Analysis of the Molecular Mechanisms Controlling Synaptic Transmission by Patch-clamp Recording in Brainstem Slices

Nihon Yakurigaku Zasshi. Folia Pharmacologica Japonica. Apr, 2003  |  Pubmed ID: 12777844

The first article describing the patch-clamp recording from neurons in the mammalian brain slice appeared in 1989. Since that article, there have been substantial scientific successes in the neuropharmacological and neurophysiological fields using this promising technique, which itself advanced largely owing to the progress in microscopic techniques such as infrared differential interference contrast (IR-DIC) video-enhanced microscopy. This article describes recent advances in the methods for the patch-clamp recording in the brainstem slices, which is now more and more important due to the increased needs in this post-genomic era for identification of the mechanisms underlying cell-to-cell communication in the central nervous system. Here we introduce some of the technical tips developed and being used in our laboratory, which include methods for making the best brainstem slices, pre-recording identification of neuron types using fluorescent tracers, markers, and green fluorescent protein (GFP) signal in transgenic mice. We also describe a method for rapid and secure drug application onto the recorded cell using electromagnetic valves, which we term the "macro Y-tube" method. These techniques may help to accelerate the understanding of the molecular mechanisms underlying dynamic regulation of central nervous function.

Differential Increases in P2X Receptor Levels in Rat Vagal Efferent Neurones Following a Vagal Nerve Section

Brain Research. Jul, 2003  |  Pubmed ID: 12788520

Extracellular ATP can influence cells via activation of P2X purinoceptors, the distribution of which can be altered in the central and peripheral nervous systems following injury or tissue damage. Here we have investigated the effect of a unilateral section of the cervical vagus nerve on the distribution of P2X(1), P2X(2), P2X(3), P2X(4) and P2X(7) receptor subunit immunoreactivity (R-IR) in the dorsal vagal motor nucleus (DVN) and the nucleus ambiguus (NA) in the medulla oblongata. As early as 2 days, and followed up to 14 days, there was a dramatic ipsilateral increase in P2X(1), P2X(2) and P2X(4)R-IR in the cell soma of vagal efferent neurones in the DVN following the nerve section, but not the NA. There were no changes in P2X(3) and P2X(7)R-IR in either nuclei. To test for possible functional consequences of increased P2X receptor levels, whole-cell patch-clamp recordings were made from DVN cells in brainstem slices 4 days following unilateral vagotomy. Application of ATP revealed large cell-to-cell variance in the current amplitude in neurones from both sectioned and control DVN. However, when ATP responses were compared to those elicited by the nicotinic acetylcholine receptor agonist carbachol, the mean ratio of the peak ATP-evoked current to the peak carbachol-evoked current was significantly larger in DVN neurones ipsilateral to the section. Thus the increase in P2XR levels in DVN cells ipsilateral to a nerve section are likely to reflect an increase in expression of functional P2XRs on the cell surface.

ATP- and Adenosine-mediated Signaling in the Central Nervous System: Synaptic Purinoceptors: the Stage for ATP to Play Its "dual-role"

Journal of Pharmacological Sciences. Feb, 2004  |  Pubmed ID: 14978346

The studies aiming to understand the function of purinoceptors in the central nervous system (CNS), which has been explored mostly in isolated and cultured cell systems, are now at the stage of identifying their physiological and pathophysiological significance in the native organs, tissues, and whole animals. The results of our recent studies made in brain slice preparations are not in full accordance with what have been demonstrated in isolated cells, mostly due to strong interplay between ATP receptors, adenosine receptors, and ecto-nucleotidases. This suggests that these proteins form coordinated regulation systems in the native tissue, controlling the local network behaviors through regulating the balance between the effects of ATP and adenosine on synaptic transmissions. We propose that this tripartite regulation system by extracellular purines may be an important target of CNS drugs.

Action Potential-independent Release of Glutamate by Ca2+ Entry Through Presynaptic P2X Receptors Elicits Postsynaptic Firing in the Brainstem Autonomic Network

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Mar, 2004  |  Pubmed ID: 15044552

P2X receptors are ATP-gated channels permeable to cations including Ca(2+). In acute slices containing the nucleus of the solitary tract, in which neuronal ATP release and ATP-elicited physiological responses are demonstrated in vivo, we recorded spontaneous action potential-independent EPSCs [miniature EPSCs (mEPSCs)]. Activation of presynaptic P2X receptors with alpha,beta-methylene ATP (alphabetamATP) triggered Ca(2+)-dependent glutamate release that was resistant to blockade of voltage-dependent calcium channels but abolished by P2X receptor antagonists. mEPSCs elicited with alphabetamATP were of larger amplitude than basal mEPSCs and resulted in postsynaptic firing caused by temporal summation of miniature events. The large-amplitude mEPSCs provoked by alphabetamATP were likely to result from highly synchronized multivesicular release of glutamate at single release sites. Neither alphabetamATP nor ATP facilitated GABA release. We conclude that this facilitated release and consequent postsynaptic firing underlie the profound autonomic responses to activation of P2X receptors observed in vivo.

