Molecules involved in the formation of synaptic connections in muscle and brain.

Synapses are highly specialized structures designed to guarantee precise and efficient communication between neurons and their target cells. Molecules of the extracellular matrix have an instructive role in the formation of the neuromuscular junction, the best-characterized synapse. In this review, the molecular mechanisms underlying these instructive signals will be discussed with particular emphasis on the receptors involved. Additionally, recent evidence for the involvement of specific adhesion complexes in the formation and modulation of synapses in the central nervous system will be reviewed. Synapses are specialized junctions between neurons and their target cells where information is transferred from the pre- to the postsynaptic cell. At most vertebrate synapses, this transfer is accomplished by the release of a specific neurotransmitter from the presynaptic nerve terminal. The release of neurotransmitter is initiated by the action potential and the subsequent influx of Ca(2+) into the presynaptic nerve terminal. This results in the rapid fusion of vesicles with the nerve membrane and the release of the neurotransmitter into the synaptic cleft. The neurotransmitter then diffuses across the cleft and binds to specific postsynaptic receptors, resulting in a change in the membrane potential of the postsynaptic cell. This can result in the generation of an action potential. The high precision of synaptic transmission requires that pre- and postsynaptic structures are both highly organized and in juxtaposition to each other. In addition, alterations in synaptic transmission are the basis of learning and memory and are likely to be accompanied by the remodeling of synaptic structures (Toni et al., 1999). Thus, the study of how synapses are formed during development is also of relevance for the understanding of the cellular and molecular processes involved in learning and memory. This review focuses on the molecular mechanisms involved in the formation and the function of synapses.     

Synaptic formations and modulations of synaptic transmissions between identified cerebellar neurons in culture.

1. Synaptic formations between a rat cerebellar granule cell and a Purkinje cell, and also between an inferior-olivary neuron and a Purkinje cell have been accomplished in culture. 2. The synaptic transmission between an inferior-olivary neuron and a Purkinje cell was far much more potent than that between a granule cell and a Purkinje cell in the culture, and the former always induced in a Purkinje cell an action potential followed by prolonged depolarization, which resembled a climbing fiber response in vivo. 3. Synaptic potentiation was induced by repetitive stimulation (2 Hz, 20 sec) of a granule cell, and the synaptic depression was induced by repetitive conjunctive stimulation of both a granule cell and an inferior-olivary neuron as in a slice preparation. 4. When repetitive stimulation of both neurons were given while the postsynaptic Purkinje cell was voltage-clamped at -80 mV, not the depression but the potentiation took place. When repetitive stimulation of a granule cell was coupled with the postsynaptic strong depolarization induced by direct outward current injection, the depression took place. These two experiments indicate that the postsynaptic depolarization during activation of a presynaptic granule cell is both necessary and sufficient to induce the depression, and that the potentiation is induced without the postsynaptic depolarization. 5. The quantal analysis on the synaptic transmission, where fluctuations of amplitudes of synaptic currents in a Purkinje cell induced by a single granule cell were measured, indicated that the synaptic potentiation involves the enhancement of transmitter release from a presynaptic granule cell and that the depression involves changes of postsynaptic receptors on a Purkinje cell.     

Shaping cellular form and function by autophagy

In addition to its familiar role in non-selective bulk degradation of cellular material, autophagy can also bring about specific changes in the structure and function of cells. Autophagy has been proposed to operate in a substrate-selective mode to carry out this function, although evidence to demonstrate selectivity has been lacking. A recent study of synapse formation in the nervous system of the nematode Caenorhabditis elegans now provides experimental evidence for substrate-selective autophagy. Synapses form when presynaptic cells contact their postsynaptic partners during development. This contact induces the assembly of synaptically-localized protein complexes in the postsynaptic cell that contain scaffolding proteins and neurotransmitter receptors. When presynaptic contact was blocked, autophagy in the postsynaptic cell was induced. Substrate selectivity was evident in this system: the gamma-aminobutyric acid type A receptor (GABA(A) receptor), an integral-membrane neurotransmitter receptor, trafficked from the cell surface to autophagosomes. By contrast, the acetylcholine receptor, a structurally-similar neurotransmitter receptor, remained on the cell surface. This result provides experimental support for the idea that autophagy can bring about changes in cell structure and behavior by degrading specific cellular proteins, particularly cell surface receptors that are often important for regulating cell growth, differentiation and function.     

