7.8 ± 3.8 ng/mL. One hundred and fifty-nine patients had organ confined disease. Fifty-six had pT3 disease (50 pT3a and 6 pT3b). Sensibilities and specificities were the following: for extracapsular extension 26% and 96%; for seminal vesicle invasion 17% and 98%; for cancer localisation 64% and 74%; for prediction of left or right side 81% and 48%. DISCUSSION: MRI showed equal performances compared to the Partin's table and the Kattan's nomogram for T stage. It showed lower performance compared to biopsy results for cancer localization (sensitivity 88%, specificity 70%). Literature provides wide ranges of results for eMRI. The results of this study were at the low limit of these ranges but reflect everyday practice. When performed, as it was in our study, eMRI did not seem to be accurate for staging. It had at best a very limited added value compared to the existing tools.

Mechanosensitive pore opening of a prokaryotic voltage-gated sodium channel.

Voltage-gated ion channels (VGICs) orchestrate electrical activities that drive mechanical functions in contractile tissues such as the heart and gut. In turn, contractions change membrane tension and impact ion channels. VGICs are mechanosensitive, but the mechanisms of mechanosensitivity remain poorly understood. Here, we leverage the relative simplicity of NaChBac, a prokaryotic voltage-gated sodium channel from Bacillus halodurans, to investigate mechanosensitivity. In whole-cell experiments on heterologously transfected HEK293 cells, shear stress reversibly altered the kinetic properties of NaChBac and increased its maximum current, comparably to the mechanosensitive eukaryotic sodium channel NaV1.5. In single-channel experiments, patch suction reversibly increased the open probability of a NaChBac mutant with inactivation removed. A simple kinetic mechanism featuring a mechanosensitive pore opening transition explained the overall response to force, whereas an alternative model with mechanosensitive voltage sensor activation diverged from the data. Structural analysis of NaChBac identified a large displacement of the hinged intracellular gate, and mutagenesis near the hinge diminished NaChBac mechanosensitivity, further supporting the proposed mechanism. Our results suggest that NaChBac is overall mechanosensitive due to the mechanosensitivity of a voltage-insensitive gating step associated with the pore opening. This mechanism may apply to eukaryotic VGICs, including NaV1.5.

Fast-activating voltage- and calcium-dependent potassium (BK) conductance promotes bursting in pituitary cells: a dynamic clamp study.

The electrical activity pattern of endocrine pituitary cells regulates their basal secretion level. Rat somatotrophs and lactotrophs exhibit spontaneous bursting and have high basal levels of hormone secretion, while gonadotrophs exhibit spontaneous spiking and have low basal hormone secretion. It has been proposed that the difference in electrical activity between bursting somatotrophs and spiking gonadotrophs is due to the presence of large conductance potassium (BK) channels on somatotrophs but not on gonadotrophs. This is one example where the role of an ion channel type may be clearly established. We demonstrate here that BK channels indeed promote bursting activity in pituitary cells. Blocking BK channels in bursting lacto-somatotroph GH4C1 cells changes their firing activity to spiking, while further adding an artificial BK conductance via dynamic clamp restores bursting. Importantly, this burst-promoting effect requires a relatively fast BK activation/deactivation, as predicted by computational models. We also show that adding a fast-activating BK conductance to spiking gonadotrophs converts the activity of these cells to bursting. Together, our results suggest that differences in BK channel expression may underlie the differences in electrical activity and basal hormone secretion levels among pituitary cell types and that the rapid rate of BK channel activation is key to its role in burst promotion.

Employing a teen advisory board to adapt an evidence-based HIV/STD intervention for incarcerated African-American adolescent women.

