Review articles
Structure suggests function:
the case for synaptic ribbons
as exocytotic nanomachines
David Lenzi1 and Henrique von Gersdorff2*
Summary
Synaptic ribbons, the organelles identified in electron
micrographs of the sensory synapses involved in vision,
hearing, and balance, have long been hypothesized to
play an important role in regulating presynaptic function
because they associate with synaptic vesicles at the
active zone. Their physiology and molecular composition
have, however, remained largely unknown. Recently, a
series of elegant studies spurred by technical innovation
have finally begun to shed light on the ultrastructure and
function of ribbon synapses. Electrical capacitance
measurements have provided sub-millisecond resolution
of exocytosis, evanescent-wave microscopy has filmed
the fusion of single 30 nm synaptic vesicles, electron
tomography has revealed the 3D architecture of the
synapse, and molecular cloning has begun to identify
the proteins that make up ribbons. These results are
consistent with the ribbon serving as a vesicle ``conveyor
belt'' to resupply the active zone, and with the suggestion
that ribbon and conventional chemical synapses have
much in common. BioEssays 23:831±840, 2001.
ß 2001 John Wiley & Sons, Inc.
Introduction
Synaptic transmission provides neurons with highly focal, fast,
and plastic signaling capabilities. Our understanding of
synaptic physiology, and especially of presynaptic mechanisms, has been anchored for many years by the vesicle
hypothesis, the grand-unified theory of synaptic transmission.
The hypothesis states that neurotransmitter is accumulated,
stored, transported, and ultimately released in quantal packets
which are thought to correspond to synaptic vesicles, the
ultrastructural hallmark of the presynaptic terminal. Ribbon
synapses constitute a specialized subclass of chemical
synapses, and are identified by an unusual presynaptic
organelle. Here, vesicles cluster not only at the active zone
1
Department of Otolaryngology-HNS, University of Virginia School of
Medicine.
2
The Vollum Institute, OHSU.
Funding agencies: HvG was supported by a Pew Biomedical Scholars
Award and a NIDCD grant.
*Correspondence to: Henrique von Gersdorff, The Vollum Institute,
OHSU, 3181 SW Sam Jackson Park Road, Portland, OR 97201-3098.
E-mail: [email protected]
BioEssays 23:831±840, ß 2001 John Wiley & Sons, Inc.
on the plasma membrane, as at conventional synapses, but
also on the surface of the ribbon, which protrudes from the
active zone into the presynaptic terminal. Ribbons are made of
osmiophilic proteinaceous material, the composition of which
is just beginning to be defined, and have a ``halo'' of synaptic
vesicles tethered to their surface (Fig. 1). Because of their
proximity to the active zone and their close association with
vesicles, it has long been supposed that these poorly
characterized organelles play an important role in the physiology of these synapses, perhaps by controlling vesicle
traffic (reviewed in Refs 1±3). The exact function of ribbons,
and whether these synapses utilize fundamentally the same
mechanisms as conventional synapses have, however,
remained elusive. In this review, we discuss recent physiological, anatomical and molecular findings to examine the most
widely discussed models of ribbon function, including the
hypothesis that ribbons shuttle vesicles to the active zone; we
also compare ribbon synapses with conventional synapses.
