Sensitivity of CaM kinase II to the frequency of Ca2+ oscillations.

The transduction of many cellular stimuli results in oscillations in the intracellular concentration of calcium ions (Ca2+). Although information is thought to be encoded in the frequency of such oscillations, no frequency decoder has been identified. Rapid superfusion of immobilized Ca2+- and calmodulin-dependent protein kinase II (CaM kinase II) in vitro showed that the enzyme can decode the frequency of Ca2+ spikes into distinct amounts of kinase activity. The frequency response of CaM kinase II was modulated by several factors, including the amplitude and duration of individual spikes as well as the subunit composition and previous state of activation of the kinase. These features should provide specificity in the activation of this multifunctional enzyme by distinct cellular stimuli and may underlie its pivotal role in activity-dependent forms of synaptic plasticity.

[Amniopatch to treat iatrogenic rupture of the fetal membranes]. / Traitement de la rupture iatrogène des membranes fœtales par "patch" amniotique.

OBJECTIVE: With the increased use of invasive fetal procedures, the number of patients facing postprocedure membrane rupture is increasing. We aimed to describe the use of platelets and fresh frozen plasma for sealing iatrogenic fetal membrane defects. PATIENTS AND METHODS: We describe the mechanisms of action of the amniopatch procedure as well as published experience. RESULTS: Amniopatch effectively sealed the fetal membranes in over two thirds of published cases (n=44). There is a risk of 17% of in utero fetal death, which may occur remotely from the procedure and is often unexplained. DISCUSSION AND CONCLUSION: In case of early onset but persistent amniotic fluid leakage following an invasive fetal procedure, amniopatch may be offered.

Intercellular calcium signaling between astrocytes and oligodendrocytes via gap junctions in culture.

To understand further how oligodendrocytes regulate brain function, the mechanism of communication between oligodendrocytes and other cell types needs to be explored. An important mode of communication between various cell types in the nervous system involves gap junctions. Astroglial cells are extensively connected through gap junctions forming the glial syncytium. Although the presence of gap junctions between oligodendrocytes and astrocytes have been well documented, evidence for gap junction-mediated calcium transfer between these two glial populations is still missing. To measure functional coupling between astrocytes and oligodendrocytes and to test whether this coupling is mediated by gap junctions we used laser photostimulation and monitored Ca(2+) propagation in cultures from transgenic animals in which oligodendrocytes express enhanced green fluorescent protein (eGFP). We show that waves of Ca(2+) spread from astrocytes to oligodendrocytes and that these waves are blocked by the broad-spectrum gap junction blocker carbenoxolone, but not the neuron-specific gap junction blocker quinine. We also show that the spread of Ca(2+) waves between astrocytes and oligodendrocytes is bi-directional. Thus, increase of Ca(2+) concentration in astrocytes triggered by surrounding neuronal activity may feed back onto different neuronal populations through oligodendrocytes.

Destruction of polymer growth substrates for cell cultures in two-photon microscopy.

The choice of the growth substrate for cell cultures used in fluorescence microscopy is guided by several factors including the type of cells studied and the type of microscopy used. Usually, cells can be cultured on either polymer or glass substrates. One type of polymer, termed Aclar, presents several attractive features: the adhesive properties are better than those of glass, the optical properties are comparable to those of glass, it is biochemically inert, unbreakable, flexible and has a high surface tension, convenient for seeding cells on the cover slip. However, here we show that when imaging with two-photon microscopy, which is based on a femtosecond pulsed laser source, local damage of the Aclar substrate occurs, starting at an average intensity of 10(5) W cm(-2) at the focal point and for exposure times insufficient to cause cell damage. This leads to the appearance of gas bubbles on cultures plated on Aclar cover slips, which perturb the imaging. By contrast, this phenomenon does not occur on borosilicate cover slips, probably because of their different physical (thermal conductivity, absorbance, melting point) and material homogeneity properties. Thus, for cell culture applications using pulsed lasers with high intensities, the use of glass is preferable to Aclar. The results also reveal that substrates can be more susceptible to thermal damage than the cells themselves.

Differential regulation of neuronal nicotinic ACh receptor subunit genes in cultured neonatal rat sympathetic neurons: specific induction of alpha 7 by membrane depolarization through a Ca2+/calmodulin-dependent kinase pathway.

We have examined the regulation of neuronal nicotinic ACh receptor (nAChR) genes and ACh-evoked currents by neonatal rat sympathetic neurons developing in culture. These neurons contain 5 nAChR transcripts: alpha 3, alpha 5, alpha 7, beta 2, and beta 4. When developing in culture, the neurons express 4 of these transcripts, alpha 3, alpha 5, beta 2, and beta 4, at levels similar to those in neurons developing in vivo: alpha 3 mRNA levels increase two- to threefold over the first week, whereas the levels for alpha 5, beta 2, and beta 4 remain essentially constant. In contrast, alpha 7 mRNA levels drop by 60-75% within the first 48 hr and remain low. We show that during the first week, the ACh-evoked current densities on these cultured neurons increase twofold and correlate well with the increase in alpha 3 mRNA levels. Depolarizing the neurons with 40 mM KCl for 1-2 d upregulates the alpha 7 gene; this specific change in alpha 7 mRNA level correlates with an increase in alpha-bungarotoxin (alpha-BTX) binding on the surface of the neurons. Depolarization has little effect on the expression of the other four transcripts, or on the magnitude or kinetics of the ACh-evoked currents. Furthermore, activators or inhibitors of protein kinase A (PKA), protein kinase C (PKC), or tyrosine kinase do not affect nAChR transcript levels in these cultured neurons. The effect of membrane depolarization on alpha 7 expression is a result of Ca2+ influx through L-type Ca2+ channels, and we show that alpha 7 is upregulated through a Ca2+/calmodulin-dependent protein kinase (CaM kinase) pathway. The identification of CaM kinase as a link between activity and neurotransmitter receptor expression may indicate a novel mechanism that underlies some forms of synaptic plasticity.

