Andrew Charles

ATP Released from Astrocytes Mediates Glial Calcium Waves

Calcium waves represent a widespread form of intercellular communication. Although they have been thought for a long time to require gap junctions, we recently demonstrated that mouse cortical astrocytes use an extracellular messenger for calcium wave propagation. The present experiments identify ATP as a major extracellular messenger in this system. Medium collected from astrocyte cultures during (but not before) calcium wave stimulation contains ATP. The excitatory effects of medium samples and of ATP are blocked by purinergic receptor antagonists and by pretreatment with apyrase; these same purinergic receptor antagonists block propagation of electrically evoked calcium waves. ATP, applied at the concentration measured in medium samples, evokes responses that are qualitatively and quantitatively similar to those evoked by those medium samples. These data implicate ATP as an important transmitter between CNS astrocytes.

Intercellular signaling in glial cells: Calcium waves and oscillations in response to mechanical stimulation and glutamate

Intercellular Ca2+ signaling in primary cultures of glial cells was investigated with digital fluorescence video imaging. Mechanical stimulation of a single cell induced a wave of increased [Ca2+]i that was communicated to surrounding cells. This was followed by asynchronous Ca2+ oscillations in some cells. Similar communicated Ca2+ responses occurred in the absence of extracellular Ca2+, despite an initial decrease in [Ca2+]i in the stimulated cell. Mechanical stimulation in the presence of glutamate induced a typical communicated Ca2+ wave through cells undergoing asynchronous Ca2+ oscillations in response to glutamate. The coexistence of communicated Ca2+ waves and asynchronous Ca2+ oscillations suggests distinct mechanisms for intra- and intercellular Ca2+ signaling. This intercellular signaling may coordinate cooperative glial function.

Mechanisms and function of intercellular calcium signaling

Intercellular Ca2+ waves initiated by mechanical or chemical stimuli propagate between cells via gap junctions. The ability of a wide diversity of cells to display intercellular Ca2+ waves suggests that these Ca2+ waves may represent a general mechanism by which cells communicate. Although Ca2+ may permeate gap junctions, the intercellular movement of Ca2+ is not essential for the propagation of Ca2+ waves. The messenger that moves from one cell to the next through gap junctions appears to be IP3 and a regenerative mechanism for IP3 may be required to effect multicellular communication. Extracellularly mediated Ca2+ signaling also exists and this could be employed to supplement or replace gap junctional communication. The function of intercellular Ca2+ waves may be the coordination of cooperative cellular responses to local stimuli.

Intercellular calcium waves in glia

Glial cells are capable of communicating increases in [Ca2+]i from a single cell to many surrounding cells. These intercellular Ca2+ waves have been observed in glia in multiple different preparations, including dissociated brain cell cultures, glial cell lines, organotypic brain slice cultures, and intact retinal preparations. They may occur spontaneously, or in response to a variety of stimuli. Ca2+ waves occurring under different conditions in different preparations may have distinctive patterns of initiation and propagation, and distinctive pharmacological characteristics consistent with the involvement of different intracellular and intercellular signaling pathways. This paper presents original data supporting a combination of gap junction and extracellular messenger‐mediated signaling in mechanically induced glial Ca2+ waves. Additional new observations provide evidence that a rapidly propagated signal may precede the glial Ca2+ wave and may mediate rapid glial‐neuronal communication. This original data is discussed in the context of a review of the literature and current concepts regarding the potential mechanisms, physiological and pathological roles of this dynamic pattern of glial intercellular signaling. GLIA 24:39–49, 1998.

Cortical spreading depression and migraine

Cortical spreading depression (CSD), a slowly propagated wave of depolarization followed by suppression of brain activity, is a remarkably complex event that involves dramatic changes in neural and vascular function. Since its original description in the 1940s, CSD has been hypothesized to be the underlying mechanism of the migraine aura. Substantial evidence from animal models provides indirect support for this hypothesis, and studies showing that CSD is common in humans with brain injury clearly demonstrate that the phenomenon can occur in the human brain. Considerable uncertainty about the role of CSD in migraine remains, however, and key questions about how this event is initiated, how it spreads, and how it might cause migraine symptoms remain unanswered. This Review summarizes current concepts of CSD and its potential roles in migraine, and addresses ongoing studies aimed at a clearer understanding of this fundamental brain phenomenon.

Glia-neuron intercellular calcium signaling.

There is increasing evidence for bidirectional communication between glial cells and neurons. In this study, calcium signaling in primary glia/neuron cultures was investigated using video fluorescence imaging and fura-2. Glial cells in culture without neurons showed occasional spontaneous intracellular Ca2+ oscillations but not intercellular Ca2+ waves. By contrast, glial cells in culture with neurons showed frequent spontaneous Ca2+ oscillations as well as propagated intercellular Ca2+ waves. These spontaneous glial intercellular Ca2+ waves often emanated from sites of contact with neurons, but were only occasionally associated with increases in neuronal Ca2+. Mechanical stimulation of a single glial cell induced a glial intercellular Ca2+ wave which was similar in its temporal and spatial characteristics to spontaneous glial Ca2+ waves. Mechanically induced glial Ca2+ waves, but not spontaneous Ca2+ waves, evoked a transient increase in [Ca2+]i or a change in the pattern of spontaneous Ca2+ oscillations in a small percentage (< 10%) of neighboring neurons. Mechanical stimulation of a single neuron consistently evoked an intercellular Ca2+ wave in neighboring glial cells. These results suggest distinct mechanisms for direct glial-neuronal and neuronal-glial communication. These signaling pathways may play important roles in both function and pathology in the central nervous system.