Ribbon synapses occur in sensory neurons such as
photoreceptors and bipolar cells of the vertebrate retina, in
pinealocytes, and in the mechanosensory hair cells of the inner
ear. In fish and amphibians, they also occur in the hair cells and
electroreceptors of the lateral line.(1) In addition, similar
structures are common in the neuromuscular junction of some
invertebrates,(4) but very little is known about them. Synaptic
ribbons are named for their appearance as electron-dense
bars in cross-section on electron micrographs. In three
dimensions, however, these structures are actually flat plates
with varied profiles (Fig. 1). In this review, we use the term
synaptic ribbon to also encompass similar presynaptic
organelles with varied shapes, including clubs(5) and spheres
(Fig. 2).(6) Despite a broad diversity in size and shape, the
comparative anatomy, common ultrastructural features and
shared physiological characteristics of these synapses suggest that they are somewhat homologous in function, and they
are known collectively as ribbon synapses. Ribbons can
assume different shapes in the same cell types between
species,(7) or even within a cell type across one tissue.(8±11)
All synaptic ribbons are osmiophilic, lack a delimiting membrane, and are surrounded by a halo of clear-core vesicles
that appear to be tethered to its surface by 1±5 thin
filaments.(6,12,13) Ribbon synapses also share physiological
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Figure 1. Ribbon synapse diversity and organization. A,B: transmission electron micrographs of skate electroreceptor (A) and frog
inner ear hair cell (B) ribbon synapse. In each, the presynaptic ribbon (arrows) is surrounded by a halo of synaptic vesicles
(arrowheads), and lies adjacent to the active zone. While the frog ribbon is spherical (see Fig. 2), the skate ribbon is a flat plate cut in
cross-section. Scale bars, 200 nm. C: The 3D organization of flat plate ribbons. The ribbon (blue) tethers a population of vesicles
(yellow) to its surface, some of which are also in contact (docked) at the plasma membrane (red). Other vesicles are shown in green. D:
At the frog neuromuscular junction, which is not a ribbon synapse, ribs connect docked vesicles to a central beam running the length of
the active zone, and connect to ion channels (green) in the plasma membrane. (A) Reprinted with permission from Ref. 85; (B) D Lenzi
and WM Roberts, unpublished; (D) Reprinted with permission from Nature, see Ref. 51.
features, occurring in cells that presumably release transmitter
(mostly glutamate) in a graded and continuous fashion
(reviewed in Ref. 14). Although a wide range of roles have
been proposed for the ribbon, the most widely discussed one
is the ribbon as a ``conveyer-belt'' that funnels vesicles
towards fusion sites at the active zone(15) that are directly
opposite to postsynaptic receptors (usually of the AMPA and
NMDA type).
In the conveyor-belt model, the ribbon supplies the continuously active synapse with release-ready vesicles by
shuttling them along its surface towards the base. Once at
the active zone, vesicles would dock and fuse with the plasma
membrane to void their contents into the synaptic cleft.
Ribbon-bound vesicles would be resupplied from the cytoplasmic pool. The hypothesis that vesicles move along the
surface of the ribbon in this manner makes several testable
predictions. During strong stimulation, the kinetics of exocytosis are likely to change as docked, ribbon-associated, and
then cytoplasmic vesicles enter the release pathway. Vesicles
on the ribbon should also be mobile, and should carry the
molecular machinery permissive to docking and fusion. We
examine these predictions in the following sections. In
addition, we review the molecular components and intricate
machinery that fine-tunes synaptic transmission at ribbon
synapses.
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BioEssays 23.9
Physiology of ribbon synapses
Synaptic vesicles, the smallest eucaryotic organelles, with
diameters of 30 to 50 nm, fuse with the plasma membrane in a
rapid (rise-times of <60 ms) and all-or-none event.(16) Among
all postsynaptic glutamate receptors, such speed can only be
captured biologically by certain subtypes of AMPA receptors
with fast rise-times, and these AMPA receptors appear to be
selectively localized just opposite to ribbons.(17,18) The small
size of synaptic vesicles may make them flexible minimum
``bits'' for information transfer at synapses and allows synaptic
boutons to be small and compact (<1 mm in diameter). A single
neuron can thus receive several thousand bouton synapses
and this greatly increases the computational capacity of neural
networks within a small CNS volume. Their small size may also
favor the rapid exocytosis of transmitter by diffusion, after the
fusion pore opens, their complete collapse into the plasma
membrane for rapid release, their quick retrieval back into the
terminal, as well as their fast and complete refilling with
neurotransmitter. The smallness of synaptic vesicles may thus
facilitate their local recycling so that bouton synapses can
operate as autonomous entities from the cell nucleus. Densecore granules, on the contrary, have to be recycled through the
Golgi apparatus in an elaborate and prolonged process. In
addition, the relatively uniform size of synaptic vesicles(6,13)
may also help to standardize quantal size.(19)
Review articles
Figure 2. Three-dimensional rendering of a frog
saccular hair cell synapse partially reconstructed
by electron tomography.(6) Approximately twothirds of a spherical ribbon (blue, arrowhead)
was captured in this reconstruction. The presynaptic ribbon was rendered as a hollow object,
and is open on the face that met the edge of the
physical section. Part of a smaller, second ribbon
is visible in the foreground, and was transected on
the opposite face of the reconstruction. Ribbontethered vesicles are yellow, and include those
docked beneath the ribbon. Other vesicles are
green, while membranous tubules and cistern are
purple. Mitochondria, both presynaptic and postsynaptic, are pale blue. The plasma membranes
of the presynaptic and postsynaptic cells are red,
and include putative sites of endocytosis (arrows)
just outside the active zone. Although surrounded
by a halo of vesicles, the smaller ribbon did not
appear to approach the plasma membrane. D
Lenzi, MH Ellisman, and WM Roberts, unpublished.
