involved in ACh synthesis, choline acetyltransferase, suggesting that the regulation of transmitter synthesis and transport may be closely related (Usdin et al. 1995). It is not known whether similar mechanisms may be involved for other transmitters.
More recently, the sequences of a GABA transporter (vGAT) (McIntire et al. 1997), and two glutamate transporters (vGlut1 and vGlut2)(Fremeau et al. 2001) have also been identified. They possess 10 (vGAT) or 8 (vGlut1 and vGlut2) potential trans-membrane domains. The closer sequence homology between the VMAT and VAChT vesicle transporters defines these proteins as a family, more distantly related to vGluts and vGAT, and to other transporters, including those neurotransmitter transporters found at the plasma membrane.
membrane fusion events in synaptic plasticity has been clearly demonstrated by the work of Kandel and his collaborators. Figure 6.7 summarizes the different stages that lead to a plastic response of a synapse and each stage that can involve membrane fusion is schematically illustrated. According to this model, modulation of transmitter release is the first step of a cascade leading to changes in gene expression and redistribution of membrane components that result in new synapse formation (Bailey et al. 1992;
Hu et al. 1993). Each step of vesicle trafficking and fusion described in the previous chapters could contribute to regulated changes of transmitter release.
6.3.2 Functional synaptic plasticity
Altered efficacy of existing synapses is often referred to as functional synaptic plasticity.
One of the most impressive example is long-term potentiation (LTP). Bliss and Lomo, discovered that brief high frequency trains of action potentials produced an increase of synaptic strength in synapses of the hippocampus that use glutamate as a transmitter (Bliss and Lomo 1973). This was the first demonstration that synaptic strength could change as a function of previous experience. LTP can last for hours or even days and seems to depend on both postsynaptic and presynaptic events. At the level of the postsynaptic cell, high levels of presynaptic stimulation result in the activation of
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1 Taget cell
Fig. 6.7 Diagram of a ‘remodeling’ synapse illustrating the possible participation of membrane fusion events during structural changes associated with learning (based on Bailey et al. 1992; Hu et al. 1993). Stimulation of the presynaptic neuron increases the fusion-dependent release of transmitter from SVs (gray circles, 1). When the stimulus is sustained, a target-derived factor (2) induces changes in gene expression in the presynaptic cell (3). In turn, this activates the endosomal pathway (4) and leads to fusion-dependent redistribution of membrane components at the sites of new growth (5,6). Black circles:
clathrin. Orange circles: adhesion molecules. Modified from Trends Neurosci., vol. 17, pp. 368–373, with permission from Elsevier Trends Journals.
glutamate receptors that are functionally blocked under normal conditions. At the level of the presynaptic cell, LTP involves an increase in transmitter release. This increase is likely to be at least partially the result of a retrograde signal originating from the postsynaptic cell. However, the possibility that covalent modifications of the pro-teins involved in vesicle trafficking and fusion could play a role is also under extensive study. For example, as mentioned in Section 6.2.4, studies on the synapsins indicate that vesicle mobilization may contribute to the number of vesicles available for release.
Regulating synapsin function may therefore have an indirect effect on synaptic strength. Also, the function of proteins such as Rab3 and RIM1 is clearly important for some forms of LTP (see Section 6.2.5). Data by Staple and colleagues (Staple et al.
1995) suggest that the relative ratio of proteins involved at different stages of transmit-ter release can vary in different synaptic boutons of the same neuron and that this variability correlates with synaptic strength.
6.3.3 Morphological synaptic plasticity
Long-term memory has been clearly associated with changes in synaptic structure in a variety of experimental systems (Wallace et al. 1991). Quantitative ultrastructural analysis indicates that four basic morphological parameters of the synapse are strongly correlated. These are total vesicle number, active zone size, presynaptic bouton volume, and postsynaptic spine volume (Pierce and Mendell 1993; Pierce and Lewin 1994).
