Anti-phase calcium oscillations in astrocytes via inositol (1, 4, 5)-trisphosphate regeneration.

Observations in cultured mouse astrocytes suggest anti-phase synchronization of cytosolic calcium concentrations in nearest neighbor cells that are coupled through gap junctions. A mathematical model is used to investigate physiologic conditions under which diffusion of the second messenger inositol (1, 4, 5)-trisphosphate (IP(3)) through gap junctions can facilitate synchronized anti-phase Ca(2+) oscillations. Our model predicts anti-phase oscillations in both cytosolic calcium and IP(3) concentrations if (a) the gap junction permeability is within a window of values and (b) IP(3) is regenerated in the astrocytes via, e.g. phospholipase C(delta). This result sheds new light on the current dispute on the mechanism of intercellular calcium signaling. It provides indirect evidence for a partially regenerative mechanism as the model excludes anti-phase synchrony in the absence of IP(3) regeneration.

Human epileptic astrocytes exhibit increased gap junction coupling.

Fluorescence Recovery After Photobleach (FRAP) was used to quantify astrocyte gap junction coupling from tissues surgically resected from medically intractable epilepsy patients. Mesial temporal lobe cases provided hippocampus, surrounding hyperexcitable parahippocampus and normal cortex for culture. Cortical tumor cases yielded astrocytoma proper, cortex margins with normal EEG activity, and hyperexcitable cortex. Cells isolated from cortex surrounding astrocytomas and the parahippocampus surrounding the hippocampus showed an increase in glutamate-induced Ca2+ oscillations and intercellular Ca2+ waves. Gap-junction coupling was more pronounced in cells isolated from hyperexcitable tissue than from normal tissues as judged by their faster and more complete fluorescence recovery from laser bleach [FRAP]. This data suggests that intractable epilepsy may be associated with alterations in glial gap junction coupling.

Astrocytes exhibit regional specificity in gap-junction coupling.

Astrocytes are coupled to each other via gap-junctions both in vivo and in vitro. Gap-junction coupling is essential to a number of astrocyte functions including the spatial buffering of extracellular K+ and the propagation of Ca2+ waves. Using fluorescence recovery after photo-bleach, we quantitatively assayed and compared the coupling of astrocytes cultured from six different central nervous system (CNS) regions in the rat: spinal cord, cortex, hypothalamus, hippocampus, optic nerve, and cerebellum. The degree of fluorescence recovery (% recovery) and time constant of recovery (tau) served as quantitative indicators of coupling strength. Gap-junction coupling differed markedly between CNS regions. Coupling was weakest in astrocytes derived from spinal cord (43% recovery, tau approximately 400 s) and strongest in astrocytes from optic nerve (91% recovery, tau approximately 226 s) and cerebellum (95% recovery, tau approximately 100 s). As indicated by the degree of recovery, coupling strength among CNS regions could be ranked as follows: spinal cord < cortex < hypothalamus < hippocampus = optic nerve = cerebellum. Gap-junction coupling also differed between CNS regions with respect to its sensitivity to inhibition by the uncoupling agent octanol. Kd values for 50% inhibition by octanol ranged from 188 microM in spinal cord astrocytes to 654 microM in hippocampal astrocytes. Sensitivity of gap-junctions to octanol could be ranked as follows: spinal cord = cortex = hypothalamus > cerebellum > optic nerve > hippocampus. The observed differences in coupling indicate differences in the number of gap-junction connections in astrocytes cultured from the six CNS regions. These differences may reflect the adaptation of astrocytes to varying functional requirements in different CNS regions.

Glutamate-induced calcium signaling in astrocytes.