The Effect of Desflurane on Neuronal Communication at a Central Synapse

Although general anesthetics are thought to modify critical neuronal functions, their impact on neuronal communication has been poorly examined. We have investigated the effect induced by desflurane, a clinically used general anesthetic, on information transfer at the synapse between mossy fibers and granule cells of cerebellum, where this analysis can be carried out extensively. Mutual information values were assessed by measuring the variability of postsynaptic output in relationship to the variability of a given set of presynaptic inputs. Desflurane synchronized granule cell firing and reduced mutual information in response to physiologically relevant mossy fibers patterns. The decrease in spike variability was due to an increased postsynaptic membrane excitability, which made granule cells more prone to elicit action potentials, and to a strengthened synaptic inhibition, which markedly hampered membrane depolarization. These concomitant actions on granule cells firing indicate that desflurane re-shapes the transfer of information between neurons by providing a less informative neurotransmission rather than completely silencing neuronal activity.     

Running to stand still

For synapses to form and function, neurotransmitter receptors must be recruited to a location on the postsynaptic cell in direct apposition to presynaptic neurotransmitter release. However, once receptors are inserted into the postsynaptic membrane, they are not fixed in place but are continually exchanged between synaptic and extrasynaptic regions, and they cycle between the surface and intracellular compartments. This article highlights and compares the current knowledge about the dynamics of acetylcholine receptors at the vertebrate peripheral neuromuscular junction and AMPA, N-methyl-D-aspartate, and gamma-aminobutyric acid receptors in central synapses.     

Interaction of postsynaptic receptor saturation with presynaptic mechanisms produces a reliable synapse

Synapses that reliably activate their postsynaptic targets typically release neurotransmitter with high probability, are not very sensitive to changes in calcium entry, and depress. We have determined the mechanisms that give rise to these characteristic features at the climbing fiber to Purkinje cell synapse. We find that saturation of presynaptic calcium entry, of presynaptic release, and of postsynaptic receptors combine to produce a postsynaptic response that is near maximal. Postsynaptic receptor saturation also accelerates recovery from depression, in part by accentuating a rapid calcium-dependent recovery phase. Thus, postsynaptic receptor saturation interacts with presynaptic mechanisms to produce highly reliable synapses that can effectively drive their targets even during sustained activation.     

Immunocytochemistry of glycine in small neurons of the granule cell areas of the guinea pig dorsal cochlear nucleus: a post-embedding ultrastructural study

The axon terminals of the acoustic nerve contact different part of the cochlear nucleus including granule cell areas. Little is known of the cell composition and neural circuits of granule cell areas present in the fusiform and upper polymorphic layers of the dorsal cochlear nucleus in the guinea pig. The present ultrastructural immunocytochemical study exploits the technique of post-embedding immunogold and silver intensification to reveal the characteristics of small neurons in granule cell areas. Few neurons (Golgi-stellate cells) use glycine as inhibitory neurotransmitter which is present in symmetric synaptic boutons with pleomorphic and flat vesicles. In contrast, most neurons (granule and unipolar brush cells) are not glycine-positive, and presumably not excitatory. Most of the large axons (mossy fibres) in granule areas are probably excitatory (glycine-negative and storing round synaptic vesicles) and contact unipolar brush cells forming large synapses or granule cell dendrites by small synapses. A few large glycinergic boutons (inhibitory) also contact unipolar brush cells. The excitatory circuit of mossy fibre-unipolar brush and granule cells may be inhibited by the glycinergic terminals from the few glycinergic cells (Golgi-stellate neurons) present within the granule cell areas. The latter are not contacted by large mossy-like glycine terminals.     

Shaping Cellular Form and Function by Autophagy

In addition to its familiar role in non-selective bulk degradation of cellular material, autophagy can also bring about specific changes in the structure and function of cells. Autophagy has been proposed to operate in a substrate-selective mode to carry out this function, although evidence to demonstrate selectivity has been lacking. A recent study of synapse formation in the nervous system of the nematode Caenorhabditis elegans now provides experimental evidence for substrate-selective autophagy. Synapses form when presynaptic cells contact their postsynaptic partners during development. This contact induces the assembly of synaptically-localized protein complexes in the postsynaptic cell that contain scaffolding proteins and neurotransmitter receptors. When presynaptic contact was blocked, autophagy in the postsynaptic cell was induced. Substrate selectivity was evident in this system: the g-aminobutyric acid type A receptor (GABA_A receptor), an integral-membrane neurotransmitter receptor, trafficked from the cell surface to autophagosomes. By contrast, the acetylcholine receptor, a structurally-similar neurotransmitter receptor, remained on the cell surface. This result provides experimental support for the idea that autophagy can bring about changes in cell structure and behavior by degrading specific cellular proteins, particularly cell surface receptors that are often important for regulating cell growth, differentiation and function.

