Human induced pluripotent stem cell (hiPSC)-derived neural stem cells (NSCs) and their differentiated neuronal/glial derivatives have been recently considered suitable to assess in vitro developmental neurotoxicity (DNT) triggered by exposure to environmental chemicals. The use of human-relevant test systems combined with in vitro assays specific for different neurodevelopmental events, enables a mechanistic understanding of the possible impact of environmental chemicals on the developing brain, avoiding extrapolation uncertainties associated with in vivo studies. Currently proposed in vitro battery for regulatory DNT testing accounts for several assays suitable to study key neurodevelopmental processes, including NSC proliferation and apoptosis, differentiation into neurons and glia, neuronal migration, synaptogenesis, and neuronal network formation. However, assays suitable to measure interference of compounds with neurotransmitter release or clearance are at present not included, which represents a clear gap of the biological applicability domain of such a testing battery. Here we applied a HPLC-based methodology to measure the release of neurotransmitters in a previously characterized hiPSC-derived NSC model undergoing differentiation towards neurons and glia. Glutamate release was assessed in control cultures and upon depolarization, as well as in cultures repeatedly exposed to some known neurotoxicants (BDE47 and lead) and chemical mixtures. Obtained data indicate that these cells have the ability to release glutamate in a vesicular manner, and that both glutamate clearance and vesicular release concur in the maintenance of extracellular glutamate levels. In conclusion, analysis of neurotransmitter release is a sensitive readout that should be included in the envisioned battery of in vitro assays for DNT testing.

Vesicular release is one of the release mechanisms of various signaling molecules. In neurons, the molecular machinery involved in vesicular release has been designed through evolution to trigger fast and synchronous release of neurotransmitters. Similar machinery with a slower kinetic and a slightly different molecular assembly allows astrocytes to release various transmitters such as adenosine triphosphate (ATP), glutamate, and D-serine. Astrocytes are important modulators of neurotransmission through gliotransmitter release. We recently demonstrated that microglia, another type of glia, release ATP to modulate synaptic transmission using astrocytes as intermediate. We now report that microglia regulate astrocytic gliotransmission through the regulation of SNARE proteins in astrocytes. Indeed, we found that gliotransmission triggered by P2Y1 agonist is impaired in slices from transgenic mice devoid of microglia. Using total internal reflection fluorescence imaging, we found that the vesicular release of gliotransmitter by astrocytes was different in cultures lacking microglia compared to vesicular release in astrocytes cocultured with microglia. Quantification of the kinetic of vesicular release indicates that the overall release appears to be faster in pure astrocyte cultures with more vesicles close to the membrane when compared to astrocytes cocultured with microglia. Finally, biochemical investigation of SNARE protein expression indicates an upregulation of VAMP2 in absence of microglia. Altogether, these results indicate that microglia seems to be involved in the regulation of an astrocytic phenotype compatible with proper gliotransmission. The mechanisms described in this study could be of importance for central nervous system diseases where microglia are activated.

Human embryonic kidney 293 (HEK293) cells stably transfected with the rat P2X2 receptor subunit were preincubated with 200 nM progesterone (HEK293-P2X2-PROG), a potent positive allosteric modulator of homomeric P2X2 receptors, and used to detect low nanomolar concentrations of extracellular ATP. Fura-2-loaded HEK293-P2X2-PROG cells were acutely plated on top of cultured DH glial cells to quantify ATP release from single DH glial cells. Application of the α1 adrenoceptor agonist phenylephrine (PHE, 20 μM) or of a low K+ (0.2 mM) solution evoked reversible increases in the intracellular calcium concentration ([Ca2+]i) in the biosensor cells. A reversible increase in [Ca2+]i was also detected in half of the biosensor cells following the interruption of general extracellular perfusion. All increases in [Ca2+]i were blocked in the presence of the P2X2 antagonist PPADS or after preloading the glial cells with the calcium chelator BAPTA, indicating that they were due to calcium-dependent ATP release from the glial cells. ATP release induced by PHE was blocked by -L-phenylalanine 2-naphtylamide (GPN) that permeabilizes secretory lysosomes and bafilomycin A1 (Baf A1), an inhibitor of the H+-pump of acidic secretory vesicles. By contrast, ATP release induced by application of a low-K+ solution was abolished by Baf A1 but not by GPN. Finally, spontaneous ATP release observed after interrupting general perfusion was insensitive to both GPN and Baf A1 pretreatment. Our results indicate that ATP is released in a calcium-dependent manner from two distinct vesicular pools and one non-vesicular pool coexisting in DH glial cells and that noradrenaline and PHE selectively target the secretory lysosome pool.