Astrocytes respond to the excitatory neurotransmitter glutamate with dynamic spatio-temporal changes in intracellular calcium [Ca2+]i. Although they share a common wave-like appearance, the different [Ca2+]i changes--an initial spike, sustained elevation, oscillatory intracellular waves, and regenerative intercellular waves--are actually separate and distinct phenomena. These separate components of the astrocytic Ca2+ response appear to be generated by two different signal transduction pathways. The metabotropic response evokes an initial spatial Ca2+ spike that can propagate rapidly from cell to cell and appears to involve IP3. The metabotropic response can also produce oscillatory intracellular waves of various amplitudes and frequencies that propagate within cells and are sustained only in the presence of external Ca2+. The ionotropic response, however, evokes a sustained elevation in [Ca2+]i associated with receptor-mediated Na+ and Ca2+ influx, depolarization, and voltage-dependent Ca2+ influx. In addition, the ionotropic response can lead to regenerative intercellular waves that propagate smoothly and nondecrementally from cell to cell, possibly involving Na+/Ca2+ exchange. All these astrocytic [Ca2+]i changes tend to appear wave-like, traveling from region to region as a transient rise in [Ca2+]i. Nevertheless, as our understanding of the cellular events that underlie these [Ca2+]i changes grows, it becomes increasingly clear that glutamate-induced Ca2+ signaling is a composite of separate and distinct phenomena, which may be distinguished not based on appearance alone, but rather on their underlying mechanisms.

Calcium excitability and oscillations in suprachiasmatic nucleus neurons and glia in vitro.

Converging lines of evidence suggest that the hypothalamic suprachiasmatic nucleus (SCN) is the site of the endogenous biological clock controlling mammalian circadian rhythms. To study the calcium responses of the cellular components that make up the clock, computer-controlled digital video and confocal scanning laser microscopy were used with the Ca2+ indicator dye fluo-3 to examine dispersed SCN cells and SCN explants with repeated sampling over time. Ca2+ plays an important second messenger role in a wide variety of cellular mechanisms from gene regulation to electrical activity and neurotransmitter release, and may play a role in clock function and entrainment. SCN neurons and astrocytes showed an intracellular Ca2+ increase in response to glutamate and 5-HT, two major neurotransmitters in afferents to the SCN. Astrocytes showed a marked heterogeneity in their response to the serial perfusion of different transmitters; some responded to both 5-HT and glutamate, some to neither, and others to only one or the other. Under constant conditions, most neurons showed irregular temporal patterns of Ca2+ transients. Expression of regular neuronal oscillations could be blocked by the inhibitory transmitter GABA. Astrocytes, on the other hand, showed very regular rhythms of cytoplasmic Ca2+ concentrations with periods ranging from 7 to 20 sec. This periodic oscillation could be initiated by in vitro application of glutamate, the putative neurotransmitter conveying visual input to the SCN critical for clock entrainment. Long-distance communication between glial cells, seen as waves of fluorescence moving from cell to cell, probably through gap junctions, was induced by glutamate, 5-HT, and ATP. These waves increased the period length of cellular Ca2+ rises to 45-70 sec. Spontaneously oscillating cells were common in culture medium, serum, or rat cerebrospinal fluid, but rare in HEPES buffer. One source for cytoplasmic Ca2+ increases was an influx of extracellular Ca2+, as seen under depolarizing conditions in about 75% of the astroglia studied. All neurotransmitter-induced Ca2+ fluxes were not dependent on voltage changes, as Ca2+ oscillations could be initiated under both normal and depolarizing conditions. Since neurotransmitters could induce a Ca2+ rise in the absence of extracellular Ca2+, the mechanisms of ultradian oscillations appear to depend on cycles of intracellular Ca2+ fluxes from Ca(2+)-sequestering organelles into the cytoplasm, followed by a subsequent Ca2+ reduction. In the adult SCN, fewer astrocytes are found than neurons, yet astrocytes frequently surround glutamate-immunoreactive axons in synaptic contact with SCN dendrites, isolating neurons from each other while maintaining contact with other astrocytes by gap junctions.(ABSTRACT TRUNCATED AT 400 WORDS)

Ca2+ and filopodial responses to glutamate in cultured astrocytes and neurons.

Neurons and glia exhibit complex homeostatic interactions via shared extracellular space which can involve metabolites, inorganic ions, and neurotransmitters. Focal application of glutamate to both human and rat central nervous system astrocytes in primary culture produced a rapid, transient increase in both cytoplasmic and nuclear Ca2+. These Ca2+ waves can propagate at up to 15-20 micron/s for long distances (millimetres) through the astrocyte syncitium. Oscillatory Ca2+ signals were frequently observed under control conditions and were enhanced by glutamate application. These Ca2+ signals were paralleled by rapid extensions of filopodia from the astrocyte cell margin and apical surface near the point of glutamate application. Focal application of glutamate to rat hippocampal neurons also elicited rapid, transient increases in intracellular Ca2+. Levels of Ca2+ signals were consistently two- to three-fold greater in pyramidal neurons cultured from CA1 than in those from CA3. Filopodial extension was extensive in CA1 neurons, but rare in CA3 neurons, and in either case observable only during the first few days of primary culture. Diversity of glial and neuronal responses to binding the glutamate receptors may reflect their roles in homeostatic interactions.

