Пресинаптическая мембрана аксона

6 Data analysis

Release of neurotransmitter (Fig. 7.4)

Once loaded with neurotransmitter, synaptic vesicles are docked at the presynaptic membrane awaiting release. Docking takes place at active zones. These consist of multi-protein complexes that tether the synaptic vesicle to the presynaptic membrane and contain high concentrations of voltage-gated calcium channels. This leads to brisk calcium influx in response to axon terminal depolarization, since the concentration of calcium is 10,000 times higher in the extracellular fluid. The focal rise in free calcium causes a number of synaptic vesicles to fuse with the presynaptic membrane, emptying their contents into the synaptic cleft by exocytosis.

Release is said to be ‘packeted’ (or quantized) since the total amount of transmitter entering the synaptic cleft is a whole-number multiple of the amount stored in a single vesicle. Transmitter release is regulated by presynaptic autoreceptors which exert negative feedback.

B Sequences of Synaptogenesis

When both pre- and postsynaptic densities are present early in development, as in Fig. 8a, they appear as continuous plaques (Aghajanian and Bloom, 1967; Jones and Revell, 1970a,b) which, later in development, undergo gradual differentiation and focalization to produce the typical paramembranous densities of the adult synaptic junction. Early workers repeatedly stressed the presence of membrane thickenings prior to the appearance of synaptic vesicles (Glees and Sheppard, 1964; Hamori and Dyachkova, 1964; Meller, 1964; Bunge et al., 1967; Larramendi, 1969; Tennyson, 1970; Alley, 1973; Stelzner et al., 1973). In all these instances, however, caution is necessary to ensure that the membrane specializations actually are precursors of synaptic junctions rather than early components of desmosome-like contacts. This also applies to the idea that puncta adhaerenta are precursors of synaptic junctions (McGraw and McLaughlin, 1980).

An alternative, therefore, is Fig. 8b with synaptic vesicles preceding membrane thickenings. While the presence of presynaptically located vesicles may not constitute an obligatory feature of immature synapses, it remains a helpful one and should not be lightly discarded. Unfortunately, once the presence of vesicles becomes an essential diagnostic feature of early synapses, it also to some extent prejudges the question of whether vesicles or membrane thickenings appear first in synaptic development.

A third alternative is Fig. 8c, in which increases in vesicle numbers and maturation of the paramembranous densities are simultaneous events (Ochi, 1967; Bodian, 1968; Jones and Revell, 1970a,b). For instance, Jones and Revell noted in rat cerebral cortex that vesicles are present when both synaptic membranes are plaquelike and undifferentiated, suggesting that synaptic vesicles appear with (or even after) undifferentiated membrane thickenings but before their differentiation into recognizable dense projections. Comparable observations have been made in other situations, including chick retina (McLaughlin, 1976) and kitten spinal trigeminal nucleus (Dunn and Westrum, 1978).

The discussion this far has assumed that the pre- and postsynaptic membranes appear concurrently (Aghajanian and Bloom, 1967; Jones and Revell, 1970a,b; Adinolfi, 1972a,b; Bloom, 1972). However, this is far from a universal phenomenon. In some studies, as shown in Fig. 8d, the postsynaptic density appears as a well-established entity before the presynaptic region with its dense projections (Poppe et al., 1973; Jones et al., 1974; West and Del Cerro, 1976; Hinds and Hinds, 1976). This phenomenon is best seen in E-PTA-stained material and has so far been described in developing guinea pig cerebral cortex (Jones et al., 1974) and in some synaptic junctions in fetal rat cerebellum (West and Del Cerro, 1976). It is possible that this appearance reflects features of the E-PTA staining reaction, in that it is not staining the presynaptic components of at least some of the immature synapses. Even if this is the case, however, other evidence points to the legitimacy of “naked” postsynaptic sites during early neuronal development.

The converse arrangement, whereby the presynaptic membrane appears prior to the postsynaptic, is far less frequently encountered (Fig. 8e). A commonly quoted illustration of this is the membrane-vesicle clusters described in the cervical spinal cord of Xenopus laevis embryos (Hayes and Roberts, 1973). A similar sequence of events has been described as part of the pattern for photoreceptor synaptogenesis in developing chick retina (McLaughlin, 1976).