Ribbon synapses are capable of fast and transient release,
as well as slow and sustained signaling.(20,21) To achieve
repeated bouts of fast and transient release of neurotransmitter, a large readily releasable pool (RRP) of vesicles would
seem to be a prerequisite. The size of the RRP of vesicles can
be directly measured with membrane capacitance measurements, which report even very small changes in cell surface
area triggered by vesicle fusion (e.g. detecting <0.1%
changes in bipolar cell terminals). Sensory neurons, with their
electrotonically compact shapes and large vesicle numbers,
are par excellence ideal for capacitance measurements.
Indeed, the cytosol of photoreceptors, bipolar cell terminals
and hair cells contains a vast number of synaptic vesicles. In
goldfish bipolar cells, for example, there are about 750,000
synaptic vesicles in a single terminal; one frog vestibular hair
cell can contain 600,000 vesicles. In goldfish bipolar cells,
however, less than 1% of these seem to be fusion competent
according to capacitance measurements. Why the total
number of synaptic vesicles is so large remains a mystery. In
goldfish bipolar cells, the total RRP is about 6,000, and there
are 110 vesicles per ribbon. By contrast, hippocampal boutontype conventional active zone synapses have a RRP of about
8±10 docked vesicles and a total pool of vesicles in the cytosol
of 200.(22) Ribbon synapses are thus associated with a larger
RRP and total vesicle population than conventional synapses,
with the possible exception of calyx-type synapses that
have hundreds of active zones, and a RRP as large as 3,500
vesicles.(23)
Unlike large dense core granules, which can fuse equally
well over extended areas of a cell, or even with themselves,
synaptic vesicle exocytosis is thought to occur at ``hot spots'',
the active zones. What is the evidence that ribbons mark hot
spots of rapid glutamate release? (1) EM immunohistochemistry shows AMPA and NMDA receptors are localized just
opposite to ribbons.(17,18) (2) Photoreceptor terminals, hair
cells, and bipolar cell terminals have been shown to directly
secrete glutamate.(24±26) (3) Calcium (Ca) channels are
closely aligned to ribbons.(11,27±29) (4) In goldfish bipolar cells,
the equivalent of 20 vesicles per ribbon fuse with the plasma
membrane following a very brief and strong stimulus (e.g. the
<1 ms Ca influx during a voltage clamp step from ‡ 60 mV to
ÿ 60 mV) ,(30) consistent with the fusion of all the docked
vesicles occupying the bottom row of each ribbon. (5) Finally,
in a recent study,(31) evanescent-wave microscopy was used
to image the surface of goldfish bipolar cell terminals, and
resolve the fusion of single, fluorescently labeled vesicles at
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the plasma membrane. Fusions occurred in ``hot spots'', and
docked vesicles were the first to fuse, consistent with the
colocalization of ribbons and sites of repeated exocytosis. By
contrast, at a conventional synapse, a single action potential
elicits the fusion of 1±3 vesicles(32) and the total releasable
pool after prolonged stimulation (an action potential train)
is about 10. Bipolar cell ribbons may thus be particularly
versatile signal transducers capable of mediating both very
fast and transient release, as well as sustained release due to
their large RRP. This ability may be just what is needed for a
cell thought to transmit, and enhance, contrast in a visual
scene.