Interestingly, co-ordinated increases in size of pre- and postsynaptic elements have been reported after LTP in the dentate gyrus. These experiments have shown expan-sion of the average active zone and apposed surface areas, as well as synaptic bouton volume (see Wallace et al. 1991). These increases imply the fusion and incorporation of new membrane into the synaptic structure. What are the mechanisms involved? There are at least two possibilities. Net changes in presynaptic surface could arise from any imbalance between the amount of membrane that is fused and then retrieved during transmitter release and vesicle recycling. Consistent with this, ultrastructural analysis of the synapses of the shibire mutant (the Drosophila homolog of dynamin, which has a defect in vesicle endocytosis, see Section 6.2.7) shows enlarged synapses. However, the observation that membrane retrieval is stimulus dependent (von Gersdorff and Matthews 1994) suggests that it may be regulated in response to previous signals received by the neuron. This would provide a mechanism of activity-dependent (or use-dependent or experience-dependent) control of membrane surface. The data showing that dynamin (see Section 6.2.7) is phosphorylated by PKC and dephospho-rylated by electrical activity (van der Bliek and Mcycrowitz 1991; Robinson et al.
1993), is also consistent with this hypothesis.
An alternative way to increase synaptic surface would be the fusion of membrane vesicles other than SVs. Pfenninger and colleagues have shown that plasmalemmal expansion involves the fusion of large clear vesicles that were identified as plasmalem-mal precursors (PPV) (Pfenninger et al. 1991). These vesicles accumulate in the growth cones of developing axons (Pfenninger and Friedman 1993). Interestingly, data
obtained with a cell-free growth cone-expansion assay suggest that PPV fusion with the plasma membrane is controlled by Ca2+influx (Lockerbie et al. 1991), thereby providing an activity-dependent mechanism of membrane expansion. If the same mechanism was maintained in adult neurons, growth of new connections, or morpho-logical changes of existing ones, could be activated by stimulating the membrane-expansion pathway. If this hypothesis is correct, the cell must be capable of regulating vesicle fusion for release and vesicle fusion for expansion through distinct mecha-nisms. Of particular relevance to these observations, is the finding that SNAP-25 is involved in both transmitter release and in axonal growth. Inhibition of SNAP-25 expression with antisense oligonucleotides prevented neurite outgrowth in PC12 cells and cortical neurons in vitro, and in chick retina neurons in vivo (Osen-Sand et al.
1993). These effects could result from inhibition of transmitter release, but it is also possible that SNAP-25 is involved in the fusion of PPV. Very recent studies support the latter interpretation. Using clostridial toxins on primary neurons in vitro, Osen-Sand et al. (1996) showed that the fusion machineries for transmitter release and axonal growth involve common but also distinct SNARES. The v-SNARE VAMP was found to be exclusively involved in transmitter release, whereas the t-SNARES SNAP-25 and syntaxin have a role in release and growth. These data suggest that transmitter release (and SV fusion) is not necessary for axonal growth and that additional v-SNARES must be involved in vesicle fusion for membrane expansion.
In addition to changes in synaptic size, the structural rearrangements that occur during morphological synaptic plasticity involve the formation of new synapses and are likely to involve the co-ordinated action of genes that regulate membrane expansion as well as vesicle storage and fusion. Consistent with this, candidate plasticity genes encod-ing products involved in the endo- and exocytotic pathways have been isolated follow-ing stimulation of N-methyl-D-aspartate receptors in rat hippocampus (Nedivi et al.
1993), during long-term facilitation in Aplysia (Hu et al. 1993) and during synapse formation in the developing chick retina (see Osen-Sand et al. 1993, and in prepara-tion). These findings demonstrate the importance of the fusion machinery during nerve terminal remodeling in the adult and suggest that the mechanisms involved are also at work during development. Studies of SNAP-25 isoform expression support this hypoth-esis. SNAP-25 exists in at least two alternatively spliced variants, differentially regulated during development (Bark 1993; Bark and Wilson 1994a; Bark et al. 1995; Boschert et al. 1996). SNAP25a mRNA is highly and transiently expressed during axonal growth, while SNAP25b is induced during synapse formation and its levels are maintained throughout adulthood (Bark 1993; Bark and Wilson 1994b; Bark et al. 1995; Boschert et al. 1996). Further to these observations, Boschert and colleagues (1996) have shown that SNAP-25a (but not b) expression is induced in the adult hippocampus following lesions that are known to induce reactive sprouting. Although indirect, these obser-vations suggest that the two SNAP-25 isoforms may have different roles in transmitter release and membrane expansion. The regulated expression of the two SNAP-25 iso-forms, and of additional SNARES, may provide a molecular framework for differential
use of the fusion machinery during specific stages of maturation of the terminal.
However, Roberts et al. (1998) have demonstrated increased SNAP-25 gene expression in hippocampal granule cells following the induction of LTP in the afferent synapses, with both isoforms being affected.