Sergiy Sylantyev

Cannabinoid- and lysophosphatidylinositol-sensitive receptor GPR55 boosts neurotransmitter release at central synapses

G protein-coupled receptor (GPR) 55 is sensitive to certain cannabinoids, it is expressed in the brain and, in cell cultures, it triggers mobilization of intracellular Ca2+. However, the adaptive neurobiological significance of GPR55 remains unknown. Here, we use acute hippocampal slices and combine two-photon excitation Ca2+ imaging in presynaptic axonal boutons with optical quantal analysis in postsynaptic dendritic spines to find that GPR55 activation transiently increases release probability at individual CA3-CA1 synapses. The underlying mechanism involves Ca2+ release from presynaptic Ca2+ stores, whereas postsynaptic stores (activated by spot-uncaging of inositol 1,4,5-trisphosphate) remain unaffected by GPR55 agonists. These effects are abolished by genetic deletion of GPR55 or by the GPR55 antagonist cannabidiol, a constituent of Cannabis sativa. GPR55 shows colocalization with synaptic vesicle protein vesicular glutamate transporter 1 in stratum radiatum. Short-term potentiation of CA3-CA1 transmission after a short train of stimuli reveals a presynaptic, Ca2+ store-dependent component sensitive to cannabidiol. The underlying cascade involves synthesis of phospholipids, likely in the presynaptic cell, but not the endocannabinoids 2-arachidonoylglycerol or anandamide. Our results thus unveil a signaling role for GPR55 in synaptic circuits of the brain.

GABA-independent GABAA receptor openings maintain tonic currents.

Activation of GABAA receptors (GABAARs) produces two forms of inhibition: phasic inhibition generated by the rapid, transient activation of synaptic GABAARs by presynaptic GABA release, and tonic inhibition generated by the persistent activation of perisynaptic or extrasynaptic GABAARs, which can detect extracellular GABA. Such tonic GABAAR-mediated currents are particularly evident in dentate granule cells in which they play a major role in regulating cell excitability. Here we show that in rat dentate granule cells in ex vivo hippocampal slices, tonic currents are predominantly generated by GABA-independent GABAA receptor openings. This tonic GABAAR conductance is resistant to the competitive GABAAR antagonist SR95531 (gabazine), which at high concentrations acts as a partial agonist, but can be blocked by an open channel blocker, picrotoxin. When slices are perfused with 200 nm GABA, a concentration that is comparable to CSF concentrations but is twice that measured by us in the hippocampus in vivo using zero-net-flux microdialysis, negligible GABA is detected by dentate granule cells. Spontaneously opening GABAARs, therefore, maintain dentate granule cell tonic currents in the face of low extracellular GABA concentrations.

Electric Fields Due to Synaptic Currents Sharpen Excitatory Transmission

The synaptic response waveform, which determines signal integration properties in the brain, depends on the spatiotemporal profile of neurotransmitter in the synaptic cleft. Here, we show that electrophoretic interactions between AMPA receptor–mediated excitatory currents and negatively charged glutamate molecules accelerate the clearance of glutamate from the synaptic cleft, speeding up synaptic responses. This phenomenon is reversed upon depolarization and diminished when intracleft electric fields are weakened through a decrease in the AMPA receptor density. In contrast, the kinetics of receptor-mediated currents evoked by direct application of glutamate are voltage-independent, as are synaptic currents mediated by the electrically neutral neurotransmitter GABA. Voltage-dependent temporal tuning of excitatory synaptic responses may thus contribute to signal integration in neural circuits.

Hemichannel-mediated release of lactate

In the central nervous system lactate contributes to the extracellular pool of readily available energy substrates and may also function as a signaling molecule which mediates communication between glial cells and neurons. Monocarboxylate transporters are believed to provide the main pathway for lactate transport across the membranes. Here we tested the hypothesis that lactate could also be released via opening of pannexin and/or functional connexin hemichannels. In acute slices prepared from the brainstem, hippocampus, hypothalamus and cortex of adult rats, enzymatic amperometric biosensors detected significant tonic lactate release inhibited by compounds, which block pannexin/connexin hemichannels and facilitated by lowering extracellular [Ca2+] or increased PCO2. Enhanced lactate release triggered by hypoxia was reduced by ∼50% by either connexin or monocarboxylate transporter blockers. Stimulation of Schaffer collateral fibers triggered lactate release in CA1 area of the hippocampus, which was facilitated in conditions of low extracellular [Ca2+], markedly reduced by blockade of connexin hemichannels and abolished by lactate dehydrogenase inhibitor oxamate. These results indicate that lactate transport across the membranes may occur via mechanisms other than monocarboxylate transporters. In the central nervous system, hemichannels may function as a conduit of lactate release, and this mechanism is recruited during hypoxia and periods of enhanced neuronal activity.

Spike-driven glutamate electrodiffusion triggers synaptic potentiation via a homer-dependent mGluR-NMDAR link.

Summary Electric fields of synaptic currents can influence diffusion of charged neurotransmitters, such as glutamate, in the synaptic cleft. However, this phenomenon has hitherto been detected only through sustained depolarization of large principal neurons, and its adaptive significance remains unknown. Here, we find that in cerebellar synapses formed on electrically compact granule cells, a single postsynaptic action potential can retard escape of glutamate released into the cleft. This retardation boosts activation of perisynaptic group I metabotropic glutamate receptors (mGluRs), which in turn rapidly facilitates local NMDA receptor currents. The underlying mechanism relies on a Homer-containing protein scaffold, but not GPCR- or Ca2+-dependent signaling. Through the mGluR-NMDAR interaction, the coincidence between a postsynaptic spike and glutamate release triggers a lasting enhancement of synaptic transmission that alters the basic integrate-and-spike rule in the circuitry. Our results thus reveal an electrodiffusion-driven synaptic memory mechanism that requires high-precision coincidence detection suitable for high-fidelity circuitries.

