Semisynthetic photoprotein reporters for tracking fast Ca(2+) transients.

Changes in the intracellular concentration of free ionized calcium ([Ca(2+)]i) control a host of cellular processes as varied as vision, muscle contraction, neuronal signal transmission, proliferation, apoptosis etc. The disturbance in Ca(2+)-signaling causes many severe diseases. To understand the mechanisms underlying the control by calcium and how disorder of this regulation relates to pathological conditions, it is necessary to measure [Ca(2+)]i. The Ca(2+)-regulated photoproteins which are responsible for bioluminescence of marine coelenterates have been successfully used for this purpose over the years. Here we report the results on comparative characterization of bioluminescence properties of aequorin from Aequorea victoria, obelin from Obelia longissima, and clytin from Clytia gregaria charged by native coelenterazine and coelenterazine analogues f, i, and hcp. The comparison of specific bioluminescence activity, stability, emission spectra, stopped-flow kinetics, sensitivity to calcium, and effect of physiological concentrations of Mg(2+) establishes obelin-hcp as an excellent semisynthetic photoprotein to keep track of fast intracellular Ca(2+) transients. The rate of rise of its light signal on a sudden change of [Ca(2+)] is almost 3- and 11-fold higher than those of obelin and aequorin with native coelenterazine, respectively, and 20 times higher than that of the corresponding aequorin-hcp. In addition, obelin-hcp preserves a high specific bioluminescence activity and displays higher Ca(2+)-sensitivity as compared to obelin charged by native coelenterazine and sensitivity to Ca(2+) comparable with those of aequorin-f and aequorin-hcp.

Bioluminescent properties of obelin and aequorin with novel coelenterazine analogues.

The main analytical use of Ca(2+)-regulated photoproteins from luminous coelenterates is for real-time non-invasive visualization of intracellular calcium concentration ([Ca(2+)]i) dynamics in cells and whole organisms. A limitation of this approach for in vivo deep tissue imaging is the fact that blue light emitted by the photoprotein is highly absorbed by tissue. Seven novel coelenterazine analogues were synthesized and their effects on the bioluminescent properties of recombinant obelin from Obelia longissima and aequorin from Aequorea victoria were evaluated. Only analogues having electron-donating groups (m-OCH3 and m-OH) on the C6 phenol moiety or an extended resonance system at the C8 position (1-naphthyl and α-styryl analogues) showed a significant red shift of light emission. Of these, only the α-styryl analogue displayed a sufficiently high light intensity to allow eventual tissue penetration. The possible suitability of this compound for in vivo assays was corroborated by studies with aequorin which allowed the monitoring of [Ca(2+)]i dynamics in cultured CHO cells and in hippocampal brain slices. Thus, the α-styryl coelenterazine analogue might be potentially useful for non-invasive, in vivo bioluminescence imaging in deep tissues of small animals.

Differences in the spinal command of ejaculation in rapid ejaculating rats.

INTRODUCTION: It has been hypothesized that lifelong premature ejaculation is part of a biological variation in the intravaginal ejaculation latency, but what causes this variation remains poorly understood. AIM: The aim of this study is to elucidate whether variations in ejaculation latencies in an experimental rat model for premature ejaculation are linked to differences in the spinal command of ejaculation. MAIN OUTCOME MEASURES: Electrical microstimulation of the spinal generator for ejaculation revealed an accelerated expulsion phase in rapid ejaculating rats. METHODS: Adult male Wistar rats were categorized as "sluggish,""normal," or "rapid" ejaculators on the basis of their ejaculation frequency in sexual mating tests. One to three weeks after selection, males were urethane anesthetized and electrically microstimulated in the spinal generator for ejaculation, evoking ejaculation. Bulbospongiosus muscle electromyographic and intraluminal vas deferens pressure were measured simultaneously, representing, respectively, the expulsion and emission phase in ejaculation. RESULTS: Electrical microstimulation of the spinal generator for ejaculation evoked ejaculation in "sluggish" (N = 9), "normal" (N = 13), and "rapid" (N = 11) ejaculating rats. Vas deferens contraction (emission phase) was evoked at different stimulation strengths, but response properties were not statistically different between "sluggish,""normal," and "rapid" ejaculator rats. Bulbospongiosus muscle contractions (expulsion phase) following microstimulation was significantly accelerated in "rapid" rats as compared with "sluggish" and "normal" rats. The total duration of bulbospongiosus muscle contractions remained unchanged between the three ejaculator groups. CONCLUSIONS: Our results provide the first scientific evidence supporting a neurophysiological difference between "rapid,""normal," and "sluggish" ejaculators, expressed as an accelerated expulsion phase in "rapid" ejaculator rats. This bridges the gap between a sexual behavior trait and the spinal command of ejaculation.

Ejaculation elicited by microstimulation of lumbar spinothalamic neurons.

