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Spontaneous calcium activity of neural progenitors is largely dependent on a paracrine signaling mechanism involving release of ATP and activation of purinergic receptors. Although it is well documented that, in mature astrocytes, cytokines modulate the expression levels of certain purinergic receptors, nothing is known about their impact during early stages of development. Here we provide evidence that conditioned medium from activated microglia and interleukin-1β, but not tumor necrosis factor-α, decrease the frequency of calcium oscillations and reduce the rate of in vitro migration of astrocyte progenitors. Such alterations were due to changes in activity of two purinergic P2 receptors, and not to the amount of released ATP. These results indicate that interleukin-1β plays an important role during early stages of CNS development, modulating calcium signaling and cell migration.

Connexin43 (Cx43) is the principal gap junction protein between astrocytes in the neonatal brain and also interconnects neural precursor cells during CNS development. In an attempt to understand global effects of expression of the Cx43 gap junction gene on development and function of the nervous system, we have compared gene expression patterns in cultured astrocytes and brains from wildtype mice with those in which Cx43 is deleted as well as in spinal cords of experimental autoimmune encepahlomyelitis (EAE) mice. One surprising result obtained from high densitity mouse cDNA studies was the large number of genes that were statistically altered in mice with decreased expression of Cx43. These altered genes encode proteins with a wide range of functions within cells, and thus deletion of normal gap junction expression appears to result in globally altered glial functions in addition to disruption of intercellular communication. Here we discuss those results in the context of the strategies and data analysis paradigms that we are using in such studies.

The electrical properties of Aplysia brasiliana myogenic heart were evaluated. Two distinct types of action potentials (APs) were recorded from intact hearts, an AP with a slow rising phase followed by a slow repolarizing phase and an AP with a ‘fast’ depolarizing phase followed by a plateau. Although these two APs differ in their rates of depolarization (2.2×0.3 V/s), both APs were abolished by the addition of Co 2+ , Mn 2+ and nifedipine or by omitting Ca 2+ from the external solution. These data suggest that a Ca 2+ inward current is responsible for the generation of both types of APs. Two outward currents activated at −40 mV membrane potential were prominent in isolated cardiac myocytes: a fast activating, fast inactivating outward current similar to the A-type K + current and a slow activating outward current with kinetics similar to the delayed rectifier K + current were recorded under voltage clamp conditions. Based on the effects of 4-AP and TEA on the electrical properties of ventricular myocytes, we suggest that the fast kinetic outward current substantially attenuates the peak values of the APs and that the slow activating outward current is involved on membrane repolarization.

Astrocytes produce and supply metabolic substrates to neurons through gap junction-mediated astroglial networks. However, the role of astroglial metabolic networks in behavior is unclear. Here, we demonstrate that perturbation of astroglial networks impairs the sleep-wake cycle. Using a conditional Cre-Lox system in mice, we show that knockout of the gap junction subunit connexin 43 in astrocytes throughout the brain causes excessive sleepiness and fragmented wakefulness during the nocturnal active phase. This astrocyte-specific genetic manipulation silenced the wake-promoting orexin neurons located in the lateral hypothalamic area (LHA) by impairing glucose and lactate trafficking through astrocytic networks. This global wakefulness instability was mimicked with viral delivery of Cre recombinase to astrocytes in the LHA and rescued by in vivo injections of lactate. Our findings propose a novel regulatory mechanism critical for maintaining normal daily cycle of wakefulness and involving astrocyte-neuron metabolic interactions.

In this issue, Chanson et al 1 present data that document the failure of airway cells expressing the mutant form of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene to down-regulate gap junction connectivity after treatment with the cytokine tumor necrosis factor-α (TNF-α). However, this response could be restored by transfecting in the wild-type CFTR gene, suggesting that a functional CFTR gene is necessary for regulation of gap junctional communication by TNF-α. The authors postulate that the persistence of gap-junction connectivity in the inflamed CF airway epithelium permits the intercellular diffusion of signaling molecules that serve to activate neighboring cells, thus contributing to the excessive inflammatory response characteristic of the CF airway epithelium. Although these conclusions are based only on estimation of gap-junction connectivity using transfer of the gap-junction permeant dye Lucifer Yellow, the data fit well with several studies that have documented regulation of gap junction connectivity in a number of different cell types by proinflammatory cytokines such as TNF-α and interleukin-1β (IL-1β). 2-8 These interesting observations raise the questions of how inflammatory cytokines might regulate gap junction connectivity and in what way expression of the mutant CFTR gene could influence this response.

