All four syntaxin isoforms associated with trafficking to the plasma membrane are expressed in the synaptic layers of the retina. Each isoform displays a unique distribution in the synaptic layers, with little colocalization of isoforms at the subcellular level, although they may be co-expressed within a cell. These findings strongly suggest that each syntaxin isoform mediates different trafficking events in the retina, and under normal conditions at least, serve different functions with little redundancy between isoforms. Syntaxins 1 and 3 are both presynaptic, but are found at different synapses. Syntaxins 2 and 4 generally do not appear to be presynaptic and are likely to have primary functions other than regulating neurotransmitter release.
Differential distribution of trafficking proteins reflects functional differences
The distribution of trafficking proteins and their isoforms reflects functional differences. The distribution of several presynaptic proteins show distributions in the retina that differ according to the functional characteristics of synaptic release at conventional and ribbon synapses. Synapsins and rabphilin are present only in conventional retinal synapses [19, 36]. Complexins III and IV and SV2B are found exclusively at ribbon synapses [20, 23]. The expression of synaptotagmin isoforms, key vesicular Ca++ sensors that regulate vesicle fusion , also can differ among conventional and ribbon synapses [16, 38]. Content of presynaptic active zone cytomatrix proteins, such as piccolo and bassoon, also may not be uniform among synapses [[17, 18], but also see ] and, at ribbon synapses at least, can be spatially segregated within an individual synapse [18, 40]. Proteins associated with endocytosis, including dynamin, clathrin and amphiphysin, also are differentially distributed among ribbon and conventional synapses in a manner consistent with differences in the mode of endocytosis [41–46]. There is considerable diversity in post-synaptic trafficking as well, as post synaptic terminals exhibit a complex synapse-specific array of transmitter receptors and subunits, signaling, scaffolding and anchoring proteins [47–49]. Thus, the differential distribution of syntaxin isoforms observed here is likely to reflect functional differences in pre-synaptic, postsynaptic, and possibly, extrasynaptic compartments within the synaptic layers of the retina.
Segregation and colocalization of syntaxin isoforms in the synaptic layers of the retina
There was no substantial colocalization of any syntaxin isoforms in either synaptic layer, although co-expression of syntaxin 1 and 2 were co-expressed in amacrine cells. Co-expression of multiple syntaxin isoforms that mediate trafficking to the plasma membrane is common, but the isoforms typically are spatially segregated to different subcellular compartments and mediate different trafficking functions [e.g., [7, 8, 50–52]; reviewed in [1, 6]]. This is consistent with the current findings for syntaxin 1 and 2 which were co-expressed in amacrine cells, but differentially distributed at the subcellular level. Thus, each syntaxin isoform is likely to mediate a unique set of cell- and/or synapse-specific vesicular trafficking events in the synaptic layers of the retina, with little functional redundancy.
The large variety of syntaxin isoforms and their localization to specific subcellular compartments is thought to contribute to the specificity of vesicular trafficking to appropriate target membranes. Syntaxins can interact promiscuously with other SNAREs in vitro, but in vivo each syntaxin isoform has preferred binding partners [4, 5, 10, 53]. Thus, the specific binding partners available to complex with syntaxin also may contribute to the regulation of membrane fusion and targeting. Presynaptic proteins that interact with syntaxins, including VAMPs and complexins, show synapse-specific distribution in the synaptic layers of the retina [20–22, 54]. Munc-13, another protein that interacts with syntaxins, also may show differential distribution among retinal synapses, although this is controversial [40, 55]. The distribution of these proteins do not precisely parallel the distribution of the various syntaxins, suggesting many possible combinations may exist that could shape the complex, synapse-specific characteristics of synaptic vesicle trafficking and exocytosis. For example, the distribution of complexin isoforms, small presynaptic proteins that bind to syntaxin and stabilize the fusion core complex , does not match syntaxin isoform distribution [[20, 54], this report]. Complexins 1 and 2 are differentially distributed among amacrine cells, which co-express syntaxin 1 and 2. Complexins 1 and/or 2 are also found in horizontal cells, which express syntaxin 4. Complexins 3 and 4 are unique to ribbon synapses but are expressed in a cell-specific manner among the ribbon synapses of photoreceptors and bipolar cells, which all contain the same syntaxin, syntaxin 3.
Isoform-specific functions of syntaxins 1 through 4 in the retina
The functional consequences of cellular and subcellular segregation of the various syntaxin isoforms in the plexiform layers of the retina are not yet known, as direct functional data are currently lacking. Potential trafficking functions that may be associated specifically with each syntaxin isoform in the retina are discussed below.
Syntaxins 1 and 3
The current study directly confirms that syntaxins 1 and 3 are the principal presynaptic syntaxins in the retina [; this report] and extend previous findings by directly demonstrating that syntaxins 1 and 3 do not colocalize and that syntaxin 3 is present in all retinal ribbon synapses.
