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Post date: May 4, 2011 6:43:02 PM
Chemical synapses are specialized sites of cell–cell contact that mediate the transmission of information between neurons, or between neurons and other cells. Synapses differ from most other cellular junctions in that they are inherently asymmetric. The flow of information is directional; in general, neurotransmitters are released from a presynaptic neuron and bind to receptors on a postsynaptic target cell (Figure 1). The presynaptic neuron and postsynaptic target cell are separated by a synaptic cleft of 10 to 20 nm. The presynaptic side contains a specialized set of proteins that synthesizes the neurotransmitter, packages it into synaptic vesicles and regulate fusion of vesicles with the plasma membrane for transmitter release. Synaptic vesicles are small (approximately 40 nm), and have a clear lumen when viewed through the electron microscope (EM). These vesicles contain neurotransmitter molecules; classic neurotransmitters include amino acids such as glutamate and γ-aminobutyric acid (GABA), monoamines such as dopamine and serotonin, and acetylcholine (ACh). These contents are released from the vesicles into the synaptic cleft through Ca++-dependent exocytosis.
In the area of synaptic contact, there is an active zone, where vesicle fusion occurs. The active zone proteins make the vesicles ready to release their contents into the synaptic cleft, through a number of steps including docking, priming and fusion (Figure 2). After fusion, the vesicular membrane and associated proteins are retrieved from the plasma membrane through clathrin-mediated endocytosis (Martin, 2002, Li and Chin, 2003).
Figure 1. A simplified schematic of a mammalian glutamatergic synapse
This highly simplified cartoon depicts the molecular interactions mediated by neuroligin and neurexin at a synapse. Neurexin binds the scaffolding proteins CASK and Mint through its C-terminal PDZ-binding motif. CASK and Mint interact with each other, as well as voltage-gated
Ca++ channels. In addition, they also bind Velli, which interacts with β-catenin. CASK also interacts with rabphilin and Band4.1. Neurexin is also capable of binding synaptotagmin, which functions as the Ca++ sensor on synaptic vesicles. On the postsynaptic side, neuroligin binds postsynaptic scaffold proteins, including PSD95 and S-SCAM, via its C-terminal PDZ-binding motif. PSD95 links neuroligin to glutamate receptors, while S-SCAM link neuroligin to cadherin-catenin based cell adhesion. Thus, neurexin on the presynaptic side facilitates the recruitment of neurotransmitter release machinery, while neuroligin on the postsynaptic side facilitates the recruitment of neurotransmitter receptor
In apposition to the presynaptic active zone, the postsynaptic cell also forms a thickening under the plasma membrane, which is called the postsynaptic density (PSD). At the postsynaptic membrane, neurotransmitters act as ligands for specific neurotransmitter receptors that signal via ion channels or G-proteins (GPCRs). The presynaptic active zones and postsynaptic densities of a given synapse are usually comparable in surface area, are aligned in precise apposition, and occupy an area of approximately 5 to 20 μm2.
Methods used to study synapse development and function include electrophysiology, neuroanatomy, neurochemistry, pharmacology, cell biology, molecular biology and genetics. Data have come from synaptosomes, explants and cell culture systems and from vertebrate and invertebrate models, including humans, mice, rats, frogs, squid, fruitflies (Drosophila melanogaster) and nematodes (Caenorhabditis elegans). The molecular biology of the synapse is conserved to a significant degree across phyla.
Synaptic vesicle proteins are initially trafficked from the Golgi apparatus to the synapse on so-called “pre-synaptic vesicles”. Trafficking of pre-synaptic vesicles is dependent on specialized motor proteins, which are members of the kinesin family. In C. elegans, the major motor protein for trafficking synaptic vesicle components is UNC-104 (Otsuka et al., 1991). There is also evidence that other proteins such as the kinesin heavy chain protein UNC-116 (Sakamoto et al., 2005) and Sunday Driver/UNC-16 (Bowman et al., 2000, Byrd et al., 2001) regulate the transport of synaptic vesicle proteins. Precisely how “pre-synaptic vesicles” differ in composition from mature synaptic vesicles and the details of the maturation process remain unclear. It is likely that the maturation process involves fusion with the cell membrane, sorting of synaptic vesicle proteins and retrieval through endocytosis.