A Dual-role Played by Extracellular ATP in Frequency-filtering of the Nucleus Tractus Solitarii Network

Advances in Experimental Medicine and Biology. 2004  |  Pubmed ID: 15602957

Blocker-resistant Presynaptic Voltage-dependent Ca2+ Channels Underlying Glutamate Release in Mice Nucleus Tractus Solitarii

Brain Research. Aug, 2006  |  Pubmed ID: 16814754

The visceral sensory information from the internal organs is conveyed via the vagus and glossopharyngeal primary afferent fibers and transmitted to the second-order neurons in the nucleus of the solitary tract (NTS). The glutamate release from the solitary tract (TS) axons to the second-order NTS neurons remains even in the presence of toxins that block N- and P/Q-type voltage-dependent Ca(2+) channels (VDCCs). The presynaptic VDCC playing the major role at this synapse remains unidentified. To address this issue, we examined two hypotheses in this study. First, we examined whether the remaining large component occurs through activation of a omega-conotoxin GVIA (omega-CgTX)-insensitive variant of N-type VDCC by using the mice genetically lacking its pore-forming subunit alpha(1B). Second, we examined whether R-type VDCCs are involved in transmitter release at the TS-NTS synapse. The EPSCs evoked by stimulation of the TS were recorded in medullary slices from young mice. omega-Agatoxin IVA (omega-AgaIVA; 200 nM) did not significantly affect the EPSC amplitude in the mice genetically lacking N-type VDCC. SNX-482 (500 nM) and Ni(2+) (100 microM) did not significantly reduce EPSC amplitude in ICR mice. These results indicate that, unlike in most of the brain synapses identified to date, the largest part of the glutamate release at the TS-NTS synapse in mice occurs through activation of non-L, non-P/Q, non-R, non-T and non-N (including its posttranslational variants) VDCCs at least according to their pharmacological properties identified to date.

Facilitation of Spontaneous Glycine Release by Anoxia Potentiates NMDA Receptor Current in the Hypoglossal Motor Neurons of the Rat

The European Journal of Neuroscience. Mar, 2007  |  Pubmed ID: 17408431

Deficiency in energy supply, such as occurs during hypoxia, anoxia, metabolic stress and mitochondrial failure, strongly affects the excitability of central neurons. Such lowered energy supply evokes various changes in spontaneous synaptic input to the hippocampal and cortical neurons. However, how this energy deprivation affects synaptic input to motor neurons, which are also vulnerable to energy deprivation, has never been addressed. Here we report for the first time the effect of metabolic stress on synaptic input to motor neurons by recording postsynaptic currents in the hypoglossal nucleus. Chemical anoxia with NaCN (1 mm) and anoxia with 95% N(2) induced a persistent inward current and a marked and robust increase in action potential-independent synaptic input. This increase was abolished by strychnine, but not by picrotoxin, CNQX or MK-801, indicating glycine release facilitation. Blockade of voltage-dependent Ca(2+) channels and extracellular Ca(2+) deprivation strongly attenuated this facilitation. The amplitude of inward currents evoked by local application of NMDA to the motor neurons in the presence of strychnine was significantly increased during NaCN application. A saturating concentration of d-serine occluded this potentiation, suggesting that released glycine activated the glycine-binding sites of NMDA receptors. By contrast, neurons in the dorsal motor nucleus of the vagus showed no detectable change in synaptic input in response to NaCN. These data suggest that increase in synaptically released glycine in response to metabolic stress may play an exacerbating role in NMDA receptor-mediated excitotoxicity in motor neurons.

[Purinergic Regulatory Complex in the Brain Synapses]

Nihon Shinkei Seishin Yakurigaku Zasshi = Japanese Journal of Psychopharmacology. Jun, 2007  |  Pubmed ID: 17633523