Our double-labeling studies confirm that the synaptic localization of syntaxin 1 is restricted to conventional synaptic terminals which show transient release characteristics and typically release an inhibitory amino acid transmitter, GABA or glycine, in the retina [15, 29]. These results are consistent with previous reports in retina [12, 23, 36, 57–59]. In contrast, syntaxin 3 was found exclusively at ribbon synapses of photoreceptors and bipolar cells, which are complex synapses organized around a lamellar synaptic ribbon and show very high, sustained rates of glutamate release, likely mediated by compound vesicle fusion [13, 14]. This is consistent with previous results . Syntaxin 3 may confer some specific advantage for rapid compound fusion of multiple vesicles for rapid transmitter release from photoreceptor terminals. Syntaxin 3 is known to localize to the membrane of secretory vesicles in the acinar cells of the pancreas and gastric parietal cells [8, 60]. The current study also directly establishes that syntaxin 3 is not present at the putative glutamatergic conventional synapses of the VGLUT3 amacrine cells [24–26], indicating that syntaxin 3 is not specifically associated with glutamatergic transmission. Thus, syntaxins 1 and 3 segregate specifically according to the architectural and functional characteristics of the synapses.
Syntaxins 1 and 3 also were found extrasynaptically. Syntaxin 1 was diffusely distributed along amacrine cell processes, consistent with previous reports indicating that syntaxin 1 is not strictly localized to the synaptic active zone [e.g., [11, 61]]. Extrasynaptic functions of syntaxin 1 in the retina are uncertain, but several possibilities exist. One possibility is a role in exocytosis of neuropeptides via dense cored vesicles, which can be released from any part of a neuron [62–65]. Such a role would be consistent with the well-known expression of a variety of neuropeptides by amacrine cells [66, 67]. Another potential function for extrasynaptic syntaxin 1 is trafficking and regulation of transporters and channels. Syntaxin 1 associates specifically with a variety of neurotransmitter transporters [e.g., [68–74]] and ion channels [e.g., [75–77]]. Extrasynaptic syntaxins also have important roles in process growth and remodeling during neural development [78–81], and might have similar roles in process remodeling or plasticity associated with normal retinal function or pathology. Extrasynaptic pools of syntaxin 3 were present in photoreceptor inner segments and the cell bodies and axons of photoreceptors and bipolar cells. The function of syntaxin 3 in the photoreceptor inner segments is unclear, but an attractive candidate function is trafficking of outer segment proteins, such as opsins, which are trafficked via vesicles to the apical portion of the inner segment for assembly of outer segment discs [82–84]. Somatic pools of syntaxin 3 may be associated with standard "housekeeping" trafficking needs.
Syntaxin 2 is expressed in amacrine cells and their processes in the IPL, similar to syntaxin 1. Syntaxin 2, however, does not colocalize with syntaxin 1, suggesting that syntaxins 1 and 2 are functionally complementary to one another despite being expressed by the same cells. Consistent with these findings, syntaxin 2 shows very little colocalization with conventional or ribbon presynaptic markers. These results indicate that syntaxin 2 must have principal functions other than presynaptic transmitter release despite its localization to the IPL.
One particularly attractive candidate function for syntaxin 2 in the IPL is trafficking of post-synaptic components, such as neurotransmitter receptors. Such a function would be consistent with the frequent apposition of syntaxin 2 to presynaptic terminals labeled for syntaxin 1 or syntaxin 3. However, this function has never been tested directly either in retina or in brain and the current study does not establish unequivocally whether syntaxin 2 is specifically localized to post-synaptic terminals. It is clear, however, that syntaxin 2 is not localized exclusively to postsynaptic terminals, as it is also found in the cell body and does not always align precisely with presynaptic markers. Thus, syntaxin 2 might serve extrasynaptic trafficking functions instead of, or in addition to, postsynaptic functions. A potential extrasynaptic function for syntaxin 2 is trafficking of proteins with neural functions that are not strictly localized to the synapse, such as transporters, ion channels or extrasynaptic transmitter receptors. Again, these functions have not been tested directly, but would be consistent with the colocalization of labeling for syntaxin 2 and GlyT1 in the IPL. Further studies to localize syntaxin 2 at the ultrastructural level would aid in resolving precisely which cellular compartments syntaxin 2 is present in within the processes of the amacrine cells.
Elsewhere in the body syntaxin 2 is known for mediating fusion of large secretory vesicles for exocytosis of proteins from non-neural cells [85–87]. By extension, syntaxin 2 might have a similar function in the IPL and mediate release of neuropeptides from amacrine cell processes via dense-cored vesicles as suggested above for syntaxin 1. Other important functions mediated by syntaxin 2 elsewhere in the body include cytokinesis  and the regulation of epithelial morphogenesis during development as a secreted, rather than an intracellular, protein [reviewed in [89, 90]]. However, it seems unlikely that syntaxin 2 would have comparable functions in the adult retina.