The precision and reliability of synaptic information transfer depend on the molecular organization of voltage-gated calcium channels (VGCCs) within the presynaptic membrane. Alternative splicing of exon 47 affects the C-terminal structure of VGCCs and their affinity to intracellular partners and synaptic vesicles (SVs). We show that hippocampal synapses expressing VGCCs either with exon 47 (CaV2.1+47) or without (CaV2.1Δ47) differ in release probability and short-term plasticity. Tracking single channels revealed transient visits (∼100 ms) of presynaptic VGCCs in nanodomains (∼80 nm) that were controlled by neuronal network activity. Surprisingly, despite harboring prominent binding sites to scaffold proteins, CaV2.1+47 persistently displayed higher mobility within nanodomains. Synaptic accumulation of CaV2.1 was accomplished by optogenetic clustering, but only CaV2.1+47 increased transmitter release and enhanced synaptic short-term depression. We propose that exon 47-related alternative splicing of CaV2.1 channels controls synapse-specific release properties at the level of channel mobility-dependent coupling between VGCCs and SVs.

Calcium diffusion in the thin 100 nm layer located between the plasma membrane and docked vesicles in the pre-synaptic terminal of neuronal cells mediates vesicular fusion and synaptic transmission. Accounting for the narrow-cusp geometry located underneath the vesicle is a key ingredient that defines the probability and the time scale of calcium diffusion to bind calcium sensors for the initiation of vesicular release. We review here the time scale, the calcium binding dynamics and the consequences for asynchronous versus synchronous release. To conclude, three-dimensional modeling approaches and the associated coarse-grained simulations can now account efficiently for the precise co-organization of vesicles and Voltage-Gated-Calcium-Channel (VGCC). This co-organization is a key determinant of short-term plasticity and it shapes asynchronous release. Moreover, changing the location of VGCC from few nanometers underneath the vesicle modifies significantly the release probability. Finally, by modifying the calcium buffer concentration, a single synapse can switch from facilitation to depression.

Auxiliary α2δ subunits are important proteins for trafficking of voltage-gated calcium channels (CaV) at the active zones of synapses. We have previously shown that the post-translational proteolytic cleavage of α2δ is essential for their modulatory effects on the trafficking of N-type (CaV2.2) calcium channels (Kadurin et al., 2016). We extend these results here by showing that the probability of presynaptic vesicular release is reduced when an uncleaved α2δ is expressed in rat neurons and that this inhibitory effect is reversed when cleavage of α2δ is restored. We also show that asynchronous release is influenced by the maturation of α2δ-1, highlighting the role of CaV channels in this component of vesicular release. We present additional evidence that CaV2.2 co-immunoprecipitates preferentially with cleaved wild-type α2δ. Our data indicate that the proteolytic maturation increases the association of α2δ-1 with CaV channel complex and is essential for its function on synaptic release.

Endothelial cells participate in extracellular ATP release elicited by mechanosensors. To characterize the dynamic interactions between mechanical and chemical factors that modulate ATP secretion by the endothelium, we assessed and compared the mechanisms participating in the spontaneous (basal) and mechanically stimulated secretion using primary cultures of rat mesentery endothelial cells. ATP/metabolites were determined in the cell media prior to (basal) and after cell media displacement or a picospritzer buffer puff used as mechanical stimuli. Mechanical stimulation increased extracellular ATP that peaked within 1 min, and decayed to basal values in 10 min. Interruption of the vesicular transport route consistently blocked the spontaneous ATP secretion. Cells maintained in media lacking external Ca2+ elicited a spontaneous rise of extracellular ATP and adenosine, but failed to elicit a further extracellular ATP secretion following mechanical stimulation. 2-APB, a TRPV agonist, increased the spontaneous ATP secretion, but reduced the mechanical stimulation-induced nucleotide release. Pannexin1 or connexin blockers and gadolinium, a Piezo1 blocker, reduced the mechanically induced ATP release without altering spontaneous nucleotide levels. Moreover, thrombin or related agonists increased extracellular ATP secretion elicited by mechanical stimulation, without modifying spontaneous release. In sum, present results allow inferring that the spontaneous, extracellular nucleotide secretion is essentially mediated by ATP containing vesicles, while the mechanically induced secretion occurs essentially by connexin or pannexin1 hemichannel ATP transport, a finding fully supported by results from Panx1-/- rodents. Only the latter component is modulated by thrombin and related receptor agonists, highlighting a novel endothelium-smooth muscle signaling role of this anticoagulant.

