Characteristics Of Synaptic Transmission Research Articles

Monitoring Interneuron-Astrocyte Signaling and Its Consequences on Synaptic Transmission.

Whole-cell patch clamp allows the characterization of synaptic transmission in neurons. It is possible to manipulate astrocytic activity and record how these glial cells affect neuronal networks. Here we describe the methodology to monitor the endogenously activation of astrocytes by inhibitory synaptic activity. Afterward, such glial activation will let us study the consequences of interneuron-astrocyte signaling on excitatory neurotransmission at hippocampal synapses.

Elementary properties of Ca(2+) channels and their influence on multivesicular release and phase-locking at auditory hair cell ribbon synapses.

Voltage-gated calcium (Cav1.3) channels in mammalian inner hair cells (IHCs) open in response to sound and the resulting Ca2+ entry triggers the release of the neurotransmitter glutamate onto afferent terminals. At low to mid sound frequencies cell depolarization follows the sound sinusoid and pulses of transmitter release from the hair cell generate excitatory postsynaptic currents (EPSCs) in the afferent fiber that translate into a phase-locked pattern of action potential activity. The present article summarizes our current understanding on the elementary properties of single IHC Ca2+ channels, and how these could have functional implications for certain, poorly understood, features of synaptic transmission at auditory hair cell ribbon synapses.

Subcellular structural plasticity caused by the absence of the fast Ca(2+) buffer calbindin D-28k in recurrent collaterals of cerebellar Purkinje neurons.

Purkinje cells (PC) control spike timing of neighboring PC by their recurrent axon collaterals. These synapses underlie fast cerebellar oscillations and are characterized by a strong facilitation within a time window of <20 ms during paired-pulse protocols. PC express high levels of the fast Ca2+ buffer protein calbindin D-28k (CB). As expected from the absence of a fast Ca2+ buffer, presynaptic action potential-evoked [Ca2+]i transients were previously shown to be bigger in PC boutons of young (second postnatal week) CB-/- mice, yet IPSC mean amplitudes remained unaltered in connected CB–/– PC. Since PC spine morphology is altered in adult CB–/– mice (longer necks, larger spine head volume), we summoned that morphological compensation/adaptation mechanisms might also be induced in CB–/– PC axon collaterals including boutons. In these mice, biocytin-filled PC reconstructions revealed that the number of axonal varicosities per PC axon collateral was augmented, mostly confined to the granule cell layer. Additionally, the volume of individual boutons was increased, evidenced from z-stacks of confocal images. EM analysis of PC–PC synapses revealed an enhancement in active zone (AZ) length by approximately 23%, paralleled by a higher number of docked vesicles per AZ in CB–/– boutons. Moreover, synaptic cleft width was larger in CB–/– (23.8 ± 0.43 nm) compared to wild type (21.17 ± 0.39 nm) synapses. We propose that the morphological changes, i.e., the larger bouton volume, the enhanced AZ length and the higher number of docked vesicles, in combination with the increase in synaptic cleft width likely modifies the GABA release properties at this synapse in CB–/– mice. We view these changes as adaptation/homeostatic mechanisms to likely maintain characteristics of synaptic transmission in the absence of the fast Ca2+ buffer CB. Our study provides further evidence on the functioning of the Ca2+ homeostasome.

PUNISHER: a high-level expression cassette for use with recombinant viral vectors for rapid and long term in vivo neuronal expression in the CNS