Addendum to:

Presynaptic Terminals Independently Regulate Synaptic Clustering and Autophagy of GABA_A Receptors in Caenorhabditis elegans

.A.M. Rowland, J.E. Richmond, J.G. Olsen, D.H. Hall and B. A. Bamber

J Neurosci 2006; 26:1711-20     

Ligand-Induced Cell Adhesion as a New Mechanism to Promote Synapse Formation

The formation of neuronal synapses is a finely organized process that involves the presynaptic assembly of the machinery responsible for neurotransmitter release and the postsynaptic recruitment of neurotransmitter receptors and scaffold proteins to the postsynaptic density (PSD). The molecular cues guiding the establishment of synaptic connections are now beginning to be identified. Recent evidences indicate that cell adhesion molecules (CAMs) participate prominently in the key steps of synapse formation, inducing trans-synaptic adhesion and promoting a precise alignment of pre- and postsynaptic terminals. This addendum describes a new mechanism of cell-cell interaction that combines features of both diffusible and membrane-bound synaptogenic factors. It particularly points out the key role played by GDNF triggering trans-homophilic binding between GFRα1 molecules and cell adhesion between GFRα1-expressing cells. In this model GFRα1 functions as a ligand-induced cell adhesion molecule (LICAM) to establish precise synaptic contacts and promote the assembly of presynaptic terminals. In this overview, I summarize the current concepts of synapse formation in the limelight of this new mechanism of ligand-induced cell adhesion.     

Signaling at the vertebrate synapse: new roles for embryonic morphogens?

The formation of synapses is critical for functional neuronal connectivity. The coordinated assembly at both sides of the synapse is fundamental for the proper apposition of the neurotransmitter release machinery on the presynaptic neuron and the clustering of neurotransmitter receptors and ion channels on the receptive postsynaptic cell. This process requires bidirectional communication between the presynaptic neuron and its postsynaptic target, another neuron, or muscle fiber. Extracellular signals such as WNT, TGF-beta, and FGF factors are emerging as key target-derived signals required for the initial stages of synaptic assembly. Studies in invertebrates are also providing new insights into the function of these signals in synaptic growth and homeostasis. During early embryonic patterning, WNT, TGF-beta, and FGF factors function as typical morphogens in a concentration-dependent manner to regulate cell fate decisions. This mode of action raises the provocative idea that these same morphogens might also provide a coordinate system for axons to establish the distance to their targets during axon guidance and synapse formation.     

Orchestration of neuronal migration by activity of ion channels,neurotransmitter receptors,and intracellular Ca2+ fluctuations

The real-time observation of cell movement in acute cerebellar slices reveals that granule cells alter their shape concomitantly with changes in the mode and rate of migration as they traverse different cortical layers. Although the origin of local environmental cues responsible for these position-specific changes in migratory behavior remains unclear, several signaling mechanisms involved in controlling granule cell movement have emerged. The onset of one such mechanism is marked by the expression of voltage-gated ion channels and neurotransmitter receptors in postmitotic cells prior to the initiation of their migration. Granule cells start their radial migration after the expression of N-type Ca²⁺ channels and the N-methyl-D -aspartate subtype of glutamate receptors on the plasmalemmal surface. Blockade of the channel or receptor activity significantly decreases the rate of cell movement, indicating that the activation of these membrane constituents provides an essential signal for the translocation of granule cells. Another signal that controls the rate of cell migration is embedded in the combined amplitude and frequency components of Ca²⁺ fluctuations in the somata of migrating granule cells. Interestingly, each phase of Ca²⁺ fluctuation controls a separate phase of saltatory movement in the granule cells: The cells move forward during the phase of transient Ca²⁺ elevation and remain stationary during the troughs. Consequently, the changes in the amplitude and frequency components of Ca²⁺ fluctuations directly affect granule cell movement: Reducing the amplitude or frequency of Ca²⁺ fluctuations slows down the speed of cell movement, while the enhancement of these components accelerates migration. These findings suggest that signaling molecules present in the local cellular milieu encountered on the migratory route control the shape and motility of granule cells by modifying Ca²⁺ fluctuations in the soma through the activation of specific ion channels and neurotransmitter receptors. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 110–130, 1998     