Central synapses release a resource-efficient amount of glutamate

Why synapses release a certain amount of neurotransmitter is poorly understood. We combined patch-clamp electrophysiology with computer simulations to estimate how much glutamate is discharged at two distinct central synapses of the rat. We found that, regardless of some uncertainty over synaptic microenvironment, synapses generate the maximal current per released glutamate molecule while maximizing signal information content. Our result suggests that synapses operate on a principle of resource optimization.

A method for direct thalamic stimulation in fMRI studies using a glass-coated carbon fiber electrode

Recent fMRI studies are of interest in exploring long-range interactions between different brain structures and the functional activation of specific brain regions by known neuroanatomical pathways. One of the experimental approaches requires the invasive implantation of an intracranial electrode to excite specific brain structures. In the present report, we describe a procedure for the production of a glass-coated carbon fiber electrode and the use of this electrode for direct activation of the brain in fMRI studies. The glass-coated carbon fiber microelectrode was implanted in the medial thalamus of anaesthetized rats and T2*-weighted gradient echo images in the sagittal plane obtained on a 4.7 T system (Biospec BMT 47/40) during electrical stimulation of the medial thalamus. The image quality obtained using this electrode was acceptable without reduction of the signal-to-noise ratio and image distortion. Cross-correlation analysis showed that the signal intensities of activated areas in the ipsilateral anterior cingulate cortex were significantly increased by about 4-5% during medial thalamus stimulation. The present study shows that glass-coated carbon fiber electrodes are suitable for fMRI studies and can be used to investigate functional thalamocingulate activation.

Sub-millisecond ligand probing of cell receptors with multiple solution exchange

The accurate knowledge of receptor kinetics is crucial to our understanding of cell signal transduction in general and neural function in particular. The classical technique of probing membrane receptors on a millisecond scale involves placing a recording micropipette with a membrane patch in front of a double-barrel (θ-glass) application pipette mounted on a piezo actuator. Driven by electric pulses, the actuator can rapidly shift the θ-glass pipette tip, thus exposing the target receptors to alternating ligand solutions. However, membrane patches survive for only a few minutes, thus normally restricting such experiments to a single-application protocol. In order to overcome this deficiency, we have introduced pressurized supply microcircuits in the θ-glass channels, thus enabling repeated replacement of application solutions within 10–15 s. This protocol, which has been validated in our recent studies and takes 20–60 min to implement, allows the characterization of ligand-receptor interactions with high sensitivity, thereby also enabling a powerful paired-sample statistical design.

Short-term synaptic plasticity in layer II/III of the rat anterior cingulate cortex.

Recent in vivo electrophysiological studies in our laboratory demonstrated medial thalamus (MT) induced short-term facilitation in the middle layers of the anterior cingulate cortex (ACC). The aim of the present study was to investigate different forms of short-term plasticity (STP) in layer II/III of the ACC in an in vitro slice preparation. Extracellular field potentials in layer II/III consisting of an early component (fAP) and a late component (fPSP) were activated by electrical stimulation of the deep layers. The fPSP and intracellularly recorded excitatory post-synaptic potential (EPSP) could be facilitated by paired-pulse stimulation at a low frequency (0.033Hz, pulse interval 20-400ms). An initial facilitation and subsequent depression were obtained when high frequency (12.5, 25 and 50Hz) tetanus stimulations were applied to the ACC slice. A post-tetanic augmentation 30s in duration was also observed. The effects of tetanic stimulation were altered in the presence of an increased or a decreased calcium concentration. Application of omega-conotoxin GVIA (CTX) in normal calcium concentration conditions decreased overall responses during tetanic stimulation similar to reducing calcium exposure. However CTX application did not increase paired-pulse facilitation (PPF) as is seen under low calcium conditions. These results indicate that calcium is involved in the formation of certain features of STP in layer II/III of the ACC and that N-type calcium channels contribute to some, but not all, components of these plastic changes. Two-site electrical stimulation testing showed that two separate presynaptic inputs can produce short-term facilitation. Our findings implicate a post-synaptic mechanism in STP in layer II/III of the ACC.

The fish community in Zmiinyi Island waters: structure and determinants

The fish fauna assemblage and its association with environmental factors in the waters around Zmiinyi Island in the Black Sea were examined based on biological and hydrological data collected in the period 2003–2011. The aims of the study were to identify the fish species composition and community parameters in the area, to determine the contribution of different environmental factors to seasonal variations of the fish assemblage, and to assess the Danube outflow-induced shaping of the fish species community on a seasonal time scale. A total of 58 species belonging to 35 families were collected; most were of Mediterranean origin. The most abundant species were Atherina boyeri, Aidablennius sphynx, Engraulis encrasicolus, Merlangius merlangus, Neogobius melanostomus, Sprattus sprattus and Trachurus mediterraneus. The fish community in the area studied displayed pronounced seasonal variations. Mediterranean species were the group which predominantly determined the directions of such variations and the overall community structure. Data analysis revealed temperature and salinity to be the most important factors influencing the community assemblage. Despite the substantial amount of freshwater runoff from the adjacent Danube Delta, seasonal variations in this factor did not cause significant fluctuations in fish fauna composition.