Fast onset and high-level neurospecific transgene expression in vivo is of importance for many areas in neuroscience, from basic to translational, and can significantly reduce the amount of vector load required to maintain transgene expression in vivo. In this study, we tested various cis elements to optimize transgene expression at transcriptional, posttranscriptional, and posttranslational levels and combined them together to create the high-level neuronal transgene expression cassette pUNISHER. Using a second-generation adenoviral vector system in combination with the pUNISHER cassette, we characterized its rate of onset of detectable expression and levels of expression compared with a neurospecific expression cassette driven by the 470-bp human synapsin promoter in vitro and in vivo. Our results demonstrate in primary neurons that the pUNISHER cassette, in a recombinant adenovirus type 5 background, led to a faster rate of onset of detectable transgene expression and higher level of transgene expression. More importantly, this cassette led to highly correlated neuronal expression in vivo and to stable transgene expression up to 30 days in the auditory brain stem with no toxicity on the characteristics of synaptic transmission and plasticity at the calyx of Held synapse. Thus the pUNISHER cassette is an ideal high-level neuronal expression cassette for use in vivo for neuroscience applications.

Voltage-Clamp Analysis of Synaptic Transmission at the Drosophila Larval Neuromuscular Junction

The Drosophila larval neuromuscular junction (NMJ) shares many structural and functional similarities to synapses in other animals, including humans. These include the basic feature of synaptic transmission as well as the molecular mechanisms regulating the synaptic vesicle cycle. Because of its large size, easy accessibility, and the well-characterized genetics, the fly NMJ remains an excellent model system for dissecting the cellular and molecular mechanisms of synaptic transmission. Although intracellular recording is particularly valuable in revealing membrane potential changes, it has several limitations. Primarily, it does not offer information on the kinetics of membrane currents associated with ion channels or synaptic receptors responsible for the potential change. Furthermore, the resting potential of the Drosophila body-wall muscle varies naturally such that the driving force also varies considerably, making it difficult to accurately compare the amplitude of minis (spontaneous miniature synaptic potentials) or evoked excitatory junction potentials (EJPs). Finally, accurate determination of quantal content based on minis and EJPs is possible only at low release conditions when nonlinear summation is not a major issue. The voltage-clamp technique can overcome these limitations by using negative feedback mechanisms to keep the cell membrane potential steady at any reasonable set points. In the large larval muscle cells of Drosophila, the two-electrode voltage-clamp (TEVC) method is used, in which one electrode monitors the cell membrane potential while the other electrode passes electric currents. This protocol introduces the application of TEVC in analysis of synaptic currents using the larval NMJ preparation.

Fabrication of Microelectrodes, Suction Electrodes, and Focal Electrodes

The Drosophila larval neuromuscular junction (NMJ) shares many structural and functional similarities to synapses in other animals, including humans. These include the basic feature of synaptic transmission, as well as the molecular mechanisms regulating the synaptic vesicle cycle. Because of its large size, easy accessibility, and well-characterized genetics, the fly NMJ remains an excellent model system for dissecting the cellular and molecular mechanisms of synaptic transmission. This protocol details some basic methods for manufacturing microelectrodes used for intracellular recording and two-electrode voltage-clamp (TEVC), and loose patch electrodes used for focal recording. In addition, a method is provided for manufacturing homemade suction electrodes used for nerve stimulation.

Electrophysiological Recording from a ‘Model’ Cell

The Drosophila larval neuromuscular junction (NMJ) shares many structural and functional similarities to synapses in other animals, including humans. These include the basic feature of synaptic transmission, as well as the molecular mechanisms regulating the synaptic vesicle cycle. Because of its large size, easy accessibility, and the well-characterized genetics, the fly NMJ remains an excellent model system for dissecting the cellular and molecular mechanisms of synaptic transmission. This protocol describes the basics of setting up and conducting electrophysiological experiments using a model cell. For the novice, a model cell provides a way to learn the operation of electrophysiology equipment and software without the anxiety of damaging living cells. This protocol also illustrates passive membrane properties such as the input resistance, capacitance, and time constant.

