Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

<p>Abstract</p> <p>Background</p> <p>Along with high affinity binding of epibatidine (<it>K</it>_d1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (<it>K</it>_d2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [³H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites.</p> <p>Results</p> <p>Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [³H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [³H]epibatidine binding after adding a large concentration of cold competitor. Fourth, nonspecific binding of a heterologous competitor changed estimates of high and low inhibition constants but did not change the ratio of those estimates.</p> <p>Conclusions</p> <p>Investigating the low affinity site of α4β2 nAChR with equilibrium binding when ligand depletion and nonspecific binding are present likely needs special attention to experimental design and data interpretation beyond fitting total binding data. Manipulation of maximum ligand and receptor concentrations and intentionally increasing ligand depletion are potentially helpful approaches.</p

Spike-Timing Precision and Neuronal Synchrony Are Enhanced by an Interaction between Synaptic Inhibition and Membrane Oscillations in the Amygdala

The basolateral complex of the amygdala (BLA) is a critical component of the neural circuit regulating fear learning. During fear learning and recall, the amygdala and other brain regions, including the hippocampus and prefrontal cortex, exhibit phase-locked oscillations in the high delta/low theta frequency band (∼2–6 Hz) that have been shown to contribute to the learning process. Network oscillations are commonly generated by inhibitory synaptic input that coordinates action potentials in groups of neurons. In the rat BLA, principal neurons spontaneously receive synchronized, inhibitory input in the form of compound, rhythmic, inhibitory postsynaptic potentials (IPSPs), likely originating from burst-firing parvalbumin interneurons. Here we investigated the role of compound IPSPs in the rat and rhesus macaque BLA in regulating action potential synchrony and spike-timing precision. Furthermore, because principal neurons exhibit intrinsic oscillatory properties and resonance between 4 and 5 Hz, in the same frequency band observed during fear, we investigated whether compound IPSPs and intrinsic oscillations interact to promote rhythmic activity in the BLA at this frequency. Using whole-cell patch clamp in brain slices, we demonstrate that compound IPSPs, which occur spontaneously and are synchronized across principal neurons in both the rat and primate BLA, significantly improve spike-timing precision in BLA principal neurons for a window of ∼300 ms following each IPSP. We also show that compound IPSPs coordinate the firing of pairs of BLA principal neurons, and significantly improve spike synchrony for a window of ∼130 ms. Compound IPSPs enhance a 5 Hz calcium-dependent membrane potential oscillation (MPO) in these neurons, likely contributing to the improvement in spike-timing precision and synchronization of spiking. Activation of the cAMP-PKA signaling cascade enhanced the MPO, and inhibition of this cascade blocked the MPO. We discuss these results in the context of spike-timing dependent plasticity and modulation by neurotransmitters important for fear learning, such as dopamine

The Acclimation of European Sea Bass (Dicentrarchus labrax) to Temperature: Behavioural and Neurochemical Responses