Ca2+ waves in astrocytes.

The glial cell is the most numerous cell type in the central nervous system and is believed to play an important role in guiding brain development and in supporting adult brain function. One type of glial cell, the astrocyte also may be an integral computational element in the brain since it undergoes neurotransmitter-triggered signalling. Here we review the role of the astrocyte in the central nervous system, emphasizing receptor-mediated Ca2+ physiology. One focus is the recent discovery that the neurotransmitter glutamate induces a variety of intracellular Ca2+ changes in astrocytes. Simple Ca2+ spikes or intracellular Ca2+ oscillations often appear spatially uniform. However, in many instances, the Ca2+ rise has a significant spatial dimension, beginning in one part of the cell it spreads through the rest of the cell in the form of a wave. With high enough agonist concentration an astrocyte syncitium supports intercellular waves which propagate from cell to cell over relatively long distances. We present results of experiments using more specific pharmacological glutamate receptor agonists. In addition to describing the intercellular Ca2+ wave we present evidence for another form of intercellular signalling. Some possible functions of a long-range glial signalling system are also discussed.

Na(+)-current expression in rat hippocampal astrocytes in vitro: alterations during development.

1. With the use of whole-cell patch-clamp recording. Na(+)-current expression was studied in hippocampal astrocytes in vitro, individually identified by filling with Lucifer yellow (LY) and staining for glial fibrillary acidic protein (GFAP) and vimentin. 2. The proportion of astrocytes that express Na+ currents in rat hippocampal cultures changes during development in vitro and decreases from approximately 75% at day 1 to approximately 30% after 10 days in culture. 3. The sodium currents expressed in astrocytes can be differentiated into two types on the basis of kinetics. At early times in culture the time course of Na+ currents is fast in both onset and decay with an average decay time constant of 1.27 ms, whereas after 6 days Na+ currents become comparatively slow and decayed with an average time constant of 1.86 ms. 4. As with the time-course of Na+ currents, the two age groups of astrocytes (i.e., days 1-5 and day 6 and older) differ with respect to their steady-state inactivation characteristics. Early after plating and up to day 5, the midpoint of the steady-state inactivation curve is close to -60 mV, as also observed in hippocampal neurons of various ages; in contrast, after 6 days in culture the curve is shifted by approximately 25 mV toward more hyperpolarized potentials with a midpoint close to -85 mV. 5. In contrast to h infinity-curves, current-voltage (I-V) curves of Na(+)-current activation were identical in all astrocytes studied and did not change with time in culture. 6. In astrocytes expressing Na+ currents, current densities (average of 35 pA/pF on day 1) decreased throughout the first 5 days and were almost abolished around days 4 and 5 in culture. Beginning on day 6, however, current densities increased again and maintained a steady level (average of 14 pA/pF) for the duration of the time period in culture (20 days). This biphasic time course closely correlates with the time course of changes in Na(+)-current kinetics and steady-state inactivation. 7. These data suggest that Na+ currents in cultured hippocampal astrocytes show characteristic changes with increasing time in culture. During the first 4-5 days in culture, hippocampal astrocytes display Na+ currents with properties similar to those of hippocampal neurons. Our data further suggest that Na+ currents with distinctive, "glial-type" characteristics are only expressed in hippocampal astrocytes after 6 days in culture.

Tubulovesicular processes emerge from trans-Golgi cisternae, extend along microtubules, and interlink adjacent trans-golgi elements into a reticulum.