Syntaxin 4 had the most restricted distribution of all the isoforms studied, and is expressed specifically in horizontal cell processes at synaptic ribbon complexes in the terminals of rods and cones, and in small puncta in the IPL and INL. Syntaxin 4 also is found in non-neural cells associated with the retinal vasculature. The functions of syntaxin 4 in the retina have never been studied, but the distribution of syntaxin 4 did not overlap with the other syntaxin isoforms, strongly suggesting non-redundant functions. The lack of colocalization between syntaxin 4 and presynaptic markers for conventional and ribbon synapses indicate that the primary function of syntaxin 4 is not likely to be pre-synaptic transmitter release. On the other hand, syntaxin 4 was often found in puncta apposed to presynaptic markers, including synaptic ribbons and the active-zone protein bassoon, suggesting potential key functions in post-synaptic trafficking.
Syntaxin 4 in the OPL was restricted to the post-synaptic processes of horizontal cells, and was not found in bipolar cell dendrites or photoreceptor terminals. The presence of syntaxin 4 at the tips of horizontal cell processes in the ribbon synaptic complexes of rods and cones is intriguing. Photoreceptors provide glutamatergic input to horizontal cell processes flanking the synaptic ribbon via AMPA receptors [91, 92]. The localization of syntaxin 4 to this site would be consistent with a role in local post-synaptic trafficking of neurotransmitter receptors and/or other signaling proteins to the horizontal cell plasma membrane, but this potentially important function has not been explored. Syntaxin 4 also might mediate trafficking of neurotransmitter transporters to the cell surface. Syntaxin 4 is critical to translocation of other transporter proteins, particularly glucose transporters, to the cell surface in response to receptor-mediated signals in non-neural cells [e.g., [93–95]] and also has been shown to mediate neurotransmitter transporter trafficking to the cell surface in cultured glioma cells .
Horizontal cell processes in the ribbon synaptic complex also provide inhibitory feedback to photoreceptors [; reviewed in ]. The manner in which this feedback is provided is controversial. Horizontal cells have been suggested to provide this feedback by several different mechanisms: via the neurotransmitter GABA ; via electrical currents created by connexin hemi-channels [100, 101], and by regulation of the pH in the synaptic cleft [98, 102, 103]. The presence of syntaxin 4 in the tips of horizontal cell processes could be interpreted as evidence for the existence of vesicular GABA release from adult horizontal cells. Support for this idea is provided by the expression of the vesicular GABA transporter and, sometimes, GAD and GABA in adult mammalian horizontal cells [104–107]. In addition, complexin 1 and/or 2, which interact specifically with syntaxins, are present in horizontal cells . However, several key components needed for vesicular synaptic release of GABA, such as presynaptic active zones, SNAP-25, VAMP, and synaptotagmin family members have not been specifically identified in mature horizontal cell processes in the ribbon synaptic complex to date.
The current study showed only weak, diffuse labeling for syntaxin 1 in the OPL, consistent with previous reports [12, 23, 36, 57–59]. In contrast, Hirano et al.  reported syntaxin 1 labeling in horizontal cell processes in the rabbit retina. This labeling may correspond to the weak syntaxin 1 labeling observed in the current and previous studies. The reasons for the relative differences in syntaxin 1 labeling intensity are not clear but may include species differences or subtle differences in labeling and visualization techniques. The high intensity of syntaxin 4 labeling relative to syntaxin 1 labeling in the OPL, however, suggests that syntaxin 4 is the predominant syntaxin for plasma membrane trafficking in horizontal cells. Resolution of the existence of GABAergic feedback from mammalian horizontal cells to photoreceptors and the potential roles of syntaxins 1 and 4 will require further investigation.
In the IPL, syntaxin 4 appears to be associated with processes from a small subset of amacrine and/or interplexiform cells, as double labeling showed no expression of syntaxin 4 in the processes of bipolar, ganglion or Müller cells. Most syntaxin 4 labeling in the IPL was concentrated in puncta at the INL/IPL border. These puncta do not arise from the dopaminergic or the CD15-positive GABAergic amacrine cells that stratify at this level of the IPL, but do appear to make contact with those cell types. The functional role of syntaxin 4 in the IPL is uncertain, but syntaxin 4 does not colocalize with presynaptic markers and is unlikely to have a major function in transmitter release. In contrast, syntaxin 4 was observed apposed to presynaptic active zones labeled for bassoon suggesting that syntaxin 4 in the IPL likely functions in postsynaptic trafficking or extrasynaptic transport functions.