This manuscript assesses priorities and challenges of adolescent females by conducting a meeting with teen advisory board (TAB) members to collect information regarding their lives and experiences pre-, during and post-incarceration in a juvenile detention facility. Multiple themes emerged regarding the impact of incarceration on young African-American females, including experiencing a loss of personal liberties, the importance of making money upon release, unfaithfulness by partners on the 'outside', substance use and lack of control over their environment upon release, including parents, peers and male sexual partners. Based on feedback from TAB members, unique barriers and challenges were identified that suggested areas where adaptations to an evidenced-based HIV/sexually transmitted disease (STD) intervention would be justified to more adequately meet the needs of this particular subgroup of young African-American women. Adaptations to the evidence-based interventions included enhancing activities related to goal setting, emotion regulation skills, decision-making, recognizing and utilizing support networks and addressing the relationship between substance use and risky sexual behavior. Future health education efforts focusing on either the creation of new HIV/STD interventions or adaptations to existing interventions should consider utilizing advisory boards with members of the priority population at the earliest stages of intervention planning.

[Prone and supine position for percutaneous nephrolithotomy: is it necessary to change the operative technique?]. / Néphrolithotomie percutanée : faut-il passer du décubitus ventral au décubitus dorsal ?

PURPOSE: To assess the safety and effectiveness of percutaneous nephrolithotomy performed in the supine position. To investigate whether the change in operative technique, between prone and supine position, is easy. METHOD: Thirty patients who underwent percutaneous nephrolithotomy, by one surgeon in one hospital, were studied retrospectively. The eight first cases were performed in prone position, and the following 22 patients were operated in supine position according to Valdivia's operative technique. Data were analyzed with t-test. We considered P<0.05 as significant. RESULTS: Surgical complication rate, transfusion rate and fever rate were the same for both groups. Success rate was 72% in supine position group and 63% in prone position group (P>0.05). These results were comparable with the data of the literature. Only operative time was shorter in supine position group with significant statistical difference (P=0.02). CONCLUSION: In our experience, percutaneous nephrolithotomy in the supine position was safe and efficient, and outcomes were similar to that in the prone position. Learning curve was easy and fast.

Parameter Optimization for Ion Channel Models: Integrating New Data with Known Channel Properties.

Ion channels play a central role in membrane physiology, but to fully understand how they operate, one must have accurate kinetic mechanisms. Estimating kinetics is not trivial when the mechanism is complex, and a large number of parameters must be extracted from data. Furthermore, the information contained in the data is often limited, and the model may not be fully determined. The solution is to reduce the number of parameters and to estimate them in such a way that they not only describe well the new data but also agree with the existing knowledge. In a previous study, we presented a comprehensive formalism for estimating kinetic parameters subject to a variety of explicit and implicit constraints that define quantitative relationships between parameters and describe specific mechanism properties. Here, we introduce the reader to the QuB software, which implements this constraining formalism. QuB features a powerful visual interface and a high-level scripting language that can be used to formulate kinetic models and constraints of arbitrary complexity, and to efficiently estimate the parameters from a variety of experimental data.

Kinetic properties and functional dynamics of sodium channels during repetitive spiking in a slow pacemaker neuron.

We examined the kinetic properties of voltage-gated Na(+) channels and their contribution to the repetitive spiking activity of medullary raphé neurons, which exhibit slow pacemaking and strong spiking adaptation. The study is based on a combination of whole-cell patch-clamp, modeling and real-time computation. Na(+) currents were recorded from neurons in brain slices obtained from male and female neonatal rats, using voltage-clamp protocols designed to reduce space-clamp artifacts and to emphasize functionally relevant kinetic features. A detailed kinetic model was formulated to explain the broad range of transient and stationary voltage-dependent properties exhibited by Na(+) currents. The model was tested by injecting via dynamic clamp a model-based current as a substitute for the native TTX-sensitive Na(+) currents, which were pharmacologically blocked. The model-based current reproduced well the native spike shape and spiking frequency. The dynamics of Na(+) channels during repetitive spiking were indirectly examined through this model. By comparing the spiking activities generated with different kinetic models in dynamic-clamp experiments, we determined that state-dependent slow inactivation contributes significantly to spiking adaptation. Through real-time manipulation of the model-based current, we established that suprathreshold Na(+) current mainly controls spike shape, whereas subthreshold Na(+) current modulates spiking frequency and contributes to the pacemaking mechanism. Since the model-based current was injected in the soma, the results also suggest that somatic Na(+) channels are sufficient to establish the essential spiking properties of raphé neurons in vitro.