These diverse developmental patterns reflect the range of ways by which pre- and postsynaptic junctional elements establish their adult organization. It is not known whether a particular pattern characterizes a particular synaptic population, or whether similar synapses are put together in different ways under differing environmental pressures.

It has long been argued that axodendritic synapses are formed prior to axo-somatic ones (Voeller et al., 1963; Jones, 1975), the synaptic vesicles being spherical rather than flattened (Oppenheim and Foelix, 1972). More recent studies substantiate these general principles in a wide range of situations, e.g., chick optic tectum (McGraw and McLaughlin, 1980), human cervical spinal cord (Okado, 1980), and mouse spinal motor neurons (Vaughn et al., 1977). Occasional exceptions do occur however, including the study of Smolen and Raisman (1980) on rat superior cervical ganglion. In this instance, axosomatic synapses are present transiently for the first 2 weeks of life. It is possible that such synapses may facilitate dendritic development by providing directive cues to the location of the developing synaptogenic field (Vaughn et al., 1977).

Early axodendritic synapses may be of two types: those with asymmetrical junctions and spherical vesicles, and those with symmetrical junctions and spherical vesicles (Devon and Jones, 1981). Neocortical development is characterized by a decrease in the symmetrical/spherical group. This trend may represent a developmental continuum of symmetrical/spherical to asymmetrical/spherical synapses (Bunge et al., 1967; Cragg, 1972; Hinds and Hinds, 1976; Dunn and Westrum, 1978), although the possibility that the asymmetrical/axodendritic terminals represent an intermediate synaptic type cannot be excluded (Adams and Jones, 1982a). The recognition of the symmetrical/spherical terminal type may provide a means of distinguishing immature terminals in adult, and especially in retarded adult, cortical tissue (Devon and Jones, 1981). In this connection it would be interesting to know whether these symmetrical/spherical terminals correspond to the immature type E synaptic junction described by Dyson and Jones (1976a) in E-PTA-stained cortical material.

Terminals with spherical vesicles are repeatedly described as being the first to appear, with flattened or pleomorphic vesicles appearing in later-developing terminals, e.g., in chick optic tectum (McGraw and McLaughlin, 1980), human cervical spinal cord (Okado, 1980), and rabbit superior colliculus and visual cortex (Mathers et al., 1978).

Quantitative approaches to the morphological categorization of developing synaptic junctions are currently confined to the characterization of E-PTA-stained junctions. The quantitative morphogenetic scheme of Dyson and Jones (1976a) assigns synaptic junctions to five categories, A–E, on the basis of variations in the organization of their presynaptic densities. Of these descriptive characteristics, type A represents a mature junctional form with well-defined and discrete dense projections, whereas, at the other end of the scale, type E is an immature form lacking recognizable presynaptic densities; types B–D probably represent intermediate forms (Fig. 3).

The approach to synaptogenesis illustrated by this study gives some idea of the manner in which synaptic ultrastructure can be traced at varying stages during maturation and adult life. By permitting quantitation of the contribution a particular morphogenetic form makes to the population at developmental intervals, it provides a model system for assessing the maturity of both individual junctions and synaptic populations.

Mini-dictionary of terms

Active zone, a specialized area on the presynaptic membrane of a nerve cell at which synaptic vesicles fuse to release neurotransmitters into the synaptic cleft and transmit signals to the next synapse or muscle.

Amyotrophic lateral sclerosis, a rare neurodegenerative disease that primarily affects the neurons responsible for controlling voluntary muscle movement.

D. melanogaster, a species of fly in the family Drosophilidae.

Frontotemporal lobar degeneration, a clinically and pathologically heterogeneous syndrome, characterized by progressive declines in behavior or language associated with the degeneration of the frontal and anterior temporal lobes.

FUS, the FET family of RNA-binding proteins that regulate gene expression, genomic integrity, and RNA processing.

GAL4-UAS-targeted expression system, a system using the yeast GAL4 protein that interacts with UAS to induce the expression of genes from any organism in tissue- and temporal-specific manners.

Long non-coding RNA, a group of non-coding RNAs longer than 200 nucleotides.

Neuromuscular junction, a synaptic connection between the terminal end of a motor nerve and a muscle.