After maturation, synaptic vesicles are loaded with neurotransmitter. The biosynthetic enzymes UNC-25/GAD (Jin et al., 1999) and CHA-1/ChAT (Okuda et al., 2000, Matthies et al., 2006, Mullen et al., 2007) make the neurotransmitters GABA and ACh respectively, whereas glutamate and glycine are amino acids which are necessary for other cellular processes, and so are abundant in the cell.
Loading of these neurotransmitters is an active process. A vacuolar-type proton pump ATPase provides a proton and electrochemical gradient whichprovides the energy required to load the neurotransmitter into the vesicle lumen. The loading process also requires vesicular neurotransmitter transporters (Rand et al., 2000); these transporters are specific for different types of transmitters. The identity of 3 of the 4 classes of these transporters was determined through the molecular characterization of C. elegans mutants. For example, UNC-17/VAChT (Alfonso et al., 1993) is the vesicular ACh transporter, and UNC-47/VGAT-1 (McIntire et al., 1997) is the vesicular GABA transporter. EAT-4/VGLUT is the vesicular glutamate transporter (Lee et al., 1999b, Bellocchio et al., 2000) in C. elegans.
After they are loaded with neurotransmitter, vesicles must dock near release sites. The molecular details of docking are not fully understood, perhaps because docking is difficult to assay. In cultures of neurons with labeled synaptic vesicles, vesicles come near the plasma membrane, and become motionless for a period of time. The vesicles then either detach or fuse (Murthy and Stevens, 1999, Zenisek et al., 2000). These data imply that docking may be a reversible step. Although many proteins on vesicles and at release sites have been identified, none of the identified protein interactions that occur between vesicle proteins and proteins at release sites appears to be responsible for docking. Mutations in rab-3 and unc-18 alter vesicle docking, or vesicle organization at release sites, but they do not completely disrupt docking (Nonet et al., 1997, Weimer et al., 2003, Weimer and Richmond, 2004). The SNARE proteins, which are thought to mediate fusion, do not appear to be involved in the docking process either (Südhof, 2004). Two massive multi-domain proteins have emerged as potential vesicle-docking proteins. Bassoon (Dieck et al., 1998) is a 420-kDa protein with two amino-terminal zinc finger domains and three coiled-coil domains. Piccolo/Aczonin (Cases-Langhoff et al., 1996, Wang et al., 1999), a 550-kDa protein, possesses two amino-terminal zinc finger domains and a carboxyl-terminal array of one PDZ and two C2 domains. These large proteins appear to be at least partially redundant. In neurons lacking both Bassoon and Piccolo, a significant reduction in synaptic vesicle clustering is observed by electron microscopy.(Mukherjee et al., 2010).
Figure 2. A simplified schematic of the mammalian synaptic vesicle cycle
The vesicle cycle has several steps. Neurotransmitter is loaded into the vesicle. The vesicle then moves to the active zone, becomes docked and is primed for fusion. Ca++ influx triggers release of neurotransmitter into the synaptic cleft, although spontaneous fusions (mEPSCs or mIPSCs) are also possible and are Ca++ independent. Vesicle proteins are then recovered from the membrane through endocytosis, and the cycle starts over again. Many of the proteins responsible for these processes have been identified.
