The endocannabinoid anandamide inhibits potassium conductance in rat cortical astrocytes.

Endocannabinoids are a family of endogenous signaling molecules that modulate neuronal excitability in the central nervous system (CNS) by interacting with cannabinoid (CB) receptors. In spite of the evidence that astroglial cells also possess CB receptors, there is no information on the role of endocannabinoids in regulating CNS function through the modulation of ion channel-mediated homeostatic mechanisms in astroglial cells. We provide electrophysiological evidence that the two brain endocannabinoids anandamide (AEA) and 2-arachidonylglycerol (2-AG) markedly depress outward conductance mediated by delayed outward rectifier potassium current (IK(DR)) in primary cultured rat cortical astrocytes. Pharmacological experiments suggest that the effect of AEA does not result from the activation of known CB receptors. Moreover, neither the production of AEA metabolites nor variations in free cytosolic calcium are involved in the negative modulation of IK(DR). We show that the action of AEA is mediated by its interaction with the extracellular leaflet of the plasma membrane. Similar experiments performed in situ in cortical slices indicate that AEA downregulates IK(DR) in complex and passive astroglial cells. Moreover, IK(DR) is also inhibited by AEA in NG2 glia. Collectively, these results support the notion that endocannabinoids may exert their modulation of CNS function via the regulation of homeostatic function of the astroglial syncytium mediated by ion channel activity.

Pathological potential of astroglia.

The pathological potential of glial cells was recognized already by Rudolf Virchow, Santiago Ramon y Cajal and Pio Del Rio-Ortega. Many functions and roles performed by astroglia in the healthy brain determine their involvement in brain diseases; as indeed any kind of brain insult does affect astrocytes, and their performance in pathological conditions, to a very large extent, determines the survival of the brain parenchyma, the degree of damage and neurological defect. Astrocytes being in general responsible for overall brain homeostasis are involved in virtually every form of brain pathology. Here we provide an overview of recent developments in identifying the role and mechanisms of the pathological potential of astroglia.

Membrane currents and morphological properties of neurons and glial cells in the spinal cord and filum terminale of the frog.

Using the patch-clamp technique in the whole-cell configuration combined with intracellular dialysis of the fluorescent dye Lucifer yellow (LY), the membrane properties of cells in slices of the lumbar portion of the frog spinal cord (n=64) and the filum terminale (FT, n=48) have been characterized and correlated with their morphology. Four types of cells were found in lumbar spinal cord and FT with membrane and morphological properties similar to those of cells that were previously identified in the rat spinal cord (Chvátal, A., Pastor, A., Mauch, M., Syková, E., Kettenmann, H., 1995. Distinct populations of identified glial cells in the developing rat spinal cord: Ion channel properties and cell morphology. Eur. J. Neurosci. 7, 129-142). Neurons, in response to a series of symmetrical voltage steps, displayed large repetitive voltage-dependent Na(+) inward currents and K(+) delayed rectifying outward currents. Three distinct types of non-neuronal cells were found. First, cells that exhibited passive symmetrical non-decaying currents were identified as astrocytes. These cells immunostained for GFAP and typically had at least one thick process and a number of fine processes. Second, cells with the characteristic properties of rat spinal cord oligodendrocytes, with passive symmetrical decaying currents and large tail currents after the end of the voltage step. These cells exhibited either long parallel or short hairy processes. Third, cells that expressed small brief inward currents in response to depolarizing steps, delayed rectifier outward currents and small sustained inward currents identical to rat glial precursor cells. Morphologically, they were characterized by round cell bodies with a number of finely branched processes. LY dye-coupling in the frog spinal cord gray matter and FT was observed in neurons and in all glial populations. All four cell types were found in both the spinal cord gray matter and FT. The glia/neuron ratio in the spinal cord was 0.78, while in FT it was 2.0. Moreover, the overall cell density was less in the FT than in the spinal cord. The present study shows that the membrane and morphological properties of glial cells in the frog and rat spinal cords are similar. Such striking phylogenetic similarity suggests a significant contribution from distinct glial cell populations to various spinal cord functions, particularly ionic and volume homeostasis in both mammals and amphibians.

Vasoactive intestinal peptide potentiates and directly stimulates catecholamine secretion from rat adrenal chromaffin cells.