Isolation of somatic Na+ currents by selective inactivation of axonal channels with a voltage prepulse.

We present a simple and effective method for isolating the somatic Na(+) current recorded under voltage clamp from neurons in brain slices. The principle is to convert the axon from an active compartment capable of generating uncontrolled axonal spikes into a passive structure by selectively inactivating axonal Na(+) channels. Typically, whole-cell currents from intact neurons under somatic voltage clamp contain a mixture of Na(+) current and axial current caused by escaped axonal spikes. We found that a brief prepulse to voltages near spike threshold evokes the axonal spike, which inactivates axonal but not somatic channels. A subsequent voltage step then evokes only somatic Na(+) current from electrotonically proximal sodium channels under good voltage-clamp control. Simulations using a neuron compartmental model support the idea that the prepulse effectively inactivates currents from the axon and isolates well controlled somatic currents. Na(+) currents recorded from cortical pyramidal neurons in slices, using the prepulse, were found to have voltage dependence nearly identical to that of currents recorded from acutely dissociated pyramidal neurons. In addition, studies in dissociated neurons show that the prepulse has no visible effect on the voltage dependence and kinetics of Na(+) currents elicited by the subsequent voltage step, only decreasing the amplitude of the currents by 10-20%. The technique was effective in several neuronal types in brain slices from male and female neonatal rats and mice, including raphé neurons, cortical pyramidal neurons, inferior olivary neurons, and hypoglossal motoneurons.

Dynamic Clamp on a Windows PC.

Dynamic clamp is a powerful tool for interfacing computational models and real cells. We describe here how to set up and carry out dynamic clamp experiments using a patch clamp amplifier, a National Instruments data acquisition card, and the freely available QuB software that operates on a PC running MS Windows.

The mechanism of MICU-dependent gating of the mitochondrial Ca2+uniporter.

Ca2+ entry into mitochondria is through the mitochondrial calcium uniporter complex (MCUcx), a Ca2+-selective channel composed of five subunit types. Two MCUcx subunits (MCU and EMRE) span the inner mitochondrial membrane, while three Ca2+-regulatory subunits (MICU1, MICU2, and MICU3) reside in the intermembrane space. Here, we provide rigorous analysis of Ca2+ and Na+ fluxes via MCUcx in intact isolated mitochondria to understand the function of MICU subunits. We also perform direct patch clamp recordings of macroscopic and single MCUcx currents to gain further mechanistic insights. This comprehensive analysis shows that the MCUcx pore, composed of the EMRE and MCU subunits, is not occluded nor plugged by MICUs during the absence or presence of extramitochondrial Ca2+ as has been widely reported. Instead, MICUs potentiate activity of MCUcx as extramitochondrial Ca2+ is elevated. MICUs achieve this by modifying the gating properties of MCUcx allowing it to spend more time in the open state.

A modeling study of T-type Ca2+ channel gating and modulation by L-cysteine in rat nociceptors.

L-cysteine (L-cys) increases the amplitude of T-type Ca(2+) currents in rat T-rich nociceptor-like dorsal root ganglia neurons. The modulation of T-type Ca(2+) channel gating by L-cys was studied by fitting Markov state models to whole-cell currents recorded from T-rich neurons. The best fitting model tested included three resting states and inactivation from the second resting state and the open state. Inactivation and the final opening step were voltage-independent, whereas transitions between the resting states and deactivation were voltage-dependent. The transition rates between the first two resting states were an order of magnitude faster than those between the second and third resting states, and the voltage-dependency of forward transitions through resting states was two to three times greater than for analogous backward transitions. Analysis with the best fitting model suggested that L-cys increases current amplitude mainly by increasing the transition rate from resting to open and decreasing the transition rate from open to inactivated. An additional model was developed that could account for the bi-exponential time course of recovery from inactivation of the currents and the high frequency of blank sweeps in single channel recordings. This model detected basically the same effects of L-cys on channel gating as the best fitting model.

