Quantification of astrocyte volume changes during ischemia in situ reveals two populations of astrocytes in the cortex of GFAP/EGFP mice.

Energy depletion during ischemia leads to disturbed ionic homeostasis and accumulation of neuroactive substances in the extracellular space, subsequently leading to volume changes in astrocytes. Confocal microscopy combined with 3D reconstruction was used to quantify ischemia-induced astrocyte volume changes in cortical slices of GFAP/EGFP transgenic mice. Twenty-minutes of oxygen-glucose deprivation (OGD) or oxygen-glucose deprivation combined with acidification (OGD(pH 6.8)) revealed the presence of two distinct astrocytic populations, the first showing a large volume increase (HR astrocytes) and the second displaying a small volume increase (LR astrocytes). In addition, changes in resting membrane potential (V(m)), measured by the patch-clamp technique, supported the existence of two astrocytic populations responding differently to ischemia. Although one group markedly depolarized during OGD or OGD(pH 6.8), only small changes in V(m) toward more negative values were observed in the second group. Conversely, acidification (ACF(pH 6.8)) led to a uniform volume decrease in all astrocytes, accompanied by only a small depolarization. Interestingly, two differently responding populations were not detected during acidification. Differences in the expression of inwardly rectifying potassium channels (Kir4.1), glial fibrillary acidic protein (GFAP), and taurine levels in cortical astrocytes were detected using immunohistochemical methods. We conclude that two distinct populations of astrocytes are present in the cortex of GFAP/EGFP mice, based on volume and V(m) changes during exposure to OGD or OGD(pH 6.8). Immunohistochemical analysis suggests that the diverse expression of Kir4.1 channels and GFAP as well as differences in the accumulation of taurine might contribute to the distinct ability of astrocytes to regulate their volume.

Melatonin inhibits prostaglandin E2- and sodium nitroprusside-induced ion secretion in rat distal colon.

Although the gastrointestinal tract is a rich source of melatonin and possesses numerous melatonin-binding sites, the role of melatonin in this tissue has not yet been fully elucidated. In this work we focused on the role of melatonin in the modulation of ion transport in rat distal colon. Whereas melatonin had no effect on colonic secretion or caused only infrequent and small changes in the short circuit current (Isc) due to its solvent ethanol, this mediator significantly modulated the secretion elicited by some secretagogues. Out of the five substances tested (prostaglandin E(2); 5-hydroxytryptamine; bethanechol; histamine; sodium nitroprusside) melatonin inhibited the effect of prostaglandin E(2) (PGE(2)) and sodium nitroprusside (SNP). Melatonin concentration-dependently decreased PGE(2)-evoked Isc and this inhibitory effect was more obvious from the mucosal side. The basal level of cAMP in colonic mucosa was not influenced by melatonin, but this drug prevented a PGE(2)-induced increase in the level of cAMP. The neurotoxin tetrodotoxin blocked the inhibitory effect of melatonin on SNP-induced Isc. Our data suggests that melatonin takes part in the modulation of colonic ion transport. The modulatory effect of melatonin on PGE(2)-induced Isc occurs directly at the level of the epithelium, whereas the effect on SNP-induced Isc is indirect and located in tetrodotoxin-sensitive enteric neurons.

Three-dimensional confocal morphometry reveals structural changes in astrocyte morphology in situ.

Neuronal activity and many pathological states in the CNS are accompanied by transient astrocytic swelling, which affects excitability, extrasynaptic transmission, and neuron-glia interactions. By using three-dimensional confocal morphometry (3DCM), we quantified the morphometric parameters of astrocytes in intact tissue. In experiments performed in brain cortex slices from transgenic GFAP/EGFP mice, we applied 3DCM to study the dynamic changes in astrocyte morphology during hypotonic stress. Our morphometric analysis showed that the effect of a 10-min application of hypotonic solution (200 mmol/kg) on the swelling of different cell compartments was dependent on the extent of the swelling of the total astrocyte volume. If the swelling of the whole cell, i.e., soma and processes, was less than approximately 10%, there were no differences between the swelling of the soma and the processes. However, if the swelling of the total cell volume was greater than 10%, the swelling of the processes was greater than the swelling of the soma. Analyzing the effect of hypotonic solution on the morphology of these astrocytes revealed that the total cell volume increased; however, certain cell compartments were distinguished in which the volume increased, whereas in other compartments cell volume decreased or apparently did not change, and the structure of some compartments was altered. Our data show that astrocytes in brain slices undergoing hypotonic stress display cell volume regulation as well as transient changes in morphology.