Exocytosis, for signaling purposes, is, however, a futile and
expensive exercise if it is not accompanied by neurotransmitter release. While empty vesicles may be fusion competent(33)
they are clearly ``mute'' entities for signal transfer. Empty
vesicles must thus have efficient transport mechanisms to
quickly refill themselves after endocytosis and should be
prevented from engaging in exocytosis until they become
refilled. Recent studies show that indeed endocytosed vesicles rapidly acidify to reestablish the pH gradients necessary
to drive inward transport of glutamate.(34) It may thus be the
case that only the vesicles that are filled with neurotransmitter,
and primed or ready for fusion can tether to the ribbons. Recent
imaging studies suggest that vesicles within the cytoplasm of
ribbon synapses collide with each other and with the plasma
membrane in a jittery 3D Brownian motion.(31) Perhaps this
occurs until they chance upon a synaptic ribbon, which
captures and confines them into 2D Brownian motion on its
surface. Ribbons may thus act as selective vesicle capturing
devices, and this may subsequently greatly increase the
probability that vesicles will dock at the active zone, and
therefore fuse in appropriate apposition to postsynaptic receptors. One can also imagine that ribbons contain molecular
motors that actively drive vesicles towards docking sites, a
picture implied by conveyor-belts. Recently, a kinesin subunit
(see below), part of a vesicular motor that drives directed
movement of vesicles along microtubules, has been localized
to photoreceptor and bipolar cell ribbons.(35) Because kinesin
can move at up to 1 mm/s,(36) vesicles could be translocated by
a molecular motor from the top of a 150 nm goldfish bipolar cell
ribbon in 150 ms, which is about the time that it takes to
release the equivalent of all the vesicles on the ribbon. On
these distance scales, however, diffusion may deliver vesicles
as fast as a motor. Indeed, imaging showed vesicles to be
delivered equally rapidly at and away from ``hot spots'', the
putative ribbon locations in bipolar cell terminals.(31)
To avoid a catastrophic shutdown in release due to massive
depletion of the RRP, synaptic vesicles must be quickly
recycled. Accordingly, vesicular membrane added to the
presynaptic plasma membrane is rapidly retrieved (time
constant t ˆ 1±2 sec) in bipolar cells and in hair cells
(t ˆ 7.5±14 sec).(37,38) In bipolar cells and hair cells, exocy-
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BioEssays 23.9
tosis and endocytosis are temporally well separated following
short depolarizations but, in photoreceptors, they appear to
occur simultaneously, although a brief imbalance can occur for
strong stimuli.(39) The precise balance of addition and
subtraction of membrane apparently keeps the presynaptic
terminal's surface area tightly controlled and on average
constant. A carefully choreographed cascade of proteins
initiates and regulates endocytosis and these proteins again
seem to be common to both ribbon and conventional
synapses. Recent findings using different FM dyes also
indicate that a rapid form of endocytosis (t ˆ 1 sec) is present
in conventional bouton-type synapses.(40) Upon stronger
stimulation, a slower form of endocytosis is evident at ribbon
and conventional synapses.(41) The locus of endocytosis has
classically been associated with sites outside of the active
zone. Recently, however, clathrin-coated endocytosis has
been shown to occur at, or adjacent to conventional active
zones(42) and near ribbons (Fig. 2) ,(6) and vesicle recycling
may bypass early endosomes.(43) These results thus suggest
that a very rapid and highly local form of endocytosis is present
at both ribbon and conventional synapses.
Microanatomy of ribbon synapses
The vesicle conveyer-belt model of ribbon function predicts
that vesicles at the base of the ribbon can dock and fuse with
the plasma membrane, that there is turnover of vesicles on the
surface of the ribbon, and that strong stimulation should
momentarily deplete the docked and ribbon-associated vesicles. At conventional synapses, vesicles are believed to dock
at the plasma membrane by specific binding, before fusion and
exocytosis. At ribbon synapses, many ultrastructural studies
have shown that vesicles tethered to the base of the ribbon
often also touch the active zone, whether adjacent to a flat
ribbon sheet(13) (Fig. 1) or beneath a spherical ribbon(6,44)
(Fig. 2). In hair cells from the frog sacculus, docked vesicles
are probably specifically bound to the membrane, as they are
far more concentrated than those in the adjacent cytoplasm.(6)
They sit within nanometers of the voltage-gated calcium
channels,(27) and are therefore likely to experience the high
calcium influx(45) that triggers exocytosis.(37) Furthermore,
omega-profiles found on the plasma membrane beneath these
ribbons(6) may correspond to vesicle fusion sites, although
confirmation of this observation will require rapid freezing
instead of chemical fixation.