Agonist and toxin sensitivities of ACh-evoked currents on neurons expressing multiple nicotinic ACh receptor subunits.

1. We have investigated the pharmacological properties of functional nicotinic acetylcholine receptors (nAChRs) on neonatal rat sympathetic neurons from the superior cervical ganglion (SCG) to learn more about the subunit composition of these receptors. These neurons express five nAChR transcripts: alpha 3, alpha 5, alpha 7, beta 2, and beta 4; this finding suggests that SCG neurons may express several different, physiologically distinct, subtypes of nAChRs. 2. To identify potential subtypes, we have characterized currents evoked by different nicotinic agonists and determined their sensitivity to blockade by alpha-bungarotoxin (alpha-BTX) and by neuronal bungarotoxin (n-BTX). From dose-response curves, we find that the ED50 for both cytisine and dimethylphenylpiperazinium (DMPP) is 20 microM and for ACh is 52 microM. n-BTX blocks the ACh-gated currents rapidly, but the kinetics for n-BTX removal is dependent on the duration of the application: brief applications were quickly reversible, whereas prolonged applications took orders of magnitude longer to reverse. 3. Using fast (ms) agonist application, we observed no rapidly desensitizing currents despite the high levels of alpha 7 in these neurons, nor did we observe any currents that could be blocked by alpha-BTX. 4. Using Xenopus oocytes expressing alpha 7 receptors, we show that choline evokes a significant current that is blocked by alpha-BTX. In contrast, choline is much less potent on alpha 3 beta 4 receptors expressed in Xenopus oocytes. Choline can also act as a weak agonist for nAChRs on rat SCG neurons, but its evoked current is not blocked by alpha-BTX. 5. Our results indicate that, when measured at the macroscopic level, most functional nAChRs on SCG neurons behave as a uniform population of receptors, at least with respect to agonist activation and toxin blockade. In comparison with known receptors expressed in heterologous systems, the physiological properties of ACh-evoked currents on SCG neurons are most similar to receptors that have coassembled with both beta 2 and beta 4.

NGF induces neonatal rat sensory neurons to extend dendrites in culture after removal of satellite cells.

Vertebrate sensory neurons have a pseudo-unipolar morphology; their somata are covered by satellite cells and lack dendrites or synaptic contacts. However, when neonatal rat sensory neurons from the nodose ganglia develop in culture in absence of satellite cells and with NGF, they form synapses among themselves. In this study, we investigated whether neonatal rat nodose neurons express dendrites under the same culture conditions. We show by Lucifer yellow injection that nodose neurons remain typically unipolar when cocultured with their ganglionic satellite cells. However, when these neurons are cultured without satellite cells, virtually all neurons acquire a multipolar morphology. Moreover, when NGF is added to satellite cell-free cultures, several neurons extend dendrites; these processes stain positively for microtubule-associated protein-2. NGF induces a 17-fold increase in dendritic outgrowth after 3 weeks but has little effect on axon number. In addition, we find that the ability of nodose neurons to extend dendrites is developmentally regulated. Furthermore, in a combined morphological and electrophysiological study, using whole-cell voltage-clamp technique with Lucifer yellow in the recording solution, we demonstrate a positive correlation between the extent of dendritic outgrowth and the density of ACh currents, suggesting that these dendrites have ACh receptors. Our results indicate that neonatal rat nodose neurons are capable of extending dendrites and that extrinsic factors can induce or suppress their extension. In addition, the results suggest that these dendrites may act as principal post-synaptic structures for synapse formation that occurs in these cultures.

Interaction with the NMDA receptor locks CaMKII in an active conformation.

Calcium- and calmodulin-dependent protein kinase II (CaMKII) and glutamate receptors are integrally involved in forms of synaptic plasticity that may underlie learning and memory. In the simplest model for long-term potentiation, CaMKII is activated by Ca2+ influx through NMDA (N-methyl-D-aspartate) receptors and then potentiates synaptic efficacy by inducing synaptic insertion and increased single-channel conductance of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. Here we show that regulated CaMKII interaction with two sites on the NMDA receptor subunit NR2B provides a mechanism for the glutamate-induced translocation of the kinase to the synapse in hippocampal neurons. This interaction can lead to additional forms of potentiation by: facilitated CaMKII response to synaptic Ca2+; suppression of inhibitory autophosphorylation of CaMKII; and, most notably, direct generation of sustained Ca2+/calmodulin (CaM)-independent (autonomous) kinase activity by a mechanism that is independent of the phosphorylation state. Furthermore, the interaction leads to trapping of CaM that may reduce down-regulation of NMDA receptor activity. CaMKII-NR2B interaction may be prototypical for direct activation of a kinase by its targeting protein.