Raphé neurons stimulate respiratory circuit activity by multiple mechanisms via endogenously released serotonin and substance P.

Brainstem serotonin (5-HT) neurons modulate activity of many neural circuits in the mammalian brain, but in many cases endogenous mechanisms have not been resolved. Here, we analyzed actions of raphé 5-HT neurons on respiratory network activity including at the level of the pre-Bötzinger complex (pre-BötC) in neonatal rat medullary slices in vitro, and in the more intact nervous system of juvenile rats in arterially perfused brainstem-spinal cord preparations in situ. At basal levels of activity, excitation of the respiratory network via simultaneous release of 5-HT and substance P (SP), acting at 5-HT(2A/2C), 5-HT(4), and/or neurokinin-1 receptors, was required to maintain inspiratory motor output in both the neonatal and juvenile systems. The midline raphé obscurus contained spontaneously active 5-HT neurons, some of which projected to the pre-BötC and hypoglossal motoneurons, colocalized 5-HT and SP, and received reciprocal excitatory connections from the pre-BötC. Experimentally augmenting raphé obscurus activity increased motor output by simultaneously exciting pre-BötC and motor neurons. Biophysical analyses in vitro demonstrated that 5-HT and SP modulated background cation conductances in pre-BötC and motor neurons, including a nonselective cation leak current that contributed to the resting potential, which explains the neuronal depolarization that augmented motor output. Furthermore, we found that 5-HT, but not SP, can transform the electrophysiological phenotype of some pre-BötC neurons to intrinsic bursters, providing 5-HT with an additional role in promoting rhythm generation. We conclude that raphé 5-HT neurons excite key circuit components required for generation of respiratory motor output.

Crystallization dynamics of a single layer complex plasma.

We report a series of complex (dusty) plasma experiments, aimed at the study of the detailed time evolution of the recrystallization process following a rapid quench of a two-dimensional dust liquid. The experiments were accompanied by large-scale (million-particle) molecular dynamics simulations, assuming Yukawa-type interparticle interaction. Both experiment and simulation show a ∝t(α) (power-law) dependence of the linear crystallite domain size as measured by the bond-order correlation length, translational correlation length, dislocation (defect) density, and a direct size measurement algorithm. The results show two stages of order formation. On short time scales, individual particle motion dominates; this is a fast process characterized by α=0.93±0.1. At longer time scales, small crystallites undergo collective rearrangement, merging into bigger ones, resulting in a smaller exponent α=0.38±0.06.

Application of ADAPT-ITT: adapting an evidence-based HIV prevention intervention for incarcerated African American adolescent females.

African American adolescent females are disproportionately affected by the HIV epidemic. Recent findings suggest that gender- and culturally appropriate HIV prevention interventions can significantly reduce HIV-associated sexual risk behaviors among this vulnerable population. Currently, there are no evidence-based interventions (EBIs) for this vulnerable subgroup. Thus, interventions specifically tailored for this subgroup are urgently needed. Effective interventions that reduce HIV risk behaviors remain one of the most powerful tools in curbing the HIV epidemic. The selected intervention (Horizons) was adapted using a coordinated and systematically guided adaptation process based on the ADAPT-ITT framework. This article serves as a starting point to support using the ADAPT-ITT model, which was beneficial when using an EBI in an alternative setting than originally created. Using this prescriptive method for adapting Horizons for incarcerated young girls proved to be a time- and cost-effective method.