Focal Recording of Synaptic Currents from Single Boutons at the Drosophila Neuromuscular Junction

The Drosophila larval neuromuscular junction (NMJ) shares many structural and functional similarities to synapses in other animals, including humans. These include the basic feature of synaptic transmission, as well as the molecular mechanisms regulating the synaptic vesicle cycle. Because of its large size, easy accessibility, and the well-characterized genetics, the fly NMJ remains an excellent model system for dissecting the cellular and molecular mechanisms of synaptic transmission. Focal recording is an extracellular method designed for the study of synaptic activity of one or a few synaptic boutons rather than the ensemble activity of all of the boutons, as occurs with intracellular recording or the two-electrode voltage-clamp (TEVC) method. This is a useful technique for investigating the properties of different motor neurons that innervate the same muscle, applying statistical analysis to discrete synaptic events, and investigating the heterogeneity of synaptic release properties among boutons.

A procedure to label inner ear afferent nerve endings for calcium imaging

Characterization of synaptic transmission between the inner ear sensory cells and primary neuron dendrites has been hampered by the limited access to the postsynaptic terminals. Because direct physiological recording of postsynaptic currents are difficult to achieve, no information regarding the synaptic and dendritic events are available. This is due to the small size of the postsynaptic afferent nerve endings that do not allow a clear identification, and thus compromise direct electrophysiological recordings of the buttons. To study the physiology of afferent nerve endings, we have developed a two-photon imaging technique in cochlear and vestibular slice preparations from neonatal rats and turtles. This technique is based on a retrograde labeling of afferent nerve endings with high-affinity calcium-sensitive dyes. Dye filling was achieved by 6 h application of the dextran–amine conjugate of calcium green-1. Calcium changes were measured in afferent nerve endings in line scan and time lap mode. To address recording in a near-physiological situation, iontophoretic application of K + was performed in the area of the stereocilia whereas glutamate was applied at the basal pole of sensory hair cells. Both types of application cause a reversible and sustained increase of Ca 2+ in the button of afferent nerve fibers. Typical recordings are presented and potential interests for pharmacological studies of inner ear sensory cell synapses are discussed.

Effect of pyroglutamylasparagine amide on plastic characteristics of synaptic transmission in the hippocampus.

Preincubation of rat hippocampal slices with 0.05-0.5 microM pyroglutamylasparagine amide improved characteristics of long-term potentiation of focal responses in the synaptic system of Schaffer collaterals-CA1 field pyramids facilitating LTP development and increasing its amplitude and duration. Presumably, the positive modulation of plastic characteristics of synaptic transmission in the hippocampusis is responsible for facilitation of learning and memory induced by pyroglutamylasparagine.

Characterization of synaptic transmission in the ventrolateral periaqueductal gray of rat brain slices

Synaptic transmission evoked by focal stimulation in the ventrolateral periaqueductal gray was characterized using the whole-cell recording technique in rat brain slices. At resting membrane potential (−62±1 mV), focal stimulation (0.05–0.1 ms, 0.03 Hz) usually evoked a 6-cyano-7-nitroquinoxaline-2,3-dione-sensitive fast excitatory postsynaptic potential and a dl-2-amino-5-phosphonopentanoic acid-sensitive slow excitatory postsynaptic potential with a bicuculline-sensitive inhibitory postsynaptic potential in between. In the presence of kynurenic acid, bicuculline-sensitive inhibitory postsynaptic currents recorded in the voltage-clamp mode displayed a reversal potential of −68±3 mV, resembling GABA A receptor-mediated inhibitory postsynaptic currents. However, no GABA B receptor-mediated inhibitory postsynaptic current was evoked, even at stronger stimulating intensity. 6-Cyano-7-nitroquinoxaline-2,3-dione-sensitive fast excitatory postsynaptic currents were isolated by dl-2-amino-5-phosphonopentanoic acid plus bicuculline and dl-2-amino-5-phosphonopentanoic acid-sensitive slow fast excitatory postsynaptic currents by bicuculline plus 6-cyano-7-nitroquinoxaline-2,3-dione. Both types of excitatory postsynaptic current reversed at potentials near 0 mV. The I– V curve of slow fast excitatory postsynaptic currents or N-methyl- d-aspartate currents displayed a negative slope at potentials more negative than −30 mV in an Mg 2+-sensitive manner. The control postsynaptic currents reversed at potentials between −50 and −35 mV, inclined to the reversal potential of GABA A, but not glutamate, receptor channels. It is concluded that, in the ventrolateral periaqueductal gray, focal stimulation elicits both inhibitory and excitatory transmission, while the former is dominant. The inhibitory transmission is mediated by GABA A but not GABA B receptors. The excitatory transmission is mediated by glutamate acting on α-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate as well as N-methyl- d-aspartate receptors.