The actions of vasoactive intestinal polypeptide (VIP) on catecholamine secretion and changes in [Ca2+]i in single rat chromaffin cells were studied using amperometry and Indo-1. Application of VIP prior to acetylcholine (ACh) or co-application of VIP and ACh enhanced secretion by 94% and 153% respectively, compared to ACh alone. [Ca2+]i was increased by 17% when VIP was preapplied and by 73% upon co-application. Exposure to VIP before stimulation with 60 mM K+ enhanced secretion by 68%, but not [Ca2+]i. VIP application prior to DMPP and nicotine had no effect on [Ca2+]i, but increased [Ca2+]i signals to muscarine by 18%. VIP co-application potentiated only [Ca2+]i responses to muscarine, by 28%. The effect of VIP on muscarine-induced [Ca2+]i signals was mimicked by 8-Br-cAMP, and both were blocked by H-89, a protein kinase A inhibitor. Long-lasting increases in secretion accompanied by a sustained rise in [Ca2+]i to VIP alone were seen in 55% of cells. Removal of Ca2+ or addition of La3+ inhibited both responses, while L-, N- and P-type Ca2+ channel blockers were ineffective. SK&F 96365 inhibited VIP-induced secretion completely and rises in [Ca2+]i by 75%. Neither 8-Br-cAMP nor 8-Br-cGMP evoked responses similar to VIP alone. Thus in rat chromaffin cells, VIP acts both directly as a neurotransmitter in provoking sustained catecholamine secretion in a cAMP-independent manner, and also by enhancing ACh-induced secretion, via a cAMP-dependent action involving muscarinic receptors.

Effect of increasing methanol concentrations on physiology and cytology of Candida boidinii.

Concentration of methanol in the medium strongly affected not only the physiology but also the cytology of Candida boidinii strain 2 cells in a methanol-limited chemostat at a constant dilution rate D 0.1/h and at low pH 3.0. The formation of large cubic peroxisomes with high alcohol oxidase (AO) activity observed at low methanol concentration (S0 3 g/L) disappeared on increasing the methanol concentration in the inflow medium. The AO activity in the cells sharply decreased, followed by accumulation of riboflavin phosphate and residual methanol in the medium. The activity of catalase was relatively stable. At methanol concentration S0 > KI (KI equal to 12 g methanol per L), which included a substantial increase in methanol dissimilation, documented by higher formaldehyde and formate dehydrogenase activities and by lower yield coefficient on methanol, the yeast cells contained large lobe-shaped peroxisomes and a smaller number of larger mitochondria. The cells formed pseudomycelium with a thick septum between the mother and daughter cells.

Focal cerebral ischemia induces the neurogenic potential of mouse Dach1-expressing cells in the dorsal part of the lateral ventricles.

The mouse Dach1 gene, involved in the development of the neocortex and the hippocampus, is expressed by neural stem cells (NSCs) during early neurogenesis, and its expression also continues in a subpopulation of cells in the dorsal part of the lateral ventricles (LV) of the adult mouse brain. In this study we aimed to elucidate the role of Dach1-expressing cells in adult neurogenesis/gliogenesis under physiological as well as post-ischemic conditions, employing transgenic mice in which the expression of green fluorescent protein (GFP) is controlled by the D6 promotor of the mouse Dach1 gene. A neurosphere-forming assay of GFP⁺ cells isolated from the dorsal part of the LV was carried out with subsequent differentiation in vitro. To elucidate the neurogenic/gliogenic potential of GFP⁺ cells in the dorsal part of the LV, in situ immunohistochemical/electrophysiological analyses of GFP⁺ cells in adult sham-operated brains (controls) and those after middle cerebral artery occlusion (MCAo) were performed. The GFP⁺ cells isolated from the dorsal part of the LV of controls formed neurospheres and differentiated solely into a glial phenotype, while those isolated after MCAo also gave rise to cells with the properties of neuronal precursors. In situ analyses revealed that GFP⁺ cells express the phenotype of adult NSCs or neuroblasts in controls as well as following ischemia. Following MCAo we found a significantly increased number of GFP⁺ cells expressing doublecortin as well as a number of GFP⁺ cells migrating through the rostral migratory stream into the olfactory bulb, where they probably differentiated into calretinin⁺ interneurons. Collectively, our results suggest the involvement of the mouse Dach1 gene in adult neurogenesis; cells expressing this gene exhibit the properties of adult NSCs or neuroblasts and respond to MCAo by enhanced neurogenesis.

The inhibitor of volume-regulated anion channels DCPIB activates TREK potassium channels in cultured astrocytes.