We investigated the possible role of ion channels and transporters in cell volume control using Aplysia brasiliana ventricular tissues exposed to a 26% hyposmotic shock, by assessing changes in wet weight, intracellular water and ionic contents. Thirty minutes after the shock, the wet weight of isolated ventricles increase about 20% above control levels and then attain near original weight within 60 min after the shock. At the time when the wet weight returned to control values, intracellular water and KCl contents are decreased by 22 and 20%, respectively. The K channel blockers, 4-AP and TEA, but not the cotransport blockers, hydrochlorothiazide and furosemide, greatly affect the magnitude of wet weight gain and the time course of weight recovery, indicating that KCl loss occur through conductive pathways. Intracellular recordings performed on ventricular myocytes during exposure to the osmotic shock showed an immediate membrane hyperpolarization and blockade of spontaneous electrical activity; diastolic membrane potential recover over time and spontaneous action potentials are completely restored 60 min after the hyposmotic shock. Because significant weight loss is observed during the exposure of ventricular tissues to 26% hypo-ionic, but isosmotic saline, it is suggested that ventricular volume restoration is accomplished by two distinct but simultaneously occurring processes: a volume-dependent and a volume-independent mechanism. Because wet weight restoration is completely prevented by exposing ventricular tissue to a Ca 2 -free hyposmotic solution, we postulate that both processes involved in A. brasiliana ventricular weight restoration are Ca 2 -dependent mechanisms. © 2000 Elsevier Science Inc. All rights reserved.

The osmoregulatory capability and swimming behavior of Liriope tetraphylla (Chamisso and Eysenhardt) were analyzed under hyposmotic conditions. These hydromedusae are shown to be osmoconforming animals that are not able to maintain constant osmotic and ionic concentrations of their body fluids when exposed to anisosmotic media. Swimming behavior (as measured by the pulsation rate and the swimming index) is transiently affected by osmotic shock, being restored 30 min after exposure. However, even when swimming activity was restored, two different swimming patterns were observed depending on the hypotonic period. Thirty minutes exposure led to an increase of the quiescent period with no changes on the swimming burst characteristics while five hours exposure to hypotonicity shortened both the quiescent period and burst durations. Such behavioral modifications were related to the changes in body fluid ionic composition (mainly to extracellular K+ levels) that occur during the osmotic adjustment process. The fact that swimming activity was impaired during hypoionic shocks and this condition caused morphological changes in the animals suggests that these early metazoans are endowed with mechanisms to compensate for ionic gradient changes induced by hyposmotic, but not for the ones caused by hypoionic condition.

The effects of either cation removal or ionic channel blockade were determined on the dose-response curve (DRC) to PCH (pigment-concentrating hormone) in Macrobrachium potiuna erythrophores. In sodium-, potassium- and calcium-free salines, the pigment-aggregating responses to PCH were depressed; in the former condition, maximal aggregation was not achieved and the slope of the regression curve determined from the DRC was significantly different from control. Tetrodotoxin, verapamil or tetraethylammonium (TEA) treatments also diminished the pigment-aggregating responses to PCH, and the slopes of the regression curves were different from control in the presence of 10 −6 M verapamil or 10 −6 M TEA. Interestingly, the DRC determined in the absence of both sodium and calcium ions was not significantly different from control. When verapamil was applied in sodium-free conditions, maximal aggregation was prevented. The erythrophore resting membrane potential ranged from −62 mV to −78 mV and did not vary during PCH-induced pigment aggregation as compared to the control. Our results suggest that transient modifications of potassium equilibrium potential may interfere with PCH signal transduction, revealing a more relevant role of potassium in the process, and that a sodium influx and an intracellular calcium mobilization are necessary to maintain a cytosolic balance between the ions for normality of PCH-induced responses.

1. 1. Attempts to detect cholinesterase activity in body wall homogenates of the sea anemone Bunodosoma caissarum were made using gasometric and colorimetric techniques. 2. 2. No cholinesterase activity was found in the homogenates using either of these techniques. 3. 3. Control experiments for cholinesterase activity were performed using crude homogenates from the polychaete Eurythoe complanata and the marine fish Bathygobius soporator. 4. 4. An evolutionary line of acetylcholine-meditated transmission at the junction level is discussed.

1. 1. In an attempt to analyse the cholinergic pre-synaptic modulatory hypothesis (Scemes and Mendes, 1986), the effects of magnesium and calcium ions and of cholinergic agonists and antagonists on the behavior of Liriope tetraphylla were studied. 2. 2. The medusae were submitted to the action of atropine in the presence of magnesium and different concentrations of calcium. 3. 3. The enhancement of the responses to atropine caused by acetylcholine and nicotine agree with the idea of the presence of nicotinic excitatory and muscarinic inhibitory receptors modulating the release of a still unknown neurotransmitter at the neuromuscular junction of the radial pathway.