The modulation of calcium currents by the activation of mGluRs. Functional implications.

Glutamatergic transmission in the central nervous system (CNS) is mediated by ionotropic, ligand-gated receptors (iGluRs), and metabotropic receptors (mGluRs). mGluRs are coupled to GTP-binding regulatory proteins (G-proteins) and modulate different second messenger pathways. Multiple effects have been described following their activation; among others, regulation of fast synaptic transmission, changes in synaptic plasticity, and modification of the threshold for seizure generation. Some of the major roles played by the activation of mGluRs might depend on the modulation of high-voltage-activated (HVA) calcium (Ca2+) currents. Some HVA Ca2+ channels (N-, P-, and Q-type channels) are signaling components at most presynaptic active zones. Their mGluR-mediated inhibition reduces synaptic transmission. The interference, by agonists at mGluRs, on L-type channels might affect the repetitive neuronal firing behavior and the integration of complex events at the somatic level. In addition, the mGluR-mediated effects on voltage-gated Ca2+ signals have been suggested to strongly influence neurotoxicity. Rather different coupling mechanisms underlie the relation between mGluRs and Ca2+ currents: Together with a fast, membrane-delimited mechanism of action, much slower responses, involving intracellular second messengers, have also been postulated. In the recent past, the relative paucity of selective agonists and antagonists for the different subclasses of mGluRs had hampered the clear definition of the roles of mGluRs in brain function. However, the recent availability of new pharmacological tools is promising to provide a better understanding of the neuronal functions related to different mGluR subtypes. The analysis of the mGluR-mediated modulation of Ca2+ conductances will probably offer new insights into the characterization of synaptic transmission and the development of neuroprotective agents.

Characteristics of synaptic transmission in reinnervating rat skeletal muscle.

Synaptic transmission, and its sensitivity to the effects of 3,4-diaminopyridine (3,4-DAP) and the phosphatase 2,3-butanedione monoxime (BDM), was examined for "crushed fiber" preparations of rat extensor digitorum longus muscle undergoing reinnervation after nerve crush. While mean quantal content (m) of endplate potentials (EPPs) was low early during reinnervation (10-24 days after nerve crush), elevation of temperature or extracellular calcium concentration restored m toward normal. However, m achieved control values for reinnervating preparations exposed to 3,4-DAP. 3,4-DAP also activated quiescent motor nerve terminals: after exposure to this drug, synaptic transmission was detected as early as 8 days after nerve crush. BDM too activated quiescent regenerating motor nerve terminals and increased m to normal. It also prolonged EPP and endplate current decay, suggesting a pre-synaptic effect on the synchrony of transmitter release and/or a post-synaptic effect on the open time of acetylcholine-gated endplate channels. While the effects of temperature, extracellular calcium, 3,4-DAP, and BDM suggest that regenerating nerve terminals can mobilise a reserve of quanta, this reserve is abnormally low, since hemicholinium-3 caused rapid rundown of EPP amplitude at repetitively stimulated regenerating endplates.

