Distribution of P2X receptors on astrocytes in juvenile rat hippocampus.

Recent evidence suggested that ATP acting via ionotropic (P2X) and metabotropic (P2Y) purinergic receptors might be involved in signaling between glial cells and within glial-neuronal networks. In contrast to their neuronal counterpart, the identity of P2X receptors in CNS glial cells is largely unknown. In the present study, antibodies recognizing the subunits P2X1-P2X7 were applied together with the astroglial marker S100beta and nuclear labeling with Hoechst 33342 to investigate semiquantitatively the distribution of the whole set of P2X receptors in astrocytes of the juvenile rat hippocampus. Expression of P2X1-P2X4, P2X6, and P2X7 subunits was observed in astrocytes of various hippocampal subregions, but the cells were completely devoid of P2X5 protein. S100beta-positive cells expressing subunits P2X3-P2X7 occurred evenly in the different subfields, while P2X1- and P2X2-positive astrocytes were distributed more heterogeneously. The staining pattern of P2X subunits also differed at the subcellular level. Antibodies against P2X2 and P2X4 labeled both astroglial cell bodies and processes. Immunoreactivity for P2X1 and P2X6 was mainly confined to somatic areas of S100beta-positive cells, whereas the subunit P2X3 was primarily localized along astroglial processes. Knowledge of the distribution of P2X receptors might provide a basis for a better understanding of their specific role in cell-cell signaling.

Identification of purinergic receptors in retinal ganglion cells.

P2X receptors are ligand-gated ion channels activated by adenosine triphosphate and expressed in a broad variety of tissues. The present study demonstrates the expression of various types of purinergic P2X receptors in identified retinal ganglion cells (RGCs) of the adult rat retina. Single-cell reverse transcription polymerase chain reaction (SC-RT-PCR) resulted in a positive amplification signal for all P2X receptor subunit mRNAs examined (P2X(3-5), P2X(7)). Immunohistochemistry with P2X(3,4) receptor subunit-specific antibodies showed a labelling of neurons in the ganglion cell layer and inner nuclear layer. Our data suggest that extracellular ATP acts directly on RGCs via several types of P2X receptors and may provide neuromodulatory influences on information processing in the retina.

Functional and molecular properties of human astrocytes in acute hippocampal slices obtained from patients with temporal lobe epilepsy.

PURPOSE: The specific role of glial cells in epilepsy is still elusive. In this study, functional properties of astrocytes were investigated in acute hippocampal brain slices obtained from surgical specimens of patients with drug-resistant temporal lobe epilepsy (TLE). METHODS: The patch-clamp technique together with a single-cell reverse transcription-polymerase chain reaction approach were used to combine functional and molecular analysis in the same individual cell in situ. RESULTS: In patients with Ammon's horn sclerosis, the glial current patterns resembled properties of immature astrocytes in rodent hippocampus. Depolarizing voltage steps activated delayed rectifier and transient K+ currents as well as tetrodotoxin-sensitive Na+ currents. Hyperpolarizing voltages elicited inward rectifier K+ currents. Comparative recordings were made in astrocytes from patients with lesion-associated TLE that lacked significant histopathological hippocampal alterations. The inward rectifier K+ current density was significantly smaller in astrocytes from the sclerotic group compared with lesion-associated TLE patients. CONCLUSIONS: During normal development of rodent brain, astroglial inward rectification gradually increases. It thus appears that astrocytes in human sclerotic tissue reexpress an immature current pattern. Reduced astroglial inward rectification in conjunction with seizure-induced shrinkage of the extracellular space may lead to impaired spatial K+ buffering. This will result in stronger and prolonged depolarization of glial cells and neurons in response to activity-dependent K+ release and may thus contribute to seizure generation and spread in this particular condition of human TLE.

Evidence for P2X(3), P2X(4), P2X(5) but not for P2X(7) containing purinergic receptors in Müller cells of the rat retina.

P2X receptors are ligand-gated ion channels activated by ATP. They are expressed in a broad variety of tissues. To date, eight P2X receptor subunits (P2X(1)-P2X(7), P2XM) have been cloned. In spite of the considerable evidence of signaling by extracellular nucleotides in other sensory systems, only few studies have been undertaken in the retina. In earlier studies, we have demonstrated that there is mRNA expression of the P2X(2-5) and P2X(7) subunits in the rat retina. In the present study, molecular biological methods were used to investigate expression of P2X receptor mRNA in freshly isolated Müller cells (MCs) of the adult rat retina (Brown Norway). A total of 36 MCs was analyzed, employing the single-cell RT-PCR. A positive amplification signal of 11/14 for P2X(3)-mRNA, 5/10 for P2X(4)-mRNA, 3/10 for P2X(5)-mRNA and 0/8 for P2X(7)-mRNA was revealed. Additionally, the astroglial identity of the cells under studied was confirmed in 10 cases by simultaneous amplification of RT-PCR products of glutamine synthetase (GS)- and P2X-mRNA. We conclude that MCs of rat retina express ionotropic P2 receptors, which, in addition to other functions, may play a key role within the recently described long range calcium signaling and the fast direct glia-neuron interactions in the rat retina.