BACKGROUND AND PURPOSE: The ethacrynic acid derivative, 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid (DCPIB) is considered to be a specific and potent inhibitor of volume-regulated anion channels (VRACs). In the CNS, DCPIB was shown to be neuroprotective through mechanisms principally associated to its action on VRACs. We hypothesized that DCPIB could also regulate the activity of other astroglial channels involved in cell volume homeostasis. EXPERIMENTAL APPROACH: Experiments were performed in rat cortical astrocytes in primary culture and in hippocampal astrocytes in situ. The effect of DCPIB was evaluated by patch-clamp electrophysiology and immunocytochemical techniques. Results were verified by comparative analysis with recombinant channels expressed in COS-7 cells. KEY RESULTS: In cultured astrocytes, DCPIB promoted the activation of a K(+) conductance mediated by two-pore-domain K(+) (K(2P) ) channels. The DCPIB effect occluded that of arachidonic acid, which activates K(2P) channels K(2P) 2.1 (TREK-1) and K(2P) 10.1 (TREK-2) in cultured astrocytes. Immunocytochemical analysis suggests that cultured astrocytes express K(2P) 2.1 and K(2P) 10.1 proteins. Moreover, DCPIB opened recombinant K(2P) 2.1 and K(2P) 10.1 expressed in heterologous system. In brain slices, DCPIB did not augment the large background K(+) conductance in hippocampal astrocytes, but caused an increment in basal K(+) current of neurons. CONCLUSION AND IMPLICATIONS: Our results indicate that the neuroprotective effect of DCPIB could be due, at least in part, to activation of TREK channels. DCPIB could be used as template to build new pharmacological tools able to increase background K(+) conductance in astroglia and neuronal cells.

Distinct effects of sonic hedgehog and Wnt-7a on differentiation of neonatal neural stem/progenitor cells in vitro.

Sonic hedgehog (Shh) and Wnt-7a are morphogens involved in embryonic as well as ongoing adult neurogenesis. Their effects on the differentiation and membrane properties of neonatal neural stem/progenitor cells (NS/PCs) were studied in vitro using NS/PCs transduced with either Shh or Wnt-7a. Eight days after the onset of in vitro differentiation the cells were analyzed for the expression of neuronal and glial markers using immunocytochemical and Western blot analysis, and their membrane properties were characterized using the patch-clamp technique. Our results showed that both Shh and Wnt-7a increased the numbers of cells expressing neuronal markers; however, quantitative immunocytochemical analysis showed that only Wnt-7a enhanced the outgrowth and the development of processes in these cells. In addition, Wnt-7a markedly suppressed gliogenesis. The electrophysiological analysis revealed that Wnt-7a increased, while Shh decreased the incidence of cells displaying a neuron-like current pattern, represented by outwardly rectifying K(+) currents and tetrodotoxin-sensitive Na(+) currents. Additionally, Wnt-7a increased cell proliferation only at the early stages of differentiation, while Shh promoted proliferation within the entire course of differentiation. Thus we can conclude that Shh and Wnt-7a interfere differently with the process of neuronal differentiation and that they promote distinct stages of neuronal differentiation in neonatal NS/PCs.

Electrophysiological characterization of neural stem/progenitor cells during in vitro differentiation: study with an immortalized neuroectodermal cell line.

Despite the accumulating data on the molecular and cell biological characteristics of neural stem/progenitor cells, their electrophysiological properties are not well understood. In the present work, changes in the membrane properties and current profiles were investigated in the course of in vitro-induced neuron formation in NE-4C cells. Induction by retinoic acid resulted in neuronal differentiation of about 50% of cells. Voltage-dependent Na+ currents appeared early in neuronal commitment, often preceding any morphological changes. A-type K+ currents were detected only at the stage of network formation by neuronal processes. Flat, epithelial- like, nestin-expressing progenitors persisted beside differentiated neurons and astrocytes. Stem/progenitor cells were gap junction coupled and displayed large, symmetrical, voltage-independent currents. By the blocking of gap junction communication, voltage-independent conductance was significantly reduced, and delayed-rectifying K+ currents became detectable. Our data indicate that voltage-independent symmetrical currents and gap junction coupling are characteristic physiological features of neural stem and progenitor cells regardless of the developmental state of their cellular environment.

Ribulose-1,5-bisphosphate carboxylase of thermophilic hydrogen-oxidizing microorganism Bacillus schlegelii.

Ribulose-1,5-bisphosphate carboxylase was isolated from thermophilic hydrogen-oxidizing Bacillus schlegelii. Molecular mass of the native enzyme is 560,000 and optimal reaction temperature is 70 degrees C. Km value for ribulose 1,5-bisphosphate is 0.27 mM. The carboxylase activity of the enzyme is dependent on Mg2+ with the optimum at 10 mM. The enzyme is an oligomer of L8S8 type with Mr of large subunits and small subunits of 56,000 and 14,000, respectively. Negatively stained enzyme has regular polygonal shape in top view, 12 nm in diameter, with central electron dense patch.