Morphological dynamics and membrane transport within the living Golgi apparatus of astrocytes labeled with NBD-ceramide were imaged using both electronically enhanced fluorescence video and laser confocal microscopy. In time-lapse recordings, continuous tubulovesicular processes are observed to emerge from trans-Golgi elements and extend along microtubules at average rates of 0.4 microns/s. In addition, discrete fluorescent particles are observed to emerge from the trans-Golgi and subsequently migrate along microtubules at comparable velocities. Frequently, tubulovesicular processes form stable connections that interlink adjacent trans-Golgi elements into an extensive reticulum. Laser photobleaching-recovery experiments reveal that tubulovesicular processes can provide direct pathways for the diffusion of membrane lipids between joined trans-Golgi elements. These results suggest that microtubule-based transport and membrane fusion can operate to interconnect certain cisternal membranes of adjacent Golgi elements within the cell.

Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling.

The finding that astrocytes possess glutamate-sensitive ion channels hinted at a previously unrecognized signaling role for these cells. Now it is reported that cultured hippocampal astrocytes can respond to glutamate with a prompt and oscillatory elevation of cytoplasmic free calcium, visible through use of the fluorescent calcium indicator fluo-3. Two types of glutamate receptor--one preferring quisqualate and releasing calcium from intracellular stores and the other preferring kainate and promoting surface-membrane calcium influx--appear to be involved. Moreover, glutamate-induced increases in cytoplasmic free calcium frequently propagate as waves within the cytoplasm of individual astrocytes and between adjacent astrocytes in confluent cultures. These propagating waves of calcium suggest that networks of astrocytes may constitute a long-range signaling system within the brain.

The excitatory neurotransmitter glutamate causes filopodia formation in cultured hippocampal astrocytes.

Can neurons induce surrounding glia to provide a more favorable microenvironment? Synapses and nerve growth cones have been shown to release neurotransmitters (Hume et al. Nature 1983;305:632-634; Kater et al. Trends Neurosci. 1988;11:315-321; Young and Poo Nature 1983;305:634-637) providing a possible mechanism for this type of control. The excitatory neurotransmitter glutamate induces an increase in the number of filopodia on the surface of astrocytes cultured from the neonatal rat hippocampus. This seems to be associated with a receptor-mediated event that is activated to a lesser degree by the quisqualate and kainate, but not NMDA receptors. In addition, time-lapse video recordings have revealed a rapid extension of filopodia from the apical margins of cells treated with glutamate. The apical margins of glutamate-treated cells studied with electron microscopy contained dense cortical actin networks that are devoid of microtubules. Coated pits are often seen to invaginate from the apical membrane in the vicinity of filopodia. A receptor-binding step may be followed by a rapid reorganization of cortical actin resulting in actin-containing filopodia. This process may be mediated by inositol lipid hydrolysis. Pyramidal neurons settled on glial cultures induced filopodia to form around the entire margin of growth cones and neurite tips suggesting that these events might occur in situ.

Sequential development of a conjunctival basophil hypersensitivity lesion in guinea pig.

We investigated the cellular profile of inflammatory infiltrate during development of a guinea pig conjunctival basophil hypersensitivity lesion at 1, 6, 24, 48, and 72 hr following injection of keyhole limpet hemocyanin (KLH). Neutrophils reached maximal accumulation at 6 hr. Eosinophils and basophils reached maximal accumulation at 24 hr. Basophil numbers remained high at 48 hr and were lower at 72 hr. The number of stromal monocytes did not vary significantly from PBS-injected tissues at any time. Mast cell number decreased at 24 hr following antigen challenge. Morphologic evidence for intense secretory activity was seen in cells at the stromal reaction site. Secretion channels and evidence of intense degranulation was seen from electron microscopy of neutrophils, eosinophils, and basophils. Inflammatory cells migrate through the basement membrane into the conjunctival epithelium. After maximal accumulation there was a decrease in the number of inflammatory cell types in the epithelium.

Conjunctival basophil hypersensitivity in the guinea pig.

We have induced a basophil hypersensitivity reaction in the upper tarsal conjunctiva of the guinea pig by methods that induce a comparable basophil hypersensitivity reaction in the flank. The inflammatory cell infiltrate in this reaction contained large numbers of basophils and eosinophils with accompanying neutrophils and monocytes. Ocular tissue can serve as a priming site for systemic immunization and also for elicitation of a secondary flare after challenge with antigen. Very few inflammatory cells were observed in the cutaneous epithelium of either primary or secondary flares. In contrast, the mucosal stroma and epithelium contained large numbers of inflammatory cells (basophils, eosinophils, and neutrophils), suggesting directed cellular movement onto the ocular surface. The lesion of ocular basophil hypersensitivity in the guinea pig has features in common with two human eye diseases, vernal conjunctivitis and contact lens-associated giant papillary conjunctivitis. We hypothesize that the acute basophil hypersensitivity reactions of the conjunctiva are transformed into chronic inflammatory and proliferative states in vernal conjunctivitis and giant papillary conjunctivitis.

