Communication between neuronal and glial cells is important for many brain functions. Astrocytes can modulate synaptic strength via Ca²⁺-stimulated release of various gliotransmitters, including glutamate and ATP. A physiological role of ATP release from astrocytes was suggested by its contribution to glial Ca²⁺-waves and purinergic modulation of neuronal activity and sleep homeostasis. The mechanisms underlying release of gliotransmitters remain uncertain, and exocytosis is the most intriguing and debated pathway. We investigated release of ATP from acutely dissociated cortical astrocytes using “sniff-cell” approach and demonstrated that release is vesicular in nature and can be triggered by elevation of intracellular Ca²⁺ via metabotropic and ionotropic receptors or direct UV-uncaging. The exocytosis of ATP from neocortical astrocytes occurred in the millisecond time scale contrasting with much slower nonvesicular release of gliotransmitters via Best1 and TREK-1 channels, reported recently in hippocampus. Furthermore, we discovered that elevation of cytosolic Ca²⁺ in cortical astrocytes triggered the release of ATP that directly activated quantal purinergic currents in the pyramidal neurons. The glia-driven burst of purinergic currents in neurons was followed by significant attenuation of both synaptic and tonic inhibition. The Ca²⁺-entry through the neuronal P2X purinoreceptors led to phosphorylation-dependent down-regulation of GABAA receptors. The negative purinergic modulation of postsynaptic GABA receptors was accompanied by small presynaptic enhancement of GABA release. Glia-driven purinergic modulation of inhibitory transmission was not observed in neurons when astrocytes expressed dn-SNARE to impair exocytosis. The astrocyte-driven purinergic currents and glia-driven modulation of GABA receptors were significantly reduced in the P2X4 KO mice. Our data provide a key evidence to support the physiological importance of exocytosis of ATP from astrocytes in the neocortex. Brain function depends on the interaction between two major types of cells: neurons transmitting electrical signals and glial cells, which control cerebral circulation and neuronal homeostasis. There is a growing evidence of the participation of astrocytes in regulating neuronal excitability and synaptic plasticity via the release of “gliotransmitters,” which include glutamate and ATP. The importance of ATP release from astrocytes was suggested by studies that demonstrated its contribution to neuronal activity and sleep homeostasis via modulation of known “purinergic” receptors. But the mechanisms underlying gliotransmitter release and the physiological significance of direct glia-to-neuron communication remain unknown and intensively debated. Here, we investigate the release of ATP from astrocytes of brain neocortex and demonstrate that astrocytes can release ATP by Ca²⁺-dependent exocytosis, most likely from synaptic-like microvesicles. We also find that vesicular release of ATP from astrocytes can directly activate excitatory signaling in the neighboring neurons, operating through purinergic P2X receptors. We saw that activation of these P2X receptors by astrocyte-driven ATP down-regulated the inhibitory synaptic signaling in the neocortical neurons. Our results imply that exocytosis of gliotransmitters is important for the communication between astrocytes and neurons in the neocortex.

MiR-132 and miR-212 are two closely related miRNAs encoded in the same intron of a small non-coding gene, which have been suggested to play roles in both immune and neuronal function. We describe here the generation and initial characterisation of a miR-132/212 double knockout mouse. These mice were viable and fertile with no overt adverse phenotype. Analysis of innate immune responses, including TLR-induced cytokine production and IFNβ induction in response to viral infection of primary fibroblasts did not reveal any phenotype in the knockouts. In contrast, the loss of miR-132 and miR-212, while not overtly affecting neuronal morphology, did affect synaptic function. In both hippocampal and neocortical slices miR-132/212 knockout reduced basal synaptic transmission, without affecting paired-pulse facilitation. Hippocampal long-term potentiation (LTP) induced by tetanic stimulation was not affected by miR-132/212 deletion, whilst theta burst LTP was enhanced. In contrast, neocortical theta burst-induced LTP was inhibited by loss of miR-132/212. Together these results indicate that miR-132 and/or miR-212 play a significant role in synaptic function, possibly by regulating the number of postsynaptic AMPA receptors under basal conditions and during activity-dependent synaptic plasticity.