The fast and precise neuron-to-neuron signalling at the synapses is one of the most crucial processes in the central nervous system (CNS) function. Recent advances in the functional and morphological analysis of the brain synapses have identified adenosine 5'-triphosphate (ATP), a ubiquitous and most important molecule in the intracellular functions, to play important roles also as an extracellular messenger at synapses. Lines of evidence accumulated until today indicate that ATP (1) is released into the extracellular space particularly from astrocytes through specific mechanisms, (2) activates specific receptors for extracellular ATP, which modifies synaptic transmission, and (3) is hydrolysed to adenosine by ecto-nucleotidases, which in turn activates specific adenosine receptors modulating synaptic transmission. We have recently shown, using the patch-clamp recording of postsynaptic membrane currents in the acute brain slice preparations in vitro, that (1) ATP activates ATP-gated Ca2+ -permeable channels (P2X receptor channels) on presynaptic terminal membrane, triggering glutamate release without action potential, and (2) adenosine, produced from ATP in the extracellular milieu, activates presynaptic G protein-coupled receptors, which reduces Ca2+ entry through voltage-dependent Ca2+ channels and suppresses action potential-dependent transmitter release. These distinct mechanisms operate in synergy in various CNS structures and form the "purinergic regulatory complex" of the synaptic transmission.

Tracking Transmitter-gated P2X Cation Channel Activation in Vitro and in Vivo

Nature Methods. Jan, 2008  |  Pubmed ID: 18084300

We present a noninvasive approach to track activation of ATP-gated P2X receptors and potentially other transmitter-gated cation channels that show calcium fluxes. We genetically engineered rat P2X receptors to carry calcium sensors near the channel pore and tested this as a reporter for P2X(2) receptor opening. The method has several advantages over previous attempts to image P2X channel activation by fluorescence resonance energy transfer (FRET): notably, it reports channel opening rather than a conformation change in the receptor protein. Our FRET-based imaging approach can be used as a general method to track, in real time, the location, regional expression variation, mobility and activation of transmitter-gated P2X channels in living neurons in vitro and in vivo. This approach should help to determine when, where and how different receptors are activated during physiological processes.

Two Forms of Astrocyte Calcium Excitability Have Distinct Effects on NMDA Receptor-mediated Slow Inward Currents in Pyramidal Neurons

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jun, 2008  |  Pubmed ID: 18579739

Astrocytes display excitability in the form of intracellular calcium concentration ([Ca(2+)](i)) increases, but the signaling impact of these for neurons remains debated and controversial. A key unresolved issue is whether astrocyte [Ca(2+)](i) elevations impact neurons or not. Here we report that in the CA1 region of the hippocampus, agonists of native P2Y(1) and PAR-1 receptors, which are preferentially expressed in astrocytes, equally elevated [Ca(2+)](i) levels without affecting the passive membrane properties of pyramidal neurons. However, under conditions chosen to isolate NMDA receptor responses, we found that activation of PAR-1 receptors led to the appearance of NMDA receptor-mediated slow inward currents (SICs) in pyramidal neurons. In stark contrast, activation of P2Y(1) receptors was ineffective in this regard. The PAR-1 receptor-mediated increased SICs were abolished by several strategies that selectively impaired astrocyte [Ca(2+)](i) excitability and function. Our studies therefore indicate that evoked astrocyte [Ca(2+)](i) transients are not a binary signal for interactions with neurons, and that astrocytes result in neuronal NMDA receptor-mediated SICs only when appropriately excited. The data thus provide a basis to rationalize recent contradictory data on astrocyte-neuron interactions.

A Genetically Targeted Optical Sensor to Monitor Calcium Signals in Astrocyte Processes

Nature Neuroscience. Jun, 2010  |  Pubmed ID: 20495558

Calcium signaling is studied as a potential form of astrocyte excitability that may control astrocyte involvement in synaptic and cerebrovascular regulation. Fundamental questions remain unanswered about astrocyte calcium signaling, as current methods can not resolve calcium in small volume compartments, such as near the cell membrane and in distal cell processes. We modified the genetically encoded calcium sensor GCaMP2 with a membrane-tethering domain, Lck, increasing the level of Lck-GCaMP2 near the plasma membrane tenfold as compared with conventional GCaMP2. Using Lck-GCaMP2 in rat hippocampal astrocyte-neuron cocultures, we measured near-membrane calcium signals that were evoked pharmacologically or by single action potential-mediated neurotransmitter release. Moreover, we identified highly localized and frequent spontaneous calcium signals in astrocyte somata and processes that conventional GCaMP2 failed to detect. Lck-GCaMP2 acts as a genetically targeted calcium sensor for monitoring calcium signals in previously inaccessible parts of astrocytes, including fine processes.