Molecular architecture of glycinergic synapses

Synapses can be considered chemical machines, which are optimized for fast and repeated exocytosis of neurotransmitters from presynaptic nerve terminals and the reliable electrical or chemical transduction of neurotransmitter binding to the appropriate receptors in the postsynaptic membrane. Therefore, synapses share a common repertoire of proteins like, e.g., the release machinery and certain cell adhesion molecules. This basic repertoire must be extended in order to generate specificity of neurotransmission and allow plastic changes, which are considered the basis of developmental and/or learning processes. Here, we focus on these complementary molecules located in the presynaptic terminal and postsynaptic membrane specializations of glycinergic synapses. Moreover, as specificity of neurotransmission in this system is established by the specific binding of the neurotransmitter to its receptor, we review the molecular properties of glycine receptor subunits and their assembly into functional glycine receptors with different functional characteristics. The past years have revealed that the molecular machinery underlying inhibitory and especially glycinergic postsynaptic membrane specializations is more complex and dynamic than previously anticipated from morphological studies. The emerging features include structural components as well as signaling modules, which could confer the plasticity required for the proper function of distinct motor and sensory functions.     

The neuron-specific K-Cl cotransporter, KCC2. Antibody development and initial characterization of the protein

The neuron-specific K-Cl cotransporter (KCC2) is hypothesized to function as an active Cl- extrusion pathway important in postsynaptic inhibition mediated by ligand-gated anion channels, like gamma-aminobutyric acid type A (GABAA) and glycine receptors. To understand better the functional role of KCC2 in the nervous system, we developed polyclonal antibodies to a KCC2 fusion protein and used these antibodies to characterize and localize KCC2 in the rat cerebellum. The antibodies specifically recognized the KCC2 protein which is an approximately 140-kDa glycoprotein detectable only within the central nervous system. The KCC2 protein displayed a robust and punctate distribution in primary cultured retinal amacrine cells known to form exclusively GABAAergic synapses in culture. In immunolocalization studies, KCC2 was absent from axons and glia but was highly expressed at neuronal somata and dendrites, indicating a specific postsynaptic distribution of the protein. In the granule cell layer, KCC2 exhibited a distinct colocalization with the beta2/beta3-subunits of the GABAA receptor at the plasma membrane of granule cell somata and at cerebellar glomeruli. KCC2 lightly labeled the plasma membrane of Purkinje cell somata. Within the molecular layer, KCC2 exhibited a distinctly punctate distribution along dendrites, indicating it may be highly localized at inhibitory synapses along these processes. The distinct postsynaptic localization of KCC2 and its colocalization with GABAA receptor in the cerebellum are consistent with the putative role of KCC2 in neuronal Cl- extrusion and postsynaptic inhibition.     

Synchronization of olfactory bulb mitral cells by precisely timed inhibitory inputs

Synchronized oscillatory activity at the gamma frequency (30-70 Hz) is thought to be important for information processing in many sensory systems. Here, I used patch-clamp recordings in neuron pairs in rat olfactory bulb slices to assess the mechanisms underlying such "gamma" activity in the olfactory system. Patterned electrical stimulation of afferents that mimicked a natural odor stimulus elicited rapidly synchronized spikes (lag < or = 5 ms) in mitral cells, along with oscillatory activity at the gamma (approximately 50 Hz) frequency. Analysis of coupling potentials, combined with dendritic sectioning, indicated that mitral cell synchrony was mainly driven by inhibitory postsynaptic potentials (IPSPs) imposed by GABAergic granule cells. Recordings in granule cell pairs indicated that granule cells were themselves synchronized by their excitatory inputs from mitral cells, providing a means to coordinate GABA release. These results demonstrate that rapid synchrony can emerge in a network through the precise back-and-forth interplay between neuronal populations.     