GABA Has Excitatory Actions on GnRH-Secreting Immortalized Hypothalamic (GT1-7) Neurons

The effects of gamma-aminobutyric acid (GABA) on clonal gonadotropin-releasing hormone (GnRH)-secreting hypothalamic (GT1-7) neurons were investigated using patch-clamp and fura-2 imaging techniques. Local application of GABA (100 microM) to GT1-7 cells voltage-clamped in the whole-cell configuration immediately increased membrane conductance and noise consistent with activation of the GABAA receptor-Cl- channel complex. Depolarization activated transient Na+ currents which were abolished by tetrodotoxin (TTX; 0.5 microM), and more sustained Ca2+ currents. Under constant current conditions, GT1-7 cells fired spontaneous action potentials, and depending on the Cl- equilibrium potential, GABA either depolarized cells, causing a rapid activation of action potentials, or hyperpolarized cells. In order to determine the effect of GABA on intact cells, the cell-attached patch configuration was used to record extracellularly. Under these conditions, application of GABA (100 microM), but not the GABAB receptor agonist baclofen (10 microM), immediately evoked multiple action potentials. Measurement of [Ca2+]i using fluorescence video microscopy and fura-2 revealed spontaneous, transient, repetitive increases in [Ca2+]i which had a periodicity ranging from 1 to 60 s. These Ca2+ oscillations were abolished by TTX (1 microM) and by the removal of extracellular Ca2+. Application of GABA (1 and 10 microM) induced an immediate increase in [Ca2+]i in all cells and increased the frequency of Ca2+ oscillations in a dose-dependent manner. The GABA-induced increase in [Ca2+]i was abolished by bicuculline and by the removal of extracellular Ca2+, and was inhibited by TTX. Baclofen (1 microM) had no effect on [Ca2+]i. These results suggest that activation of GABAA receptors has an excitatory action on GnRH-secreting immortalized hypothalamic neurons caused by a Cl(-)-dependent depolarization. GABA has been reported to increase GnRH secretion; a direct stimulatory action of the neurotransmitter on GABAA receptors of GnRH-secreting hypothalamic neurons may be responsible for this effect.

Intercellular Calcium Waves in Neurons

Spontaneous intercellular Ca2+ waves were observed in groups of neurons in two different culture preparations: primary mouse cortical neurons and GT1-1 immortalized neurons. Waves of increased intracellular Ca2+ concentration propagated at rates of 100-200 microns/s over as many as 200 cells and were abolished by the removal of extracellular calcium, by nimodipine, by tetrodotoxin, and by the gap junction inhibitor octanol. A sister clone of the GT1 line, GT1-7 neurons, showed no intercellular Ca2+ waves and were found to have a significantly lower level of connexin26 mRNA than the GT1-1 line. Although we cannot definitively rule out a role for synaptic communication, we propose that intercellular Ca2+ waves in cultured neurons are generated by Ca2+ influx caused primarily by the propagation of depolarization via gap junctions. Intercellular Ca2+ signaling via gap junctions may represent an important mechanism for nonsynaptic neuronal signaling.

Extracellular Calcium Sensing by Glial Cells: Low Extracellular Calcium Induces Intracellular Calcium Release and Intercellular Signaling

Abstract: Glial cells in primary mixed cultures or purified astrocyte cultures from mouse cortex respond to reduced extracellular calcium concentration ([Ca2+]e) with increases in intracellular calcium concentration ([Ca2+]i) that include single‐cell Ca2+ oscillations and propagated intercellular Ca2+ waves. The rate and pattern of propagation of low [Ca2+]e‐induced intercellular Ca2+ waves are altered by rapid perfusion of the extracellular medium, suggesting the involvement of an extracellular messenger in Ca2+ wave propagation. The low [Ca2+]e‐induced Ca2+ response is abolished by thapsigargin and by the phospholipase antagonist U73122. The low [Ca2+]e‐induced response is also blocked by replacement of extracellular Ca2+ with Ba2+, Zn2+, or Ni2+, and by 100 µM La3+. Glial cells in lowered [Ca2+]e(0.1–0.5 mM) show an increased [Ca2+]i response to bath application of ATP, whereas glial cells in increased [Ca2+]e (10–15 mM) show a decreased [Ca2+]i response to ATP. These results show that glial cells possess a mechanism for coupling between [Ca2+]e and the release of Ca2+ from intracellular stores. This mechanism may be involved in glial responses to the extracellular environment and may be important in pathological conditions associated with low extracellular Ca2+ such as seizures or ischemia.

Substance P stimulates IL-1 production by astrocytes via intracellular calcium

There is increasing evidence that local substance P (SP) exacerbates peripheral inflammations, partly by stimulating production of inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha). SP may play similar roles in certain central nervous system inflammations. Multiple sclerosis plaques, for example, form around veins which are innervated by unmyelinated SP-containing fibers, and astrocytes in multiple sclerosis plaques stain for SP. We tested whether SP could stimulate IL-1 and TNF alpha production by cultured astrocytes and whether calcium was the second messenger in this process. We found that both SP and the calcium ionophore A23187 raised intracellular calcium ([Ca2+]i) and stimulated IL-1 production in astrocytes. SP also nonsignificantly increased TNF alpha production by astrocytes. Treatment with dibromo BAPTA/AM, an intracellular calcium buffer, blocked SP-induced IL-1 production. These findings indicate that SP induces IL-1 production by astrocytes and uses calcium as a second messenger. Our results indicate local SP may play a role in multiple sclerosis and certain other central nervous system inflammations.