Several lines of evidence also suggest that there is turnover
of ribbon-tethered vesicles. At photoreceptor,(46) retinal
bipolar cell(47) and hair cell(48) synapses, extracellular markers
taken up by endocytosis move through the vesicle cycle to
label ribbon-tethered vesicles. In turtle photoreceptors, the
labeling of ribbon-associated vesicles correlates with synaptic
activity,(46) implying that labeled ones replace unlabeled ones
during synaptic transmission. More recent studies have
compared the amplitude of measured exocytosis and the
Review articles
number of ribbon-associated vesicles. In goldfish bipolar cell
terminals, capacitance measurements have shown exocytosis to slow after the fusion of the equivalent of 1,100
vesicles,(49,50) and then plateau after the fusion of 4,600
more.(13) These estimates correspond very well to the number
of vesicles calculated to occupy the 45±65 ribbons per
terminal. Using transmission electron microscopy, each ribbon was estimated to carry 110 vesicles, of which 22 were
also docked at the active zone.(13) These results are
therefore consistent with the interpretation that docked and
ribbon-associated vesicles are in the release pathway. In this
cell, docked vesicles may not, however, be replenished
exclusively from the ribbon, but perhaps from the cytoplasm
as well.(49)
In hair cells, similar capacitance measurement studies
have shown that exocytosis also slows after an initial fast
phase in mouse cochlear hair cells, but then continues at a
constant rate for up to at least one second in mouse(38) and two
seconds in frog saccular hair cells.(37) While the ultrastructure
of the mouse hair cell synapses is not known in detail, electron
tomography has been used to map the frog hair cell synapses
at high resolution.(6) Electron tomography uses images of a
thick tissue section acquired at different tilt angles to reconstruct in three dimensions the ultrastructure of the synapse.
The method is analogous to performing a CAT scan (Computerized Axial Tomography) with an electron microscope,
and provided z-axis resolution fine enough (1±3 nm) to sample
individual synaptic vesicles in about a dozen planes. The
method revealed the spherical shape and average diameter
( 470 nm) of the ribbon, as well as the average number and
distribution of docked, tethered, and cytoplasmic vesicles. In
conjunction with the previous capacitance measurements, this
study showed that neither the docked nor the ribbon-tethered
vesicles were numerous enough to account for continuous
bouts of exocytosis and that, unlike in goldfish bipolar
cells, replenishment of the docked and ribbon-associated
vesicles is probably not rate limiting. This may indicate
functional differences between bipolar cell and hair cell
ribbon synapses. This difference is probably not due to ribbon
shape, however, as they are spheres in frog sacculus, but
plates in both goldfish bipolar cells and hair cells of the mouse
cochlea.
Recently, electron tomography has also been used to
reconstruct in three dimensions the conventional active zone
of the frog neuromuscular junction (NMJ).(51) This tour-deforce study clearly shows that the vesicles at the active zone
are docked in two rows with a central electron-dense ``beam'' in
between. Extending from this beam are ``ribs'' that contact a
docked vesicle at 4±6 points and appear to help anchor it at the
active zone (Fig. 1). This architecture is very reminiscent of the
ribbon, with the ``beam'' playing an analogous role to the ribbon
itself. In addition, the ``ribs'' also make contact with the
intramembrane particles at the active zone thought to be the
Ca and Ca-activated K (KCa) channels that are aligned also in
two rows at the active zone. Interestingly, ribbon active zones
also contain rows of intramembrane particles thought to be Ca
and KCa channels.(27) Thus, it appears that the NMJ active
zone ultrastructure may be more similar to the ribbon ultrastructure than previously thought.