Expression of purinergic receptors in bipolar cells of the rat retina.

P2X receptors are ligand-gated ion channels which are activated by excitatory neurotransmitter ATP. Despite considerable evidence of signaling by extracellular nucleotides in other sensory systems, P2X receptors in the visual system have only rarely been studied, and almost nothing is known about their functional significance in the retina. To determine whether ATP plays a role in the modulation of vertical retinal signal pathways, we examined the expression of P2X receptor mRNA in freshly isolated bipolar cells of the rat retina (Brown Norway, P25) using the single-cell RT-PCR technique. Positive amplification signals were found in about 33% of the bipolar cells for P2X(3), P2X(4) and P2X(5) but not for P2X(7) mRNA. We conclude that at least a subpopulation of bipolar cells in the rat retina expresses ionotropic P2 receptors of the P2X type and that these possibly exert a neuromodulatory influence on information processing in the retina.

GABAA receptor agonists modulate K+ currents in adult hippocampal glial cells in situ.

Glial cells are known for their role in development and expression of GABA receptors. However, there seems to be a lack of in situ studies characterizing GABA receptor expression and function in glial cells from early development to adulthood. Consequently, we examined GABA receptor expression on rat hippocampal glial cells in both neonatal and adult slices using the whole-cell patch-clamp technique. Glial cells in adult and neonatal slices exhibit responses to muscimol (1 mM; GABAA), but not baclofen (1 mM; GABAB), demonstrating that receptor electrophysiology remains qualitatively similar in glial cells throughout development. Adult muscimol current densities however, do show a decrease in size to approximately 36% of the neonatal response. Muscimol responses were found to be sensitive to bicuculline, suggesting that they are mediated by GABAA receptors. In addition to receptor currents, muscimol causes a concomitant long-term blockade of outward K+ currents in glial cells of both neonatal and adult slices. Comparisons of percentage peak blockade in adult and neonatal glial cells show no significant difference. However, when comparing average absolute conductance blockade, we see that adult glial cells display a significantly smaller response than neonatal and cultured astrocytes. Therefore, although the percentage blockade of outward currents remains consistent throughout development, neonatal glial cells display a larger physiological effect. Thus, it can be concluded that, although the complex GABA response in glial cells is affected by development, the receptor current and secondary blockade are a basic mechanism for neuronal-glial interaction throughout life.

Qualitative analysis of membrane currents in glial cells from normal and gliotic tissue in situ: down-regulation of Na+ current and lack of P2 purinergic responses.

To date, the electrophysiological properties of glial cells located in reactive scar tissue are unknown. To address this issue two subtypes of hippocampal glial cells, located in thin vital slices of normal or gliotic brain tissue, were analysed for their voltage controlled ion channels using the patch-clamp technique. Reactive gliosis was induced in adult rats by a single peritoneal injection of kainic acid. The intensity of the following seizures was rated ascending from 1 to 6. Rats which exhibited seizures of level 3 or higher showed, within three days, a marked loss of pyramidal cells (60% in CA1 and CA3) and an increase in the density of glial fibrillary acidic protein immunostaining, representing an apparent increase in the number and size of astrocytes in all layers of the hippocampal CA1 subfield. Reactive and normal astrocytes of one subtype, electrophysiologically characterized by time-independent potassium currents, did not significantly differ in membrane potential and potassium conductivity. Glutamine synthetase-positive, but mostly glial fibrillary acidic protein-negative, glial cells (presumably representing immature astrocytes) were also included in this study. This subtype of glial cells showed several voltage- and time-dependent potassium currents and, under control conditions, tetrodotoxin-sensitive voltage-gated Na+ channels, which were almost completely lost after reactive gliosis. Another part of this study focuses on the sensitivity of reactive and control glial cells for extracellular ATP. Several in vitro studies suggest that P2 purinergic receptors on glial cells could trigger the induction of reactive gliosis. In contrast to results described on cultured astrocytes, we found in situ that hippocampal glial cells were not sensitive to ATP or stable P2 receptor agonists in control or in gliotic brain slices. In summary, the presence of at least two different subtypes of hippocampal astrocytes was demonstrated for control as well as for gliotic brain tissue. A dramatic down-regulation of tetrodotoxin-sensitive sodium channels in one subpopulation of reactive astrocytes was shown. This result supports the hypothesis that the presence of active neurons could be required to maintain glial voltage-gated sodium channels. Furthermore, we conclude that there is no longtime expression of P2 purinoceptors on hippocampal astrocytes in situ, and therefore the involvement of astrocytic ATP receptors in the genesis of reactive gliosis is unlikely.