BACKGROUND AND PURPOSE Astrocytes of the mouse neocortex express functional NMDA receptors, which are not blocked by Mg²⁺ ions. However, the pharmacological profile of glial NMDA receptors and their subunit composition is far from complete.EXPERIMENTAL APPROACH We tested the sensitivity of NMDA receptor‐mediated currents to the novel GluN2C/D subunit‐selective antagonist UBP141 in mouse cortical astrocytes and neurons. We also examined the effect of memantine, an antagonist that has substantially different affinities for GluN2A/B and GluN2C/d‐containing receptors in physiological concentrations of extracellular Mg²⁺_.KEY RESULTS UBP141 had a strong inhibitory action on NMDA receptor‐mediated transmembrane currents in the cortical layer II/III astrocytes with an IC₅₀ of 2.29 µM and a modest inhibitory action on NMDA‐responses in the pyramidal neurons with IC₅₀ of 19.8 µM. Astroglial and neuronal NMDA receptors exhibited different sensitivities to memantine with IC₅₀ values of 2.19 and 10.8 µM, respectively. Consistent with pharmacological differences between astroglial and neuronal NMDA receptors, NMDA receptors in astrocytes showed lower Ca²⁺ permeability than neuronal receptors with P_Ca/P_Na ratio of 3.4.CONCLUSIONS AND IMPLICATIONS The biophysical and pharmacological properties of the astrocytic NMDA receptors strongly suggest that they have a tri‐heteromeric structure composed of GluN1, GluN2C/D and GluN3 subunits. The substantial difference between astroglial and neuronal NMDA receptors in their sensitivity to UBP141 and memantine may enable selective modulation of astrocytic signalling that could be very helpful for elucidating the mechanisms of neuron‐glia communications. Our results may also provide the basis for the development of novel therapeutic agents specifically targeting glial signalling.

Recent studies highlighted the importance of astrocyte-secreted molecules, such as ATP, for the slow modulation of synaptic transmission in central neurones. Biophysical mechanisms underlying the impact of gliotransmitters on the strength of individual synapse remain, however, unclear. Here we show that purinergic P2X receptors can bring significant contribution to the signalling in the individual synaptic boutons. ATP released from astrocytes facilitates a recruitment of P2X receptors into excitatory synapses by Ca²⁺-dependent mechanism. P2X receptors, co-localized with NMDA receptors in the excitatory synapses, can be activated by ATP co-released with glutamate from pre-synaptic terminals and by glia-derived ATP. An activation of P2X receptors in turn leads to down-regulation of postsynaptic NMDA receptors via Ca²⁺-dependent de-phosphorylation and interaction with PSD-95 multi-protein complex. Genetic deletion of the PSD-95 or P2X4 receptors obliterated ATP-mediated down-regulation of NMDA receptors. Impairment of purinergic modulation of NMDA receptors in the PSD-95 mutants dramatically decreased the threshold of LTP induction and increased the net magnitude of LTP. Our findings show that synergistic action of glia- and neurone-derived ATP can pre-modulate efficacy of excitatory synapses and thereby can have an important role in the glia-neuron communications and brain meta-plasticity.

Cortical astrocytes express fast ionotropic receptors for glutamate and ATP, although their role in neurone-glia communication remains controversial. Stimulation of neuronal afferents in mice neocortex triggers complex glial synaptic currents (GSCs) mediated by NMDA, P2X and AMPA receptors and glutamate transporters. In addition, astrocytes demonstrate spontaneous ‘miniature’ GSCs resulting from quantal release of neurotransmitters. Here, we demonstrate that maturation and aging of the brain of mice (from 1 to 21 months) affect the density of ionotropic receptors in astrocytes and their role in GSCs generation. The AMPA-receptor-mediated component is the largest in young animals and progressively declines with age. The P2X and NMDA components of GSC are smallest in young, maximal in adult (3 and 6 months old) and once more decrease in old mice, probably reflecting the remodelling of neuronal-glial circuitry. Our results demonstrate that fast synaptic transmission between neurones and astrocytes in neocortex that may be involved in information processing in neuronal-glial networks undergoes remodelling during brain maturation and aging.