Effects of ethimizol on frog neuromuscular junction

The effects were studied of ethimizol, a substance activating memory processes, on features of synaptic transmission during experiments on frog cutaneous pectoris muscle. It was found that the presynaptic action of ethimizol consists of raising the frequency of miniature potentials, when used at a concentration of 0.5–10 mM, and modulating quantal content of synaptic transmission due to changes in binomial quantal release parameters p and n when 0.5–2 mM ethimizol was used. This substance facilitated transmission at synapses with a low initial level of transmitter release. This substance facilitated transmission at synapses with a low initial level of transmitter release. Ethimizol was also found to have a postsynaptic action, consisting of reducing amplitude at a concentration of 5–10 mM and prolonging synaptic currents and potentials when concentrations of 0.5–10 mM were used. The latter effect produced a considerable increase in the time integral of endplate potentials. The postsynaptic action of ethimizol is perhaps seen in its effects on features of postsynaptic ionic channels. The effects of ethimizol are discussed with a view to how it may act within the central nervous system as a nonspecific modulator.

An intracellular study of synaptic transmission and dendritic morphology in sympathetic neurons of the chick embryo

The characteristics of synaptic transmission in whole embryonic avian sympathetic ganglia have been examined by intracellular recording. Neurons in lumbar paravertebral ganglia of chick embryos exhibit both fast nicotinic excitatory postsynaptic potentials (EPSPs) and non-cholinergic slow EPSPs. Fast nicotinic transmission is mediated by at least 3-5 convergent preganglionic inputs and can be detected at the earliest embryonic stage examined (Stage 38; 12 days of incubation). Two types of non-cholinergic slow EPSPs have been observed and distinguished by their time course and the resulting changes in input resistance. One of these slow synaptic potentials is mimicked by exogenously applied substance P, but not by exogenous luteinizing hormone-releasing hormone (LH-RH). Muscarinic agonists also evoke slow depolarizations in the ganglia, mediated at least in part by inhibition of the M-current. Intracellular labeling with horseradish peroxidase reveals cells with 5-10 primary dendrites at Stage 42 (16 days of incubation), the earliest stage to exhibit slow EPSPs. The active and passive membrane properties of avian sympathetic neurons, including the presence of the M-current, generally resemble those of adult mammalian and amphibian sympathetic neurons. Functional activity in chick sympathetic neurons is present at a developmental stage where both biochemical and morphological indices of synapse maturation are at low levels. Since this progression has also been observed in the avian ciliary ganglion, it may be of general relevance to neuronal development.

Characteristics of synaptic transmission between photoreceptors and horizontal cells

The hypothesis is further developed according to which darkness promotes and light abolishes liberation of a transmitter, from presynaptic terminals of photoreceptors, which depolarizes horizontal cells. Results indicate that the depolarization of horizontal cells by short electrical pulses passed through the retina {anode on the receptor surface, cathode in the vitreous) is the result of action of the current on presynaptic endings of receptors. The depolarization of these endings evidently leads to liberation of transmitter from the receptor. Experiments with electrical stimulation of the retina show that treatmentwith potassium cyanide disturbs synaptie transmission between receptor and horizontal cell. A potential is established in the horizontal cells, equal to their membrane potential in bright light, and this is evidently the true resting potential of these cells. Evidence in support of this hypothesis is the relative stability of the membrane potential of horizontal cells in light during changes of temperature. In darkness at higher temperatures, the membrane potential is significantly increased, possibly due to an increase in the rate of decomposition of the depolarizing transmitter. Evidence for this is given by an increase in steepness of the leading edge of the response of horizontal cells to electrical stimulation when the temperature is increased.

The biologically relevant parameter in nerve impulse trains.

An experiment is described, the results of which indicate that impulse rate is the information carrying parameter of impulse trains. The demodulation of such information occurs by low-pass filtering. This conceptual model is compatible with all the features of synaptic transmission. The implications of this model for the transmission of information in integrative neurons is discussed.

Reverberator Inhibitory Circuits in the Lateral Geniculate Nucleus of the Rat

A PROMINENT feature of synaptic transmission through the lateral geniculate nucleus (LGN) in the cat1–3 and in the rat4 is the large and prolonged depression of transmission which occurs following a single shock to the optic nerve. In the work reported here an attempt was made to discover the cause of this depression.