The nature of surround-induced depolarizing responses in goldfish cones

Cones in the vertebrate retina project to horizontal and bipolar cells and the horizontal cells feedback negatively to cones. This organization forms the basis for the center/surround organization of the bipolar cells, a fundamental step in the visual signal processing. Although the surround responses of bipolar cells have been recorded on many occasions, surprisingly, the underlying surround-induced responses in cones are not easily detected. In this paper, the nature of the surround-induced responses in cones is studied. Horizontal cells feed back to cones by shifting the activation function of the calcium current in cones to more negative potentials. This shift increases the calcium influx, which increases the neurotransmitter release of the cone. In this paper, we will show that under certain conditions, in addition to this increase of neurotransmitter release, a calcium-dependent chloride current will be activated, which polarizes the cone membrane potential. The question is, whether the modulation of the calcium current or the polarization of the cone membrane potential is the major determinant for feedback-mediated responses in second-order neurons. Depolarizing light responses of biphasic horizontal cells are generated by feedback from monophasic horizontal cells to cones. It was found that niflumic acid blocks the feedback-induced depolarizing responses in cones, while the shift of the calcium current activation function and the depolarizing biphasic horizontal cell responses remain intact. This shows that horizontal cells can feed back to cones, without inducing major changes in the cone membrane potential. This makes the feedback synapse from horizontal cells to cones a unique synapse. Polarization of the presynaptic (horizontal) cell leads to calcium influx in the postsynaptic cell (cone), but due to the combined activity of the calcium current and the calcium-dependent chloride current, the membrane potential of the postsynaptic cell will be hardly modulated, whereas the output of the postsynaptic cell will be strongly modulated. Since no polarization of the postsynaptic cell is needed for these feedback-mediated responses, this mechanism of synaptic transmission can modulate the neurotransmitter release in single synaptic terminals without affecting the membrane potential of the entire cell.     

From the stochasticity of molecular processes to the variability of synaptic transmission

The variability of the postsynaptic response following a single action potential arises from two sources: the neurotransmitter release is probabilistic, and the postsynaptic response to neurotransmitter release has variable timing and amplitude. At individual synapses, the number of molecules of a given type that are involved in these processes is small enough that the stochastic (random) properties of molecular events cannot be neglected. How the stochasticity of molecular processes contributes to the variability of synaptic transmission, its sensitivity and its robustness to molecular fluctuations has important implications for our understanding of the mechanistic basis of synaptic transmission and of synaptic plasticity.     

Methods for patch clamp capacitance recordings from the calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.     

Analytical description of the activation of multi-state receptors by continuous neurotransmitter signals at brain synapses.

Chemical synaptic transmission is a fundamental component of interneuronal communications in the central nervous system (CNS). Discharge of a presynaptic vesicle containing a few thousand molecules (a quantum) of neurotransmitter into the synaptic cleft generates a transmitter concentration signal that drives postsynaptic ion-channel receptors. These receptors exhibit multiple states, with state transition kinetics dependent on neurotransmitter concentration. Here, a novel and simple analytical approach for describing gating of multi-state receptors by signals with complex continuous time courses is used to describe the generation of glutamate-mediated quantal postsynaptic responses at brain synapses. The neurotransmitter signal, experienced by multi-state N-methyl-D-aspartate (NMDA)- and L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors at specific points in a synaptic cleft, is approximated by a series of step functions of different intensity and duration and used to drive a Markovian, multi-state kinetic scheme that describes receptor gating. Occupancy vectors at any point in time can be computed interatively from the occupancy vectors at the times of steps in transmitter concentration. Multi-state kinetic schemes for both the low-affinity AMPA subtype of glutamate receptor and for the high-affinity NMDA subtype are considered, and expected NMDA and AMPA components of synaptic currents are calculated. The amplitude of quantal responses mediated by postsynaptic receptor clusters having specific spatial distributions relative to foci of quantal neurotransmitter release is then calculated and related to the displacement between the center of the postsynaptic receptor cluster and the focus of synaptic vesicle discharge. Using this approach we show that the spatial relation between the focus of release and the center of the postsynaptic receptor cluster affects synaptic efficacy. We also show how variation in this relation contributes to variation in synaptic current amplitudes.     

Assembly of signaling machinery at the postsynaptic membrane.

The postsynaptic membrane and the subsynaptic cell compartment are specialized for inter- and intracellular signaling. Recent work has focused on the role of synaptic activity in regulating the surface distribution of neurotransmitter receptors. In addition, several components of secondary signaling pathways involved in the long-term regulation of synaptic efficacy have been identified.