Glial depolarization evokes a larger potassium accumulation around oligodendrocytes than around astrocytes in gray matter of rat spinal cord slices.

The cell membrane of astrocytes and oligodendrocytes is almost exclusively permeable for K+. Depolarizing and hyperpolarizing voltage steps produce in oligodendrocytes, but not in astrocytes, decaying passive currents followed by large tail currents (Itail) after the offset of a voltage jump. The aim of the present study was to characterize the properties of Itail in astrocytes, oligodendrocytes, and their respective precursors in the gray matter of spinal cord slices. Studies were carried out on 5- to 11-day-old rats, using the whole-cell patch clamp technique. The reversal potential (Vrev) of Itail evoked by membrane depolarization was significantly more positive in oligodendrocytes (-31.7+/-2.58 mV, n = 53) than in astrocytes (-57.9+/-2.43 mV, n = 21), oligodendrocyte precursors (-41.2+/-3.44 mV, n = 36), or astrocyte precursors (-52.1+/-1.32 mV, n = 43). Analysis of the Itail (using a variable amplitude and duration of the de- and hyperpolarizing prepulses as well as an analysis of the time constant of the membrane currents during voltage steps) showed that the Itail in oligodendrocytes arise from a larger shift of K+ across their membrane than in other cell types. As calculated from the Nernst equation, changes in Vrev revealed significantly larger accumulation of the extracellular K+ concentration ([K+]e) around oligodendrocytes than around astrocytes. The application of 50 mM K+ or hypotonic solution, used to study the effect of cell swelling on the changes in [K+]e evoked by a depolarizing prepulse, produced in astrocytes an increase in [K+]e of 201% and 239%, respectively. In oligodendrocytes, such increases (22% and 29%) were not found. We conclude that K+ tail currents, evoked by a larger accumulation of K+ in the vicinity of the oligodendrocyte membrane, could result from a smaller extracellular space (ECS) volume around oligodendrocytes than around astrocytes. Thus, in addition to the clearance of K+ from the ECS performed by astrocytes, the presence of the K+ tail currents in oligodendrocytes indicates that they might also contribute to efficient K+ homeostasis.

Role of gamma-aminobutyric acid in early neuronal development: studies with an embryonic neuroectodermal stem cell clone.

gamma-Aminobutyric acid (GABA) has been known to function as an autocrine/paracrine signal molecule in addition to its well-known inhibitory neurotransmitter function. Studies on the developing brain and on primary brain cell cultures provided evidence for a variety of GABA functions in periods preceding the formation of synapses. The exact role of GABA in the early neural development, however, is still not well understood. In this study, one-cell-derived NE-4C neuroectodermal stem cells were induced to form neurons and astrocytes in vitro, and the role of GABA was investigated in defined phases of neurogenesis. Noninduced NE-4C cells contained GABA, expressed GABA(A)R alpha subunits, and carried functional GABA(A) ion channels. A moderate cytoplasmic GABA content was detected during the entire period of differentiation. By the time of the formation of differentiated neurons, neuron-like cells with both high and low GABA content were clearly distinguishable. HPLC analysis indicated that NE-4C cells released GABA into their fluid environment during all stages of neuronal development. By using the patch-clamp technique, GABA-evoked currents were recorded during the entire proliferation/differentiation period, whereas a GABA-evoked increase in intracellular Ca(2+) was detected only during the maturation of postmitotic neuronal precursors. Bicuculline blocked both the ion currents and the [Ca(2+)](i) increase in response to GABA. Neuron formation was facilitated by GABA through GABA(A) ion channels during postmitotic differentiation, but not earlier during the phases of cell fate commitment. Although the data clearly demonstrate an early responsiveness to GABA, understanding the significance of GABA influence in early neural cell fate decisions will require further investigation.

Effect of osmotic stress on potassium accumulation around glial cells and extracellular space volume in rat spinal cord slices.

