The presence of adrenergic and histaminergic receptors in Bergmann glial cells from cerebellar slices from mice aged 20-25 days was determined using fura-2 Ca2+ microfluorimetry. To measure the cytoplasmic concentration of Ca2+ ([Ca2+]i), either individual cells were loaded with the Ca2+-sensitive probe fura-2 using the whole-cell patch-clamp technique or slices were incubated with a membrane permeable form of the dye (fura-2/AM) and the microfluorimetric system was focused on individual cells. The monoamines adrenalin and noradrenalin (0.1-10 microM) and histamine (10-100 microM) triggered a transient increase in [Ca2+]i. The involvement of the alpha1-adrenoreceptor was inferred from the observations that monoamine-triggered [Ca2+]i responses were locked by the selective alpha1-adreno-antagonist prazosin and were mimicked by the alpha1-adreno-agonist phenylephrine. The monoamine-induced [Ca2+]i signals were not affected by beta- and alpha2-adrenoreceptor antagonists (propranolol and yohimbine), and were not mimicked by beta- and alpha2-adrenoreceptor agonists (isoproterenol and clonidine). Histamine-induced [Ca2+]i responses demonstrated specific sensitivity to only H1 histamine receptor modulators. [Ca2+]i responses to monoamines and histamine did not require the presence of extracellular Ca2+ and they were blocked by preincubation of slices with thapsigargin (500 nM), indicating that the [Ca2+]i responses were recorded after application of aspartate, bradykinin, dopamine, GABA, glycine, oxytocin, serotonin, somatostatin, substance P, taurine or vasopressin. We conclude that cerebellar Bergmann glial cells are endowed with alpha1-adrenoreceptors and H1 histamine receptors which induce the generation of intracellular [Ca2+]i signals via activation of Ca2+ release from inositol-1,4,5-trisphosphate-sensitive intracellular stores.
To evaluate the expression of components of the endothelin (ET) system in single Purkinje neurons and Bergmann glial cells in situ, patch-clamp recording was combined with a multiplex RT-PCR approach. Cerebellar slices were rapidly isolated from 20- to 28-day-old mice. Cells were characterized morphologically and electrophysiologically and cell contents were aspirated and immediately reverse-transcribed. The cDNA was used as a template in a multiplex PCR reaction containing primers specific for ET-1, ET-2, and ET-3, ET-converting enzyme 1 (ECE-1) and ECE-2, and ETA and ETB receptors. The resulting PCR products were used as templates in a second PCR reaction containing only one pair of nested primers. Specific single bands were obtained from positive cells, which was confirmed by DNA sequencing of the PCR products. Of the 25 Purkinje neurons assayed, 84% were positive for ECE-1 mRNA and 68% for ECE-2 mRNA. No ET and ETA receptor mRNAs were detected, and only one cell was positive for ETB receptor mRNA. In Bergmann glial cells, ETB receptor mRNA was predominant. A total of 68% of the 25 cells assayed were positive. Sixteen percent were positive for ETA receptor mRNA, 8% for ECE-1 mRNA, and 12% for ECE-2 mRNA. Again, no ET mRNAs were detected. These results confirm the role of the ETB receptor in Bergmann glial cells and provide evidence for expression of ECE-1 and ECE-2 in Purkinje neurons.
Hypoxic damage to the central nervous system triggers morphological and functional responses in astrocytes (AC). When isolated from the cerebral microenvironment and placed in cell culture, hypoxia promotes astrocytic alterations indicative of dedifferentiation. To investigate the effect of hypoxia on AC morphology, we performed studies on cultured AC pretreated with dibutyrylcAMP. These treated cells resemble AC in vivo, assuming a stellate morphology as observed by phase contrast microscopy. Exposure to hypoxia (0.3% oxygen tension) for 24 h induced a flat and polygonal shape in most of the cells as opposed to normoxic controls. A candidate factor to mediate this response is endothelin-1 (ET-1), a peptide produced by ischemic AC in vivo. The role of the astrocytic ET system during hypoxia, therefore, was investigated. Exogenous application of ET-1 mimicked the effect of hypoxia on the astrocytic morphology. The effects of hypoxia and exogenous ET-1 on the morphology were inhibited by the nonselective ET receptor antagonists PD142893 and PD145065. The ET peptide levels in the culture supernatants of AC increased about 1.5-fold after 24 h of hypoxia as measured by radioimmunoassay. Northern blot analyses revealed a threefold up-regulation of prepro ET-1 mRNA and a concomitant downregulation of ET A receptor and ET B receptor mRNAs. However, calcium responses were still inducible by exogenous ET-1. These results indicate that hypoxia triggers an autocrine loop in AC, resulting in a morphological transformation. This response is independent from neuronal damage and is based on activation of the astrocytic ET system. Key words: cell morphology • astrocytes • cell morphology • cultured cells • culture media • endothelin-1 • endothelin converting enzyme ypoxia is a very common pathophysiological condition of the brain, which is either isolated, for example, in neonatal asphyxia, or in combination with substrate deprivation, as in ischemia. Many authors have discussed the morphology of astrocytes (AC) in a brain that has been subjected to ischemia or hypoxia. Loss of astrocytic processes during global cerebral ischemia was first recognized by Alzheimer in 1910 (1). He noted that AC could undergo a process he termed "clasmatodendrosis", which results in these cells assuming an H amoeboid shape with a clear reduction in surface area. Clasmatodendrosis can be observed as early as 30 min following occlusion of the middle cerebral artery in the rat (2). On the ultrastructural level, astrocytic processes disappear from between adjacent dendrites with the possible consequence of increased neurotransmitter levels at synapses of compromised neurons (3,4). During ischemia these alterations might favor excitotoxic neuronal death.Considering the clinical relevance of factors influencing the outcome of ischemia, it is of great importance to elucidate the mechanisms evoking these early astrocytic responses, as pharmacological intervention might be capable of prolonging postischemic neuronal survival. Cell culture models provide a va...
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