Astrocytes regulate brain extracellular pH via a neuronal activity-dependent bicarbonate shuttle.

Brain cells continuously produce and release protons into the extracellular space, with the rate of acid production corresponding to the levels of neuronal activity and metabolism. Efficient buffering and removal of excess H+ is essential for brain function, not least because all the electrogenic and biochemical machinery of synaptic transmission is highly sensitive to changes in pH. Here, we describe an astroglial mechanism that contributes to the protection of the brain milieu from acidification. In vivo and in vitro experiments conducted in rodent models show that at least one third of all astrocytes release bicarbonate to buffer extracellular H+ loads associated with increases in neuronal activity. The underlying signalling mechanism involves activity-dependent release of ATP triggering bicarbonate secretion by astrocytes via activation of metabotropic P2Y1 receptors, recruitment of phospholipase C, release of Ca2+ from the internal stores, and facilitated outward HCO3- transport by the electrogenic sodium bicarbonate cotransporter 1, NBCe1. These results show that astrocytes maintain local brain extracellular pH homeostasis via a neuronal activity-dependent release of bicarbonate. The data provide evidence of another important metabolic housekeeping function of these glial cells.

Optical monitoring of glutamate release at multiple synapses in situ detects changes following LTP induction.

Information processing and memory formation in the brain relies on release of the main excitatory neurotransmitter glutamate from presynaptic axonal specialisations. The classical Hebbian paradigm of synaptic memory, long-term potentiation (LTP) of transmission, has been widely associated with an increase in the postsynaptic receptor current. Whether and to what degree LTP induction also enhances presynaptic glutamate release has been the subject of debate. Here, we took advantage of the recently developed genetically encoded optical sensors of glutamate (iGluSnFR) to monitor its release at CA3-CA1 synapses in acute hippocampal slices, before and after the induction of LTP. We attempted to trace release events at multiple synapses simultaneously, by using two-photon excitation imaging in fast frame-scanning mode. We thus detected a significant increase in the average iGluSnFR signal during potentiation, which lasted for up to 90 min. This increase may reflect an increased amount of released glutamate or, alternatively, reduced glutamate binding to high-affinity glutamate transporters that compete with iGluSnFR.

Serotonin 5-HT4 receptor boosts functional maturation of dendritic spines via RhoA-dependent control of F-actin.

Activity-dependent remodeling of excitatory connections underpins memory formation in the brain. Serotonin receptors are known to contribute to such remodeling, yet the underlying molecular machinery remains poorly understood. Here, we employ high-resolution time-lapse FRET imaging in neuroblastoma cells and neuronal dendrites to establish that activation of serotonin receptor 5-HT4 (5-HT4R) rapidly triggers spatially-restricted RhoA activity and G13-mediated phosphorylation of cofilin, thus locally boosting the filamentous actin fraction. In neuroblastoma cells, this leads to cell rounding and neurite retraction. In hippocampal neurons in situ, 5-HT4R-mediated RhoA activation triggers maturation of dendritic spines. This is paralleled by RhoA-dependent, transient alterations in cell excitability, as reflected by increased spontaneous synaptic activity, apparent shunting of evoked synaptic responses, and enhanced long-term potentiation of excitatory transmission. The 5-HT4R/G13/RhoA signaling thus emerges as a previously unrecognized molecular pathway underpinning use-dependent functional remodeling of excitatory synaptic connections.

Polymer microchamber arrays for geometry-controlled drug release: a functional study in human cells of neuronal phenotype.

Polyelectrolyte multilayer (PEM) microchambers can provide a versatile cargo delivery system enabling rapid, site-specific drug release on demand. However, experimental evidence for their potential benefits in live human cells is scarce. Equally, practical applications often require substance delivery that is geometrically constrained and highly localized. Here, we establish human-cell biocompatibility and on-demand cargo release properties of the PEM or polylactic acid (PLA)-based microchamber arrays fabricated on a patterned film base. We grow human N2A cells (a neuroblastoma cell line widely used for studies of neurotoxicity) on the surface of the patterned microchamber arrays loaded with either a fluorescent indicator or the ubiquitous excitatory neurotransmitter glutamate. The differentiating human N2A cells show no detrimental effects on viability when growing on either PEM@PLA or PLA-based arrays for up to ten days in vitro. Firstly, we use two-photon (2P) excitation with femtosecond laser pulses to open individual microchambers in a controlled way while monitoring release and diffusion of the fluorescent cargo (rhodamine or FITC fluorescent dye). Secondly, we document the increases in intracellular Ca2+ in local N2A cells in response to the laser-triggered glutamate release from individual microchambers. The functional cell response is site-specific and reproducible on demand and could be replicated by applying glutamate to the cells using a pressurised micropipette. Time-resolved fluorescence imaging confirms the physiological range of the glutamate-evoked intracellular Ca2+ dynamics in the differentiating N2A cells. Our data indicate that the nano-engineering design of the fabricated PEM or PLA-based patterned microchamber arrays could provide a biologically safe and efficient tool for targeted, geometrically constrained drug delivery.