Conjunctival basophil hypersensitivity lesions in guinea pigs. Analysis of upper tarsal epithelium.

The upper tarsal conjunctival epithelium was analyzed for inflammatory cell profile and accompanying morphological changes in a guinea pig system with histopathology resembling two human ocular diseases: vernal conjunctivitis and contact lens-associated giant papillary conjunctivitis (GPC). Female Hartley strain guinea pigs were immunized intradermally on day 0 with 200 micrograms keyhole limpet hemocyanin (KLH) and challenged on day 6 with varying doses of KLH by injection beneath the conjunctival epithelium of one lid and phosphate-buffered saline in the contralateral lid. Tissues containing the reaction site were examined by light microscopy. The 50 micrograms dose of KLH elicited the maximal accumulation of basophils and eosinophils. These values were significantly higher than in the PBS-injected control. Injection of KLH, PBS, or insertion of a sterile needle into unsensitized animals, and uninjected tissue served as additional controls. Neutrophils were significantly higher in the epithelium of the traumatized tissue (repeated needle insertions) than in the uninjected control. Basophils and mast cells were rarely found in the epithelium of unsensitized animals. Epithelial thickening, quantified by a Zeiss Videoplan2 Image Analysis system (Zeiss, West Germany), was greatest in the traumatized tissue, followed by the KLH-challenged tissue of sensitized animals. These values were significantly greater than that of the PBS-injected lid or of naive animals, uninjected or KLH-injected. These results indicate that epithelial changes can be induced by both antigen and trauma. Such epithelial changes may have a role in both vernal conjunctivitis and giant papillary conjunctivitis.

Characterization of a localized basophil hypersensitivity lesion in guinea pig conjunctiva.

Basophils accumulate in response to antigen challenge in cutaneous basophil hypersensitivity (CBH) reactions. Two ocular diseases, vernal conjunctivitis and contact-lens-associated conjunctivitis, are also characterized by this histopathology. We have refined a model previously developed in guinea pig conjunctiva by precisely defining the site of antigen injection and correlating the site with the clinical and histologic changes. Guinea pigs were primed by an intradermal injection of keyhole limpet hemocyanin (KLH) in the flank and challenged (Day 6) by injection of a small bolus of KLH just under the conjunctival epithelium. Twenty-four hours later histologic examination showed a perivascular infiltrate of inflammatory cells containing large numbers of basophils. Eosinophils, neutrophils, macrophages, and mast cells were also seen. Serial sections of the reaction site showed discrete boundaries. At all sites examined in antigen-challenged tissues, there were significantly more basophils than in control-injected conjunctiva. Insertion of a sterile needle or injection of PBS or KLH into normal conjunctiva induced a significant increase in neutrophils and some macrophages. Injection of graded doses of antigen into the conjunctiva of primed animals, resulted in a dose-dependent increase in basophils up to 50 micrograms KLH (optimal dose).

Gender-related differences in the morphology of the lacrimal gland.

Previous research has demonstrated that distinct, gender-related differences exist in the morphology of the rat lacrimal gland. The purpose of the present study was to determine whether this sexual dimorphism is unique to the rat, or extends as well to other species. Lacrimal glands were collected from adult male and female rats, mice, guinea pigs, rabbits and humans (biopsies). Tissues were processed for light microscopy and examined with a Zeiss Videoplan II image analysis system. For morphometric determinations, we measured the area of approximately 50 glandular acini per animal for a total count of greater than 244 acini per gender per species. Our results demonstrated that significant gender-related differences exist in lacrimal glands of rats, mice, guinea pigs, rabbits, and humans. In all species analyzed, acinar area in lacrimal glands of males was larger than that of females. These findings suggest that gender differences in lacrimal gland morphology may be a general phenomenon in a variety of species.