In rat brain and spinal cord slices, the local extracellular accumulation of K(+), as indicated by K(+) tail currents (I(tail)) after a depolarization step, is greater in the vicinity of oligodendrocytes than that of astrocytes. It has been suggested that this may reflect a smaller extracellular space (ECS) around oligodendrocytes compared to astrocytes [Chvátal et al. [1997] J. Neurosci. Res. 49:98-106; [1999] J. Neurosci. Res. 56:493-505). We therefore compared the effect of osmotic stress in spinal cord slices from 5-11-day-old rats on the changes in reversal potentials (V(rev)) of I(tail) measured by the whole-cell patch-clamp technique and the changes in ECS volume measured by the real-time iontophoretic method. Cell swelling induced by a 20 min perfusion of hypoosmotic solution (200 mmol/kg) decreased the ECS volume fraction from 0.21 +/- 0.01 to 0.15 +/- 0.02, i.e., by 29%. As calculated from V(rev) of I(tail) using the Nernst equation, a depolarizing prepulse increased [K(+)](e) around astrocytes from 11.0 to 44.7 mM, i.e., by 306%, and around oligodendrocytes from 26.1 to 54.9 mM, i.e., by 110%. The ECS volume fraction decrease had the same time course as the changes in V(rev) of I(tail). Cell shrinkage in hyperosmotic solution (400 mmol/kg) increased ECS volume fraction from 0.24 +/- 0.02 to 0.32 +/- 0.02, i.e., by 33%. It had no effect on [K(+)](e) evoked by a depolarizing prepulse in astrocytes, whereas in oligodendrocytes [K(+)](e) rapidly decreased from 52 to 26 mM, i.e., by 50%. The increase in ECS volume was slower than the changes in [K(+)](e). These data demonstrate that hypoosmotic solution has a larger effect on the ECS volume around astrocytes than around oligodendrocytes and that hyperosmotic solution affects the ECS volume around oligodendrocytes only. This indicates that increased K(+) accumulation in the vicinity of oligodendrocytes could be due to a restricted ECS. Oligodendrocytes in the CNS are therefore most likely surrounded by clusters of "compacted" ECS, which may selectively affect the diffusion of neuroactive substances in specific areas and directions and facilitate spatial K(+) buffering.

Effect of elevated K(+), hypotonic stress, and cortical spreading depression on astrocyte swelling in GFAP-deficient mice.

Glial fibrillary acidic protein (GFAP) is the main component of intermediate filaments in astrocytes. To assess its function in astrocyte swelling, we compared astrocyte membrane properties and swelling in spinal cord slices of 8- to 10-day-old wild-type control (GFAP(+/+)) and GFAP-knockout (GFAP(-/-)) mice. Membrane currents and K(+) accumulation around astrocytes after a depolarizing pulse were studied using the whole-cell patch-clamp technique. In vivo cell swelling was studied in the cortex during spreading depression (SD) in 3 to 6-month-old animals. Swelling-induced changes of the extracellular space (ECS) diffusion parameters, i.e., volume fraction alpha and tortuosity lambda, were studied by the real-time iontophoretic tetramethylammonium (TMA(+)) method using TMA(+)-selective microelectrodes. Morphological analysis using confocal microscopy and quantification of xy intensity profiles in a confocal plane revealed a lower density of processes in GFAP(-/-) astrocytes than in GFAP(+/+) astrocytes. K(+) accumulation evoked by membrane depolarization was lower in the vicinity of GFAP(-/-) astrocytes than GFAP(+/+) astrocytes, suggesting the presence of a larger ECS around GFAP(-/-) astrocytes. Astrocyte swelling evoked by application of 50 mM K(+) or by hypotonic solution (HS) produced a larger increase in [K(+)](e) around GFAP(+/+) astrocytes than around GFAP(-/-) astrocytes. No differences in alpha and lambda in the spinal cord or cortex of GFAP(+/+) and GFAP(-/-) mice were found; however, the application of either 50 mM K(+) or HS in spinal cord, or SD in cortex, evoked a large decrease in alpha and an increase in lambda in GFAP(+/+) mice only. Slower swelling in GFAP(-/-) astrocytes indicates that GFAP and intermediate filaments play an important role in cell swelling during pathological states.

Expression of an outward-rectifying potassium channel from maize mRNA and complementary RNA in Xenopus oocytes.

Injection of Xenopus oocytes with poly(A)+ mRNA isolated from different plants (maize, cucumber, and squash) results in the appearance of a voltage- and time-dependent, potassium-selective, outward current that is similar to the outward-rectifying potassium current recorded in many higher plant cells. Maize shoots were found to be especially enriched in mRNA encoding such activity. A cDNA library of maize shoot mRNA was constructed in the vector lambda ZAPII and was used to synthesize RNA complementary to the cDNA (cRNA). Injection of the cRNA gave rise to an outward-rectifying potassium current with properties similar to the currents obtained by poly(A)+ mRNA injection. These results demonstrate that higher plant mRNA can be properly translated into a product that produces a voltage-regulated potassium channel in the plasma membrane of Xenopus oocytes. Thus, Xenopus oocytes can be used as a heterologous expression system for the functional identification and isolation of plant ion channel genes as well as for the study of structure-function relationship of plant ion channels.