RNA interference, a post-transcriptional genetic mechanism of eukaryotes that suppresses gene expression and in which double-stranded RNA cleaved into small fragments initiates the degradation of a complementary mRNA sequence.

Ubiquilin, a protein that regulates protein homeostasis (proteostasis).

Structure and Mechanism of Action of BTs:

BTs are synthesized as single-chain polypeptides and are activated by proteolytic enzymes in a cleaving process (Fig. 18-4). The cleaved heavy chain is responsible for high-affinity docking of the neurotoxin to the presynaptic nerve terminal receptor, enabling the internalization of the bound toxin into the cell.⁵⁰ The light chain is a zinc-dependent endopeptidase that cleaves membrane proteins responsible for docking acetylcholine vesicles on the inner side of the nerve terminal membrane. The cleavage of these proteins irreversibly precludes fusion of the vesicles with the nerve membrane, thereby preventing release of neurotransmitters into the neuromuscular junction.⁵¹

Type A BT appears to be the most potent of the subtypes, and, when injected clinically, has the longest duration of action. While type B and F have limited clinical use, and others are the subject of further study, the multiple differences thus far observed suggest that the subtypes are not interchangeable.

Pharmacological effect of BTs occurs in three stages: binding, internalization, and proteolysis.⁵²

Synapses

Future directions

Nicotine’s toxicity is mediated by nAChR subunits located in pre- or postsynaptic membranes. In addition to membrane internalization, nAChRs have a half-life of up to 10 days when membrane-bound (Benowitz et al., 2009; Benowitz et al., 1982). Nicotine exerts its various toxicities through different subunits of nAChRs, which are located in the pre- or post-synaptic membranes. Nicotine increases the expression and half-life of surface membrane nAChRs (Kuryatov et al., 2005; Nashmi et al., 2003). However, the process or mechanism involved in the degradation or elimination of nAChRs-binding or free nicotine in the synaptic cleft, as well as its half-life, is unknown. The role of nAChRs subunits in nicotine intoxication in various nervous systems is unclear. Future research should focus on synaptic nicotine degradation and nAChR subunits involved in nicotine toxicity.

C Nucleation of Synaptic Scaffolds by Adhesion Molecules

The mechanical stability of synapses and the precise apposition of pre- and postsynaptic membrane domains implies that adhesion molecules might bridge the synaptic cleft and thereby keep both sides of the synapse in register. Ultrastructural studies revealed the presence of different sites of cell-cell adhesion at the synapse—including the linkage across the synaptic cleft, as well as at puncta adherentia that flank the synaptic cleft. A third site of cellular adhesion at central synapses are the end feet of glial cells that tightly enclose the synaptic junctions.

Most attention in recent years has been focused on neuronal adhesion molecules that directly bridge the synaptic cleft and that might instruct the assembly of synaptic structures. Several such adhesion molecules have been identified. The synaptogenic activity of these adhesive factors is thought to rely on the nucleation of cytoplasmic scaffolds at the pre- and postsynaptic side, which subsequently facilitate the recruitment of other cytoplasmic and cell surface molecules to an incipient synaptic connection.

In vitro experiments with cultured neurons revealed that exposing axonal or dendritic processes to specific adhesive triggers is sufficient to induce pre- and postsynaptic structures. One extensively studied adhesion system with synapse-inducing function is the neuroligin-neurexin complex. Neuroligins and neurexins are heterophilic adhesion proteins that form a trans-synaptic complex (Ichtchenko et al. 1995; Nguyen and Südhof 1997; Song et al. 1999). Cytoplasmic sequences in neuroligin-1 target this protein exclusively to the synaptic sites in dendrites and ensure exclusion from the axons (Dresbach et al. 2004; Iida et al. 2004; Rosales et al. 2005). This strict polarity of neuroligin-1 provides this heterophilic adhesion system with the required directionality for the assembly of asymmetric synaptic structures. Contact of axons with neuroligin-1 ectopically expressed in a nonneuronal cell induces clustering of axonal neurexins and the cytoplasmic scaffolding molecule CASK (Scheiffele et al. 2000; Dean et al. 2003). These neuroligin-induced axonal contacts then mature into functional presynaptic release sites, including active zone components and clusters of synaptic vesicles. Conversely, contact of dendrites with neurexin-1 expressing cells leads to the formation of neuroligin clusters and postsynaptic structures, such as the accumulation of the scaffolding protein PSD95 and NMDA receptors (Graf et al. 2004). These experiments suggest that the neuroligin-neurexin adhesion system can nucleate pre- and postsynaptic structures. To a large extent these structures are thought to assemble around scaffolding components that bind to the cytoplasmic tails of neuroligins and neurexins (see Figure 4.1). For example, postsynaptic neuroligin-1 binds to PSD95, which in turn can recruit NMDA-receptors and other scaffolding components such as GKAP, Homer, and Shank (Irie et al. 1997; Meyer et al. 2004; Prange et al. 2004; Chih et al. 2005).