Astrocytes are highly complex cells with many emerging putative roles in brain function. Of these, gliotransmission (active information transfer from glia to neurons) has probably the widest implications on our understanding of how the brain works: do astrocytes really contribute to information processing within the neural circuitry? \"Positive evidence\" for this stems from work of multiple laboratories reporting many examples of modulatory chemical signaling from astrocytes to neurons in the timeframe of hundreds of milliseconds to several minutes. This signaling involves, but is not limited to, Ca2+-dependent vesicular transmitter release, and results in a variety of regulatory effects at synapses in many circuits that are abolished by preventing Ca2+ elevations or blocking exocytosis selectively in astrocytes. In striking contradiction, methodologically advanced studies by a few laboratories produced \"negative evidence,\" triggering a heated debate on the actual existence and properties of gliotransmission. In this context, a skeptics\' camp arose, eager to dismiss the whole positive evidence based on a number of assumptions behind the negative data, such as the following: (1) deleting a single Ca2+ release pathway (IP3R2) removes all the sources for Ca2+-dependent gliotransmission; (2) stimulating a transgenically expressed Gq-GPCR (MrgA1) mimics the physiological Ca2+ signaling underlying gliotransmitter release; (3) age-dependent downregulation of an endogenous GPCR (mGluR5) questions gliotransmitter release in adulthood; and (4) failure by transcriptome analysis to detect vGluts or canonical synaptic SNAREs in astrocytes proves inexistence/functional irrelevance of vesicular gliotransmitter release. We here discuss how the above assumptions are likely wrong and oversimplistic. In light of the most recent literature, we argue that gliotransmission is a more complex phenomenon than originally thought, possibly consisting of multiple forms and signaling processes, whose correct study and understanding require more sophisticated tools and finer scientific experiments than done until today. Under this perspective, the opposing camps can be reconciled and the field moved forward. Along the path, a more cautious mindset and an attitude to open discussion and mutual respect between opponent laboratories will be good companions.Dual Perspectives Companion Paper: Multiple Lines of Evidence Indicate That Gliotransmission Does Not Occur under Physiological Conditions, by Todd A. Fiacco and Ken D. McCarthy.

Childhood lead (Pb2+) intoxication is a public health problem of global proportion. Lead exposure during development produces multiple effects on the central nervous system including impaired synapse formation, altered synaptic plasticity, and learning deficits. In primary hippocampal neurons in culture and hippocampal slices, Pb2+ exposure inhibits vesicular release and reduces the number of fast-releasing sites, an effect associated with Pb2+ inhibition of NMDA receptor-mediated trans-synaptic Brain-Derived Neurotrophic Factor (BDNF) signaling. The objective of this study was to determine if activation of TrkB, the cognate receptor for BDNF, would rescue Pb2+-induced impairments of vesicular release. Rats were chronically exposed to Pb2+ prenatally and postnatally until 50 days of age. This chronic Pb2+ exposure paradigm enhanced paired-pulse facilitation of synaptic potentials in Schaffer collateral-CA1 synapses in the hippocampus, a phenomenon indicative of reduced vesicular release probability. Decreased vesicular release probability was confirmed by both mean-variance analysis and direct 2-photon imaging of vesicular release from hippocampal slices of rats exposed to Pb2+in vivo. We also found a Pb2+-induced impairment of calcium influx in Schaffer collateral-CA1 synaptic terminals. Intraperitoneal injections of Pb2+ rats with the TrkB receptor agonist 7,8-dihydroxyflavone (5 mg/kg) for 14-15 days starting at postnatal day 35, reversed all Pb2+-induced impairments of presynaptic transmitter release at Schaffer collateral-CA1 synapses. This study demonstrates for the first time that in vivo pharmacological activation of TrkB receptors by small molecules such as 7,8-dihydroxyflavone can reverse long-term effects of chronic Pb2+ exposure on presynaptic terminals, pointing to TrkB receptor activation as a promising therapeutic intervention in Pb2+-intoxicated children.

Dynamic alteration of the synaptic strength is one of the most important processes occurring in the nervous system. Combination of electrophysiology, confocal imaging and molecular biology led to significant advances in this research field. Yet, a progress in this area, in particular in studies of changes in the quantal behavior of central synapses and impact of glial cells on individual synapses, is hampered by technical difficulties of resolving small quantal synaptic currents. In this paper we will show how the technique of non-enzymatic vibro-dissociation, which enables to isolate living neurons avoiding artifacts of cell culture and preserving functional synapse, can be used to obtain a valuable information on fine details and mechanisms of synaptic plasticity. In particular, we will describe our recent results on Ca2+-dependent modulation of the postsynaptic AMPA and NMDA receptors in the individual synaptic boutons.