Astrocytes secrete ATP by exocytosis from synaptic-like vesicles, activating neuronal P2X receptors, which contribute to postsynaptic GABA receptor down-regulation, ultimately mediating the communication between astrocytes and neurons required for brain function.

Chemical transmission between neurons and glial cells is an important element of integration in the CNS -801). These NMDA-evoked currents were linearly dependent on membrane potential and were not affected by extracellular magnesium at concentrations up to 10 mM. Electrical stimulation of axons in layer IV-VI induced a complex inward current in astrocytes situated in the cortical layer II, part of which was sensitive to MK-801 at holding potential Ϫ80 mV and was not affected by the AMPA glutamate receptor antagonist NBQX. The fast miniature spontaneous currents were observed in cortical astrocytes in slices as well. These currents exhibited both AMPA and NMDA receptor-mediated components. We conclude that cortical astrocytes express functional NMDA receptors that are devoid of Mg 2ϩ block, and these receptors are involved in neuronal-glial signal transmission.

Adenosine triphosphate (ATP) acts as a fast excitatory transmitter in several regions of the central nervous system (CNS) including the medial habenula, dorsal horn, locus coeruleus, hippocampus, and somatosensory cortex. Postsynaptic actions of ATP are mediated through an extended family of P2X receptors, widely expressed throughout the CNS. ATP is released via several pathways, including exocytosis from presynaptic terminals and diffusion through large transmembrane pores (e.g., hemichannels, P2X(7) receptors, or volume-sensitive chloride channels) expressed in astroglial membranes. In presynaptic terminals, ATP is accumulated and stored in the synaptic vesicles. In different presynaptic terminals, these vesicles may contain ATP only or ATP and another neurotransmitter [e.g., gamma-amino-butyric acid (GABA) or glutamate]; in the latter case, two transmitters can be coreleased. Here, we discuss the mechanisms of vesicular release of ATP in the CNS and present our own data, which indicate that in central neuronal terminals, ATP is primarily stored and released from distinct pool of vesicles; the release of ATP is not synchronized either with GABA or with glutamate.

Transient currents occur at rest in cortical neurones that reflect the quantal release of transmitters such as glutamate and γ-aminobutyric acid (GABA). We found a bimodal amplitude distribution for spontaneously occurring inward currents recorded from mouse pyramidal neurones in situ, in acutely isolated brain slices superfused with picrotoxin. Larger events were blocked by glutamate receptor (AMPA, kainate) antagonists; smaller events were partially inhibited by P2X receptor antagonists suramin and PPADS. The decay of the larger events was selectively prolonged by cyclothiazide. Stimulation of single intracortical axons elicited quantal glutamate-mediated currents and also quantal currents with amplitudes corresponding to the smaller spontaneous inward currents. It is likely that the lower amplitude spontaneous events reflect packaged ATP release. This occurs with a lower probability than that of glutamate, and evokes unitary currents about half the amplitude of those mediated through AMPA receptors. Furthermore, the packets of ATP appear to be released from vesicle in a subset of glutamate-containing terminals.

ATP plays an important role in signal transduction between neuronal and glial circuits and within glial networks. Here we describe currents activated by ATP in astrocytes acutely isolated from cortical brain slices by non-enzymatic mechanical dissociation. Brain slices were prepared from transgenic mice that express enhanced green fluorescent protein under the control of the human glial fibrillary acidic protein promoter. Astrocytes were studied by whole-cell voltage clamp. Exogenous ATP evoked inward currents in 75 of 81 astrocytes. In the majority (ϳ65%) of cells, ATP-induced responses comprising a fast and delayed component; in the remaining subpopulation of astrocytes, ATP triggered a smoother response with rapid peak and slowly decaying plateau phase. The fast component of the response was sensitive to low concentrations of ATP (with EC 50 of ϳ40 nM). All ATP-induced currents were blocked by pyridoxal-phosphate-6-azophenyl-2Ј,4Ј-disulfonate (PPADS); they were insensitive to ivermectin. Quantitative real-time PCR demonstrated strong expression of P2X 1 and P2X 5 receptor subunits and some expression of P2X 2 subunit mRNAs. The main properties of the ATP-induced response in cortical astrocytes (high sensitivity to ATP, biphasic kinetics, and sensitivity to PPADS) were very similar to those reported for P2X 1/5 heteromeric receptors studied previously in heterologous expression systems.