Interestingly, the neuroligin-2 isoform, which differs in its cytoplasmic sequences from neuroligin-1, is found primarily at GABAergic synapses. Extracellular aggregation of neuroligin-2 results in not only the recruitment of PSD95, but also of gephrin, a scaffolding protein specific to inhibitory synapses (Graf et al. 2004; Varoqueaux et al. 2004). Selective interaction of excitatory and inhibitory postsynaptic scaffolding proteins with different neuroligin cytoplasmic tail sequences therefore might contribute to the nucleation of specific postsynaptic scaffolds. However, interactions between extracellular domains that may contribute to this process have not been ruled out.

Similar experiments revealed synaptogenic activities for the cell adhesion molecule SynCAM, a homophilic Ig-domain protein (Biederer et al. 2002). Interestingly, SynCAM and neuroligin-1 differ in their cytoplasmic interactions with scaffolding proteins. These differences may explain the differential recruitment of NMDA and AMPA-type glutamate receptors to neuroligin-1- and SynCAM-induced synapses, respectively (Sara et al. 2005). Neuroligin-1 recruits PSD95 and NMDA-receptors through the type I PDZ-binding motif in neuroligin, whereas SynCAM contains a type II PDZ-binding motif that contributes to the recruitment of functional AMPA-receptors to synapses. The importance of these selective scaffolding interactions was confirmed in experiments showing that a chimaeric protein, which includes neuroligin-1 containing the cytoplasmic sequences of SynCAM, is capable of stimulating AMPA receptor recruitment (Sara et al. 2005).

Another presumptive cell adhesion molecule that has been specifically linked to the synaptic recruitment of AMPA receptors through interaction with scaffolding molecules is Dasml (Shi et al. 2004). However, the extracellular binding partners of this protein are currently unknown.

It is likely that several adhesion molecules that act through mechanisms similar to the neuroligin-neurexin complex and SynCAM will cooperate in the nucleation of synaptic scaffolds and the subsequent recruitment of neurotransmitter receptors. Candidate molecules include the nectins, a family of homophilic Ig-domain proteins that are similar to SynCAM (Satoh-Horikawa et al. 2000; Mizoguchi et al. 2002). The integration of multiple adhesive signals likely occurs at the level of the cytoplasmic scaffold. There is substantial crosstalk between different scaffolding components; for example, the CASK/Mint/MALS complex that binds to neurexins is linked to beta-catenin, the cytoplasmic binding partner of cadherins (see Figure 4.1; Hata et al. 1996; Perego et al. 2000; Bamji et al. 2003). Such crosstalk might be employed for the sequential recruitment of different adhesive factors to a growing adhesion site as has been observed for cadherins and nectins in epithelial cells (Takahashi et al. 1999; Tachibana et al. 2000).

When does Ca²⁺ enter the presynaptic element in response to a presynaptic spike?

Acetylcholine Is Degraded and Then Recycled

Each action potential fuses perhaps 300 or so synaptic vesicles with the presynaptic membrane. Continuous excitation and subsequent fusion of these large numbers of vesicles with the presynaptic membrane would expand the area of the membrane and deplete it of vesicles. To avoid this, the presynaptic cell rapidly recycles the vesicles. Through an elaborate mechanism involving specialized proteins such as clathrin and dynamin, vesicles are pinched off and endocytosed. The empty vesicles are refilled with acetylcholine through a transport channel that exchanges acetylcholine for H⁺ ions. The H⁺ ions are accumulated within the vesicles through a vacuolar H-ATPase in the vesicle membrane.