Developmental regulation of Na+ and K+ conductances in glial cells of mouse hippocampal brain slices.

The relative contribution of voltage activated Na+ and K+ currents to the whole cell current pattern of hippocampal glial cells was analyzed and compared during different stages of postnatal maturation. The patch-clamp technique was applied to identified cells in thin brain slices obtained from animals between postnatal day 5 and 35 (p5-35). We focused on a subpopulation of glial cells in the CA1 stratum radiatum which most probably represents a pool of immature astrocytes, termed "complex" cells. These cells could not be labelled by O1/O4 antibodies, but some of the older cells were positively stained for glial fibrillary acidic protein (GFAP). In the early postnatal days, the current pattern of the "complex" cells was dominated by two types of K+ outward currents: a delayed rectifier and a transient component. In addition, all cells expressed significant tetrodotoxin (TTX)-sensitive Na+ currents. During maturation, the contribution of delayed rectifier and A-type currents significantly decreased. Furthermore, almost all cells after p20 lacked Na+ currents. This down-regulation of voltage gated Na+ and K+ outward currents was accompanied by a substantial increase in passive and inward rectifier K+ conductances. We found increasing evidence of electrical coupling between the "complex" cells with continued development. It is concluded that these developmental changes in the electrophysiological properties of "complex" glial cells could be jointly responsible for the well known impaired K+ homeostasis in the early postnatal hippocampus.

Properties of GABA and glutamate responses in identified glial cells of the mouse hippocampal slice.

In this study, the patch-clamp technique was applied to brain slices to test for the presence of GABAA and glutamate receptors in glial cells of an intact tissue preparation, the hippocampus from 9-12 day old mice. Two types of glial cells were studied in the CA1 stratum pyramidale, termed passive and complex cells, which were distinct by their characteristic pattern of voltage-dependent currents. Both cell types were previously identified as glial by combining electrophysiology with ultrastructural inspection (Steinhüser et al., 1992, Eur J Neurosci 4:472-484). A subpopulation of passive cells was positive, all complex cells were negative for immunocytochemical staining against glial fibrillary acidic protein, a marker of mature astrocytes. In both cell types, GABA activated currents compatible with GABAA-receptor mediated responses. The glutamate response in complex and in most of the passive cells was mediated by a ligand-gated ion channel and closely matched the pharmacology of the kainate receptor. Activation of glutamate receptors led to a transient decrease of the resting K+ conductance in complex cells and to an irreversible decrease in the passive cells. In three passive cells, glutamate-activated currents were most likely dominated by an electrogenic uptake. In a small group of passive cells NMDA-activated currents were observed. This study provides evidence that glial cells from an intact tissue express receptors for the most abundant transmitters in the central nervous system, glutamate, and GABA.

Kainate activates Ca(2+)-permeable glutamate receptors and blocks voltage-gated K+ currents in glial cells of mouse hippocampal slices.

Glial cells in the CA1 stratum radiatum of the hippocampus of 9- to 12-day-old mice show intrinsic responses to glutamate due to the activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptors. In the present study we have focused on a subpopulation of the hippocampal glial cells, the "complex" cells, characterized by voltage-gated Na+ and K+ channels. Activation of glutamate receptors in these cells led to two types of responses, the activation of a cationic conductance, and a longer-lasting blockade of voltage-gated K+ channels. In particular, the transient (inactivating) component of the outwardly rectifying K+ current was diminished by kainate. Concomitantly, as described in Bergmann glial cells, kainate also elevated cytosolic Ca2+. This increase was due to an influx via the glutamate receptor itself. In contrast to Bergmann glial cells, the cytosolic Ca2+ increase was not a link to the K+ channel blockade, since the blockade occurred in the absence of the Ca2+ signal and, vice versa, an increase in cytosolic Ca2+ induced by ionomycin did not block the transient K+ current. We conclude that glutamate receptor activation leads to complex and variable changes in different types of glial cells; the functional importance of these changes is as yet unresolved.