Following docking, priming makes vesicles capable of fusing rapidly in response to a local Ca++ influx. Soluble n-ethylmaleimide-sensitive fusion protein attachment proteins receptors (SNARE proteins) are thought to mediate fusion of membranes. Target membranes have target SNARES (t-SNARES) whereas vesicles have vesicle SNARES (v-SNARES). During the priming process, SNARE complexes are partially assembled. UNC-13 and RIM may participate in the priming process. It has been proposed that UNC-13 stimulates the change of the t-SNARE syntaxin from a closed conformation to an open conformation (Hammarlund et al., 2007), which in turn may stimulate the assembly of a v-SNARE / t-SNARE complex. RIM also appears to regulate priming, but is not essential for this step (Ashery et al., 2009, Südhof, 2004, Brose, 2008).
Vesicle fusion is mediated directly by the SNAREs and driven by the energy provided by the SNARE (SNAP receptor) complex, which is thought to act as the receptor for soluble fusion factors. Soluble NSF attachment proteins (SNAPs) bind and recruit NSF (n-ethylmaleimide-sensitive fusion protein) to the SNARE complex, hydrolyze ATP, and complete vesicle priming. Local Ca++ influx into the presynaptic cell then leads to the rapid completion of membrane fusion and release of the neurotransmitter cargo (Bennett and Scheller, 1994, Südhof, 1995, Südhof, 2004, Brose, 2008). The trigger for this event is the Ca++-sensing synaptic vesicle protein, synaptotagmin.
During vesicle fusion, the synaptic vesicle membrane and proteins are transiently inserted into the plasma membrane at the active zone. Therefore, these vesicular proteins must be retrieved to generate new synaptic vesicles. This retrieval is accomplished through clathrin-dependent endocytosis. Proteins that contribute to this process include clathrin, endophilin, synaptojanin, synaptotagmin, stonin, dynamin and AP180 (Reviewed in (Smith et al., 2008)).
After neurotransmitters are released into the synaptic cleft through vesicle fusion, they diffuse across the synaptic cleft and interact with receptors on the postsynaptic membrane. There are two major classes of receptors: ligand gated ion channels and GPCRs (G-protein coupled receptor).
Ligand gated ion channels on the postsynaptic side bind neurotransmitter molecules and respond by opening, thus allowing ions to flow in (or out, in the case of inhibitory channels and depending on the ion involved), which changes the local transmembrane potential of the cell. The resultant alteration in voltage is called a postsynaptic potential. In general, the effect is excitatory when it is a depolarizing current and inhibitory when it is a hyperpolarizing current. The type of neurotransmitter and receptors employed at the synapse determine whether a synapse is excitatory or inhibitory (Purves, 2008, Siegel, 2006).
Neurotransmitters can also act as ligands for GPCRs, causing a conformational change in the receptor, which allows it to act as a guanine nucleotide exchange factor (GEF). The GPCR can then activate an associated heteromeric G-protein by exchanging its bound GDP for a GTP. The G-protein's α subunit, together with the bound GTP, can then dissociate from the β and γ subunits to further affect intracellular signaling proteins, or target functional proteins directly, depending on the α subunit type. Various receptors of this type bind a variety of different neurotransmitters, including serotonin, dopamine, GABA, or glutamate in the mammalian CNS. GPCGs can regulate behavior and mood, and also regulate the mammalian autonomic (sympathetic and parasympathetic) nervous systems. Therefore, GPCRs control many functions of the body such as blood pressure, heart rate and the digestive processes (Purves, 2008, Siegel, 2006).
To terminate signaling, neurotransmitter must be removed from the synaptic cleft. Transmitters such as glutamate, GABA, dopamine and serotonin are cleared from the synapse by specific plasma membrane transporters (Richerson and Wu, 2003, Huang and Bergles, 2004, Melikian, 2004, Iversen, 2006). Other transmitters, including acetylcholine and some neuropeptides, are degraded by specific enzymes in the synaptic cleft. For example, acetylcholinesterase inactivates acetylcholine by breaking it down into choline and acetate at the neuromuscular junction (Bulger et al., 1982, Johnson et al., 1988) or in a cholinergic nerve-nerve synaptic cleft. Choline can then be recovered through a plasma membrane choline transporter, and may be used for synthesis of new acetylcholine.
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