Studies on fish behavioural and neurophysiological responses to water temperature change may contribute to an improved understanding of the ecological consequences of global warming. We investigated behavioural and neurochemical responses to water temperature in European sea bass (Dicentrarchus labrax) acclimated to three temperatures (18, 22 and 28°C). After 21 d of acclimation, three groups of 25 fish each were exposed to four behavioural challenges (foraging, olfactory, aversive and mirror tests). The expression of choline acetyltransferase (ChAT) was then analysed by Western blotting in CNS homogenates (from a subset of the same fish) as a marker for cholinergic system activity. In both foraging and olfactory tests, fish acclimated to 28°C exhibited significantly higher arousal responses than fish acclimated to lower temperatures. All specimens showed fright behaviour in the aversive test, but the latency of the escape response was significantly less in the fish at 28°C. Finally, the highest mirror responsiveness was exhibited by the fish acclimated to 22°C. As in the case of cholinergic neurotransmission, significantly higher ChAT levels were detected in the telencephalon, diencephalon, cerebellum and spinal cord of fish acclimated to 22 or 28°C in comparison with those maintained at 18°C. Lower ChAT levels were detected in the mesencephalon (optic tectum) at 22 and 28°C than at 18°C. These data indicate that neuronal functions are affected by water temperature. Increases or decreases in ChAT expression can be related to the functional modulation of brain and spinal cord centres involved in behavioural responses to temperature change. Overall, the results of this study suggest that the environmental temperature level influences behaviour and CNS neurochemistry in the European sea bass.Studies on fish behavioural and neurophysiological responses to water temperature change may contribute to an improved understanding of the ecological consequences of global warming. We investigated behavioural and neurochemical responses to water temperature in European sea bass (Dicentrarchus labrax) acclimated to three temperatures (18, 22 and 28 degrees C). After 21d of acclimation, three groups of 25 fish each were exposed to four behavioural challenges (foraging, olfactory, aversive and mirror tests). The expression of choline acetyltransferase (ChAT) was then analysed by Western blotting in CNS homogenates (from a subset of the same fish) as a marker for cholinergic system activity. In both foraging and olfactory tests, fish acclimated to 28 degrees C exhibited significantly higher arousal responses than fish acclimated to lower temperatures. All specimens showed fright behaviour in the aversive test, but the latency of the escape response was significantly less in the fish at 28 degrees C. Finally, the highest mirror responsiveness was exhibited by the fish acclimated to 22 degrees C. As in the case of cholinergic neurotransmission, significantly higher ChAT levels were detected in the telencephalon, diencephalon, cerebellum and spinal cord of fish acclimated to 22 or 28 degrees C in comparison with those maintained at 18 degrees C. Lower ChAT levels were detected in the mesencephalon (optic tectum) at 22 and 28 degrees C than at 18 degrees C. These data indicate that neuronal functions are affected by water temperature. Increases or decreases in ChAT expression can be related to the functional modulation of brain and spinal cord centres involved in behavioural responses to temperature change. Overall, the results of this study suggest that the environmental temperature level influences behaviour and CNS neurochemistry in the European sea bass

Lawson criterion for ignition exceeded in an inertial fusion experiment

For more than half a century, researchers around the world have been engaged in attempts to achieve fusion ignition as a proof of principle of various fusion concepts. Following the Lawson criterion, an ignited plasma is one where the fusion heating power is high enough to overcome all the physical processes that cool the fusion plasma, creating a positive thermodynamic feedback loop with rapidly increasing temperature. In inertially confined fusion, ignition is a state where the fusion plasma can begin "burn propagation" into surrounding cold fuel, enabling the possibility of high energy gain. While "scientific breakeven" (i.e., unity target gain) has not yet been achieved (here target gain is 0.72, 1.37 MJ of fusion for 1.92 MJ of laser energy), this Letter reports the first controlled fusion experiment, using laser indirect drive, on the National Ignition Facility to produce capsule gain (here 5.8) and reach ignition by nine different formulations of the Lawson criterion

Oncoplastic breast consortium recommendations for mastectomy and whole breast reconstruction in the setting of post-mastectomy radiation therapy

Aim Demand for nipple- and skin- sparing mastectomy (NSM/SSM) with immediate breast reconstruction (BR) has increased at the same time as indications for post-mastectomy radiation therapy (PMRT) have broadened. The aim of the Oncoplastic Breast Consortium initiative was to address relevant questions arising with this clinically challenging scenario. Methods A large global panel of oncologic, oncoplastic and reconstructive breast surgeons, patient advocates and radiation oncologists developed recommendations for clinical practice in an iterative process based on the principles of Delphi methodology. Results The panel agreed that surgical technique for NSM/SSM should not be formally modified when PMRT is planned with preference for autologous over implant-based BR due to lower risk of long-term complications and support for immediate and delayed-immediate reconstructive approaches. Nevertheless, it was strongly believed that PMRT is not an absolute contraindication for implant-based or other types of BR, but no specific recommendations regarding implant positioning, use of mesh or timing were made due to absence of high-quality evidence. The panel endorsed use of patient-reported outcomes in clinical practice. It was acknowledged that the shape and size of reconstructed breasts can hinder radiotherapy planning and attention to details of PMRT techniques is important in determining aesthetic outcomes after immediate BR. Conclusions The panel endorsed the need for prospective, ideally randomised phase III studies and for surgical and radiation oncology teams to work together for determination of optimal sequencing and techniques for PMRT for each patient in the context of BR