ATP receptors participate in synaptic transmission and intracellular calcium signaling in the hippocampus by providing a component of the excitatory input to CA1 pyramidal neurons. The activation of P2X purinoreceptors generates calcium influx that does not require cell depolarization, but this response desensitizes at increased rates of stimulation. Here we show that inhibition of P2X receptors dramatically facilitates the induction of long-term potentiation (LTP). High-frequency stimulation (HFS) (1 sec) induced LTP in CA1, whereas brief HFS (0.2 sec) caused only short-term potentiation. However, when P2X receptors were inhibited by PPADS (pyridoxal phosphate-6-azophenyl-2'-4'-disulphonic acid) or desensitized by the nonhydrolyzable ATP analog alpha,beta-methyleneATP, brief HFS reliably induced LTP. Inhibition of P2X receptors had no facilitatory effect on LTP when NMDA receptors were blocked. We hypothesized that P2X receptors affect the threshold for LTP by altering Ca2+-dependent inactivation of NMDA receptors. In isolated pyramidal CA1 neurons and hippocampal slices, activation of P2X receptors did cause inhibition of NMDA receptor-mediated current. We suggest that, by controlling the background calcium and thus the activity of NMDA receptors at low firing frequencies, P2X receptors act as a dynamic low-frequency filter so that weak stimuli do not induce LTP.

Communication between neuronal and glial cells is thought to be very important for many brain functions. Acting via release of gliotransmitters, astrocytes can modulate synaptic strength. The mechanisms underlying ATP release from astrocytes remain uncertain with exocytosis being the most intriguing and debated pathway. We have demonstrated that ATP and d-serine can be released from cortical astrocytes in situ by a SNARE-complex-dependent mechanism. Exocytosis of ATP from astrocytes can activate post-synaptic P2X receptors in the adjacent neurons, causing a downregulation of synaptic and extrasynaptic GABA receptors in cortical pyramidal neurons. We showed that release of gliotransmitters is important for the NMDA receptor-dependent synaptic plasticity in the neocortex. Firstly, induction of long-term potentiation (LTP) by five episodes of theta-burst stimulation (TBS) was impaired in the neocortex of dominant-negative (dn)-SNARE mice. The LTP was rescued in the dn-SNARE mice by application of exogenous non-hydrolysable ATP analogues. Secondly, we observed that weak sub-threshold stimulation (two TBS episodes) became able to induce LTP when astrocytes were additionally activated via CB-1 receptors. This facilitation was dependent on activity of ATP receptors and was abolished in the dn-SNARE mice. Our results strongly support the physiological relevance of glial exocytosis for glia–neuron communications and brain function.

Communication between neuronal and glial cells is thought to be very important for many brain functions. Acting via release of gliotransmitters, astrocytes can modulate synaptic strength. The mechanisms underlying gliotransmission remain uncertain with exocytosis being the most intriguing and debated pathway. We demonstrate that astroglial α1-adrenoreceptors are very sensitive to noradrenaline (NA) and make a significant contribution to intracellular Ca2+-signaling in layer 2/3 neocortical astrocytes. We also show that astroglial α1-adrenoreceptors are prone to desensitization upon prolonged exposure to NA. We show that within neocortical slices, α-1adrenoreceptors can activate vesicular release of ATP and D-serine from cortical astrocytes which initiate a burst of ATP receptor-mediated currents in adjacent pyramidal neurons. These purinergic currents can be inhibited by intracellular perfusion of astrocytes with Tetanus Toxin light chain, verifying their origin via astroglial exocytosis. We show that α1 adrenoreceptor-activated release of gliotransmitters is important for the induction of synaptic plasticity in the neocortex:long-term potentiation (LTP) of neocortical excitatory synaptic potentials can be abolished by the selective α1-adrenoreceptor antagonist terazosin. We show that weak sub-threshold theta-burst stimulation (TBS) can induce LTP when astrocytes are additionally activated by 1 μM NA. This facilitation is dependent on the activation of neuronal ATP receptors and is abolished in neocortical slices from dn-SNARE mice which have impaired glial exocytosis. Importantly, facilitation of LTP by NA can be significantly reduced by perfusion of individual astrocytes with Tetanus Toxin. Our results strongly support the physiological importance of astroglial adrenergic signaling and exocytosis of gliotransmitters for modulation of synaptic transmission and plasticity.