Synaptic Remodeling Depends on Signaling between Serotonin Receptors and the Extracellular Matrix.

Rewiring of synaptic circuitry pertinent to memory formation has been associated with morphological changes in dendritic spines and with extracellular matrix (ECM) remodeling. Here, we mechanistically link these processes by uncovering a signaling pathway involving the serotonin 5-HT7 receptor (5-HT7R), matrix metalloproteinase 9 (MMP-9), the hyaluronan receptor CD44, and the small GTPase Cdc42. We highlight a physical interaction between 5-HT7R and CD44 (identified as an MMP-9 substrate in neurons) and find that 5-HT7R stimulation increases local MMP-9 activity, triggering dendritic spine remodeling, synaptic pruning, and impairment of long-term potentiation (LTP). The underlying molecular machinery involves 5-HT7R-mediated activation of MMP-9, which leads to CD44 cleavage followed by Cdc42 activation. One important physiological consequence of this interaction includes an increase in neuronal outgrowth and elongation of dendritic spines, which might have a positive effect on complex neuronal processes (e.g., reversal learning and neuronal regeneration).

Astrocytic GABA transporter activity modulates excitatory neurotransmission.

Astrocytes are ideally placed to detect and respond to network activity. They express ionotropic and metabotropic receptors, and can release gliotransmitters. Astrocytes also express transporters that regulate the extracellular concentration of neurotransmitters. Here we report a previously unrecognized role for the astrocytic GABA transporter, GAT-3. GAT-3 activity results in a rise in astrocytic Na+ concentrations and a consequent increase in astrocytic Ca2+ through Na+/Ca2+ exchange. This leads to the release of ATP/adenosine by astrocytes, which then diffusely inhibits neuronal glutamate release via activation of presynaptic adenosine receptors. Through this mechanism, increases in astrocytic GAT-3 activity due to GABA released from interneurons contribute to 'diffuse' heterosynaptic depression. This provides a mechanism for homeostatic regulation of excitatory transmission in the hippocampus.

Depletion of extracellular Ca²âº prompts astroglia to moderate synaptic network activity.

Over the past decade, rapid signal exchange between astroglia and neurons across the interstitial space emerged as an essential element of synaptic circuit functioning in the brain. How and where exactly this exchange occurs in various physiological scenarios and the underlying cellular cascades remain a subject of intense study. The excitatory neurotransmitter glutamate and the inhibitory neurotransmitter γ-aminobutyric acid are thought to be the primary signal carriers that are regularly dispatched by active synapses to engage target receptors and transporters on the surface of astrocytes. New evidence identifies another ubiquitous messenger, extracellular calcium ions (Ca(2+)), which can report neural network activity to astroglia. Astrocytes in the hippocampus can respond to activity-induced partial Ca(2+) depletion in the extracellular space by generating prominent intracellular Ca(2+) waves. The underlying Ca(2+) sensing mechanism is proposed to involve the opening of the hemichannel connexin 43 in astrocytes, which in turn triggers the release of adenosine triphosphate to boost the activity of inhibitory interneurons, thus potentially providing negative feedback to tame excessive excitatory activity of neural circuits.

Long-term potentiation depends on release of D-serine from astrocytes.

Long-term potentiation (LTP) of synaptic transmission provides an experimental model for studying mechanisms of memory. The classical form of LTP relies on N-methyl-D-aspartate receptors (NMDARs), and it has been shown that astroglia can regulate their activation through Ca(2+)-dependent release of the NMDAR co-agonist D-serine. Release of D-serine from glia enables LTP in cultures and explains a correlation between glial coverage of synapses and LTP in the supraoptic nucleus. However, increases in Ca(2+) concentration in astroglia can also release other signalling molecules, most prominently glutamate, ATP and tumour necrosis factor-alpha, whereas neurons themselves can synthesize and supply D-serine. Furthermore, loading an astrocyte with exogenous Ca(2+) buffers does not suppress LTP in hippocampal area CA1 (refs 14-16), and the physiological relevance of experiments in cultures or strong exogenous stimuli applied to astrocytes has been questioned. The involvement of glia in LTP induction therefore remains controversial. Here we show that clamping internal Ca(2+) in individual CA1 astrocytes blocks LTP induction at nearby excitatory synapses by decreasing the occupancy of the NMDAR co-agonist sites. This LTP blockade can be reversed by exogenous D-serine or glycine, whereas depletion of D-serine or disruption of exocytosis in an individual astrocyte blocks local LTP. We therefore demonstrate that Ca(2+)-dependent release of D-serine from an astrocyte controls NMDAR-dependent plasticity in many thousands of excitatory synapses nearby.