Ronald Jabs

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 S100β 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. S100β‐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 S100β‐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. GLIA 36:11–21, 2001.

Synaptic transmission onto hippocampal glial cells with hGFAP promoter activity

Glial cells increasingly gain importance as part of the brains communication network. Using transgenic mice expressing green fluorescent protein (EGFP) under the control of the human GFAP promoter, we tested for synaptic input to identified glial cells in the hippocampus. Electron microscopic inspection identified synapse-like structures with EGFP-positive postsynaptic compartments. Sub-threshold stimulation to Schaffer collaterals resulted in stimulus-correlated, postsynaptic responses in a subpopulation of EGFP-positive cells studied with the patch-clamp technique in acute slices. This cell population can be recognized by its distinct morphology and has been termed GluR cells in a preceding study. These cells are distinct from the classical astrocytes due to their antigen profile and functional properties, but also lack characteristic features of oligodendrocytes or neurons. GluR cells also received spontaneous synaptic input. Stimulus-correlated and spontaneous responses were quantitatively analysed by ascertaining amplitude distributions, failure rates, kinetics as well as pharmacological properties. The data demonstrate that GABAergic and glutamatergic neurons directly synapse onto GluR cells and suggest a low number of neuronal release sites. These data demonstrate that a distinct type of glial cells is integrated into the synaptic circuit of the hippocampus, extending the finding that synapse-based brain information processing is not a property exclusive to neurons.

Functional and Molecular Properties of Human Astrocytes in Acute Hippocampal Slices Obtained from Patients with Temporal Lobe Epilepsy

Summary: 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).

Kainate activates Ca2+-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 α-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.

Astrocytic function and its alteration in the epileptic brain

Currently available anticonvulsant drugs and complementary therapies are insufficient to control seizures in about a third of epileptic patients. Thus, there is an urgent need for new treatments that prevent the development of epilepsy and control it better in patients already afflicted with the disease. A prerequisite to reach this goal is a deeper understanding of the cellular basis of hyperexcitability and synchronization in the affected tissue. Epilepsy is often accompanied by massive reactive gliosis. Although the significance of this alteration is poorly understood, recent findings suggest that modified astroglial function may have a role in the generation and spread of seizure activity. Here we summarize properties of astrocytes as well as their changes that can be associated with epileptic tissue. The goal is to provide an understanding of the current knowledge of these cells with the long‐term view of providing a foundation for the development of novel hypotheses about the role of glia in epilepsy.

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.

Lack of P2X receptor mediated currents in astrocytes and GluR type glial cells of the hippocampal CA1 region

Purinergic signalling plays a major role in intercellular communication between neurons and glial cells. Glial cells express metabotropic receptors for ATP and adenosine, the latter being activated after ATP cleavage through extracellular ecto‐ATPase activity. Ionotropic receptors for extracellular ATP, so called P2X receptors, might contribute to neuron–glia signalling. However, experimental evidence for the presence of these receptors in glial cells is less convincing so far. In a previous study, immunohistochemistry was used to identify P2X1–4,6,7 receptor protein in S100β‐positive hippocampal glial cells. Applying patch clamp and fast application techniques, here we challenged the question of the functional expression of these receptors. Time correlated membrane currents served as test criterion for receptor function, since P2X receptor activation leads to the opening of unspecific cation channels in a millisecond time scale. Agonists were applied via short pressure puffs, with a fast concentration clamp method and through UV flash triggered photolysis of caged ATP. Two types of murine hippocampal macroglial cells, both labelled by the expression of green fluorescence protein driven by the human glial fibrillary acid protein promoter, were analysed in acute brain slices and in freshly dissociated cell suspensions. Surprisingly, ATP or related agonists completely failed to activate currents. Additionally, changes in spontaneously occurring glial postsynaptic currents were never observed. These results have been verified using rat and human hippocampal tissue as well as investigating cells from P2X7 knock out mice. It is concluded that in acute preparations, astroglial cells of the hippocampal CA1 subfield do not express functional P2X receptors.

Evidence for P2X3, P2X4, P2X5 but not for P2X7 containing purinergic receptors in Müller cells of the rat retina

Abstract 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 (P2X1–P2X7, 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 P2X2–5 and P2X7 subunits in the rat retina. In the present study, molecular biological methods were used to investigate expression of P2X receptor mRNA in freshly isolated Muller 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 P2X3-mRNA, 5/10 for P2X4-mRNA, 3/10 for P2X5-mRNA and 0/8 for P2X7-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.

Gray Matter NG2 Cells Display Multiple Ca2+-Signaling Pathways and Highly Motile Processes

NG2 cells, the fourth type of glia in the mammalian CNS, receive synaptic input from neurons. The function of this innervation is unknown yet. Postsynaptic changes in intracellular Ca2+-concentration ([Ca2+]i) might be a possible consequence. We employed transgenic mice with fluorescently labeled NG2 cells to address this issue. To identify Ca2+-signaling pathways we combined patch-clamp recordings, Ca2+-imaging, mRNA-transcript analysis and focal pressure-application of various substances to identified NG2-cells in acute hippocampal slices. We show that activation of voltage-gated Ca2+-channels, Ca2+-permeable AMPA-receptors, and group I metabotropic glutamate-receptors provoke [Ca2+]i-elevations in NG2 cells. The Ca2+-influx is amplified by Ca2+-induced Ca2+-release. Minimal electrical stimulation of presynaptic neurons caused postsynaptic currents but no somatic [Ca2+]i elevations, suggesting that [Ca2+]i elevations in NG2 cells might be restricted to their processes. Local Ca2+-signaling might provoke transmitter release or changes in cell motility. To identify structural prerequisites for such a scenario, we used electron microscopy, immunostaining, mRNA-transcript analysis, and time lapse imaging. We found that NG2 cells form symmetric and asymmetric synapses with presynaptic neurons and show immunoreactivity for vesicular glutamate transporter 1. The processes are actin-based, contain ezrin but not glial filaments, microtubules or endoplasmic reticulum. Furthermore, we demonstrate that NG2 cell processes in situ are highly motile. Our findings demonstrate that gray matter NG2 cells are endowed with the cellular machinery for two-way communication with neighboring cells.

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.