Monitoring Astrocyte Calcium Microdomains with Improved Membrane Targeted GCaMP Reporters

Neuron Glia Biology. Dec, 2010  |  Pubmed ID: 21205365

Astrocytes are involved in synaptic and cerebrovascular regulation in the brain. These functions are regulated by intracellular calcium signalling that is thought to reflect a form of astrocyte excitability. In a recent study, we reported modification of the genetically encoded calcium indicator (GECI) GCaMP2 with a membrane-tethering domain, Lck, to generate Lck-GCaMP2. This GECI allowed us to detect novel microdomain calcium signals. The microdomains were random and ‘spotty’ in nature. In order to detect such signals more reliably, in the present study we further modified Lck-GCaMP2 to carry three mutations in the GCaMP2 moiety (M153K, T203V within EGFP and N60D in the CaM domain) to generate Lck-GCaMP3. We directly compared Lck-GCaMP2 and Lck-GCaMP3 by assessing their ability to monitor several types of astrocyte calcium signals with a focus on spotty microdomains. Our data show that Lck-GCaMP3 is between two- and four-times better than Lck-GCaMP2 in terms of its basal fluorescence intensity, signal-to-noise and its ability to detect microdomains. The use of Lck-GCaMP3 thus represents a significantly improved way to monitor astrocyte calcium signals, including microdomains, and will facilitate detailed exploration of their molecular mechanisms and physiological roles.

Bulk Loading of Calcium Indicator Dyes to Study Astrocyte Physiology: Key Limitations and Improvements Using Morphological Maps

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jun, 2011  |  Pubmed ID: 21697385

Calcium signaling has been studied in astrocyte cell bodies using bulk loading of calcium indicator dyes, and astrocytes are known to display intracellular calcium transients. An assumption in recent data on the neuronal impact of somatic astrocyte calcium transients has been that bulk loading reflects signaling in relevant astrocyte compartments such as processes. We assessed bulk loading using Sholl analysis (Sholl, 1953) of astrocytes loaded with common calcium indicator dyes and compared these data with Sholl analysis of astrocyte morphology. In the CA1 region of the hippocampus from rats, we found that bulk loading of calcium indicator dyes only reports on calcium signals within the soma and in the most proximal processes, leaving ∼90% of the area of an astrocyte and its extensive processes unsampled. By using morphological reconstructions as "maps" after the imaging session, we present simple procedures that remedy these shortfalls and permit reliable detection of calcium transients in distal astrocyte processes. The data thus reveal limitations in the interpretation of astrocyte calcium imaging data gathered with bulk loading and provide refinements to minimize these shortcomings.

[Synaptic Regulation by Astrocytes]

Nihon Yakurigaku Zasshi. Folia Pharmacologica Japonica. Oct, 2011  |  Pubmed ID: 21986065

Neuronal P2X2 Receptors Are Mobile ATP Sensors That Explore the Plasma Membrane when Activated

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Nov, 2011  |  Pubmed ID: 22090499

ATP-gated ionotropic P2X2 receptors are widely expressed in neurons. Although the electrophysiological properties of P2X2 receptors have been extensively studied, little is known about the plasma membrane lateral mobility of P2X2 receptors or whether receptor mobility is regulated by ATP. Here we used single-molecule imaging with simultaneous whole-cell voltage-clamp recordings to track quantum dot-labeled P2X2 receptors in the dendrites of rat hippocampal neurons to explore P2X2 receptor mobility and its regulation. We find that plasma membrane P2X2 receptor lateral mobility in dendrites is heterogeneous but mostly Brownian in nature, consisting of mobile and slowly mobile receptor pools. Moreover, lateral mobility is P2X2 subunit and cell specific, is increased in an activation-dependent manner, and is regulated by cytosolic VILIP1, a calcium binding protein. Our data provide the first direct measures of P2X receptor mobility and show that P2X2 receptors are mobile ATP sensors, sampling more of the dendritic plasma membrane in response to ATP.

TRPA1 Channels Regulate Astrocyte Resting Calcium and Inhibitory Synapse Efficacy Through GAT-3

Nature Neuroscience. Jan, 2012  |  Pubmed ID: 22158513

Astrocytes contribute to the formation and function of synapses and are found throughout the brain, where they show intracellular store-mediated Ca(2+) signals. Here, using a membrane-tethered, genetically encoded calcium indicator (Lck-GCaMP3), we report the serendipitous discovery of a new type of Ca(2+) signal in rat hippocampal astrocyte-neuron cocultures. We found that Ca(2+) fluxes mediated by transient receptor potential A1 (TRPA1) channels gave rise to frequent and highly localized 'spotty' Ca(2+) microdomains near the membrane that contributed appreciably to resting Ca(2+) in astrocytes. Mechanistic evaluations in brain slices showed that decreases in astrocyte resting Ca(2+) concentrations mediated by TRPA1 channels decreased interneuron inhibitory synapse efficacy by reducing GABA transport by GAT-3, thus elevating extracellular GABA. Our data show how a transmembrane Ca(2+) source (TRPA1) targets a transporter (GAT-3) in astrocytes to regulate inhibitory synapses.