BACKGROUND: Neuroanatomical and lesion studies have identified lumbar spinothalamic (LSt) neurons to be essential for ejaculation, but their precise role remains elusive. OBJECTIVE: To assess the role of LSt neurons as a spinal pattern generator for ejaculation (SGE) and their action on anatomical structures involved in the two ejaculation phases, the emission and expulsion of semen. DESIGN: The bulbospongiosus muscle (BSM) was implanted with electrodes and the seminal vesicle (SV) or vas deferens (VD) lumen catheterized in adult anaesthetized rats. Spinal exposure at the fourth lumbar segment (L4) allowed lowering an electrode stereotaxically into area VII/X for brief (300-500ms) electrical stimulation of LSt neurons, while recording BSM-EMG and intraluminal SV or VD pressure. RESULTS: Brief electrical microstimulation in the LSt neuron area evoked the expulsion of semen in 17 of 17 rats, with motile spermatozoa in 10 of 17 rats. After stimulation, SV/VD luminal pressure directly rose and fell, followed by rhythmic BSM contractions lasting approximately 25s. Acute T8-T9 spinalization (n=4) did not alter the activation pattern of the BSM-EMG response. Injection of the GABA(A)-receptor agonist muscimol, inhibiting neuronal activity into the LSt neuron area after LSt neuron microstimulation (n=5), stopped BSM contractions in midstream. CONCLUSIONS: Electrical microstimulation of LSt neurons activates the entire sequence of ejaculation in rats in a coordinated fashion, ie the emission (SV/VD contraction) followed by expulsion (rhythmic BSM contractions) of living spermatozoa. Midcourse interruption of ejaculation following intraspinal muscimol injection establishes that LSt neurons are the SGE. This could help to identify spinal pharmacological targets for the treatment of ejaculatory disorders and provide the rationale for intraspinal stimulation to treat anejaculation in infertile spinal cord injured (SCI) patients.

Facilitating sensory responses in developing mouse somatosensory barrel cortex.

The sensory responses in the barrel cortex of mice aged postnatal day (P)7-P12 evoked by a single whisker deflection are smaller in amplitude and spread over a smaller area than those measured in P13-P21 mice. However, repetitive 10-Hz stimulation or paired pulse whisker stimulation in P7-P12 mice evoked facilitating sensory responses, contrasting with the depressing sensory responses observed in P13-P21 mice. This facilitation occurred during an interval ranging 300-1,000 ms after the first stimulus and was measured using whole cell recordings, voltage-sensitive dye imaging, and calcium-sensitive dye imaging. The facilitated responses were not only larger in amplitude but also propagated over a larger cortical area. The facilitation could be blocked by local application of pharmacological agents reducing cortical excitability. Local cortical microstimulation could substitute for the first whisker stimulus to produce a facilitated sensory response. The enhanced sensory responses evoked by repetitive sensory stimuli in P7-P12 mice may contribute to the activity-dependent specification of the developing cortical circuits. In addition, the facilitating sensory responses allow long integration times for sensory processing compatible with the slow behavior of mice during early postnatal development.

Regulation of AMPA receptor lateral movements.

An essential feature in the modulation of the efficacy of synaptic transmission is rapid changes in the number of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors at post-synaptic sites on neurons. Regulation of receptor endo- and exocytosis has been shown to be involved in this process. Whether regulated lateral diffusion of receptors in the plasma membrane also participates in receptor exchange to and from post-synaptic sites remains unknown. We analysed the lateral mobility of native AMPA receptors containing the glutamate receptor subunit GluR2 in rat cultured hippocampal neurons, using single-particle tracking and video microscopy. Here we show that AMPA receptors alternate within seconds between rapid diffusive and stationary behaviour. During maturation of neurons, stationary periods increase in frequency and length, often in spatial correlation with synaptic sites. Raising intracellular calcium, a central element in synaptic plasticity, triggers rapid receptor immobilization and local accumulation on the neuronal surface. We suggest that calcium influx prevents AMPA receptors from diffusing, and that lateral receptor diffusion to and from synaptic sites acts in the rapid and controlled regulation of receptor numbers at synapses.

Differential and long-lasting alterations of high-voltage activated calcium currents in CA1 and dentate granule neurons after status epilepticus.

The effects on high-voltage activated (HVA) calcium currents were examined in hippocampal CA1 cells and dentate gyrus (DG) granule neurons, 2 days (short-term; ST) and 2-3 months (long-term; LT) after electrically induced, limbic electrographic and behavioural seizures in rats. Whole-cell voltage-clamp recordings in dissociated CA1 neurons of LT rats showed a decrease in the sustained HVA calcium current amplitude and a faster inactivation of the current both in rats that had experienced a status epilepticus (post-SE rats) and those in which the stimulation did not lead to SE (non-SE rats). In CA1 neurons of LT-SE rats this resulted in a reduced Ca2+ entry through the HVA channels. Perforated-patch voltage-clamp recordings in dissociated DG granule neurons of LT-SE rats showed an increased sustained HVA current amplitude compared to controls and non-SE rats, leading to an increased Ca2+ entry via HVA calcium channels. Two days after SE, we observed an increased Ca2+ entry for a defined depolarization, although the change in HVA current amplitude and inactivation rate did not reach significance. We also observed a decrease in calbindin-D28k staining in DG post-SE neurons, but this change was not associated with a change in HVA current inactivation. The opposite changes in neuronal Ca2+ entry through HVA channels in CA1 vs. DG cells depended strongly on whether rats had experienced SE and later spontaneous seizure activity. These changes are likely to contribute to regionally different effects on local network excitability.