Additional evidence for the mobility of ribbon-associated
vesicles comes from examining stimulus-dependent changes
in vesicle distributions.(52) Electron tomography has recently
revealed depletion of docked and tethered vesicles at a ribbon
synapse.(53) Frog saccular hair cell synapses were stimulated
in high-potassium or inhibited in zero-calcium salines and then
reconstructed to compare their vesicle distributions. While
depolarization depleted an average of 70% of docked and
cytoplasmic vesicles seen in controls, ribbon-tethered vesicle
numbers fell by 40%. For each vesicle class, concentrations
bracketed those found in the previous study(6) where exocytosis was neither stimulated nor inhibited, a result consistent
with tonic activity of this synapse. The statistically significant
depletion of these vesicle populations indicates that they are
mobile, and is consistent with a functional role for vesicles
tethered to the ribbon and those docked beneath it. Ribbons
could not be completely stripped of vesicles, however, even
using the long treatments (30 minutes) of these studies, and
depletion was not evident in initial observations using conventional electron microscopy.(6) This finding, along with the
greater depletion of docked and cytoplasmic populations,
argues that the rate-limiting step of exocytosis lies downstream of vesicle capture by the ribbon, and is perhaps the
docking process. If so, then ribbons may speed exocytosis by
maintaining a high vesicle concentration at release sites,
ensuring that docking is not slowed by a drop in cytoplasmic
vesicle reserves.
If vesicles do indeed move along synaptic ribbons, then
vesicles might be propelled by passive diffusion, or active
molecular motors. Whatever the mechanism, movement is
likely to involve the tethers that link vesicles to the ribbon.
Tethers are probably not artifacts of chemical fixation, as they
are evident at slam-frozen photoreceptor synapses (3±5
tethers per vesicle).(12) Vesicles bind tightly, as ribbons retain
their monolayer of vesicles after subcellular fractionation(54)
or in mechanical lysed cells;(6) transiently bound vesicles
would be lost after these treatments, and vesicles therefore
probably do not bind and unbind repeatedly. Tethers are also
resistant to high concentration of calcium (4 mM) in lysed
cells,(6) indicating that if calcium mobilizes ribbon-associated
vesicles,(49) it probably does so without disrupting the tethers.
Little is known about the molecular composition of tethers,
although they are probably not made of actin. The actindisrupting compound cytochalasin D fails to strip vesicles from
pinealocyte ribbons(55) or disrupt vesicle cycling in retinal
bipolar cells,(56) and tethers were not labeled by an antibody to
actin.(12) Tethers may, however, include proteins related to
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molecular motors. A monoclonal antibody to the kinesin
polypeptide KIF3A labels ribbons and some adjacent vesicles
by immunogold electron microscopy(35) in rat photoreceptors,
and by immunofluorescence in amphibian, avian, and mammalian retinae. Although an intriguing result, other subunits
required to form a functional motor have not yet been localized
to ribbons, and microtubules, kinesin's substrate, are not
known to be present near ribbons. Physiological evidence
suggests a role for another molecular motor, myosin, in
mobilizing synaptic vesicles. Inhibiting myosin ATPase activity
reduces capacitance measurements of exocytosis in chick
cochlear hair cells.(57) The ultrastructural localization of
myosin motors remains unknown, however.
An alternative hypothesis to the translocation of vesicles is
the protrusion of the presynaptic cell into the postsynaptic
membrane, bringing the presynaptic plasmalemma adjacent
to all the vesicles on each side of the ribbon.(49) Vesicles would
then not have to move in order to dock and fuse. Activitydependent morphological changes of this type have been
observed in electroreceptors,(58) but this mechanism might
hinder the reloading of docking sites, and would be restricted to
flat-ribbon synapses, as spherical ones have never been
observed to evaginate the plasma membrane very far.
Vesicles tethered to the top of these ribbons could not approach the active zone without moving.
Molecular components of ribbon synapses
A significant advance of the past decade has been the
identification and sequencing of many synaptic proteins,
including most, if not all, of those found in synaptic vesicles.
Although the function of many of these proteins remains
unknown, immunohistochemistry has been widely used to
describe their expression patterns at many synapses, including those with ribbons (reviewed in Ref. 29). Table 1 summarizes their expression patterns in photoreceptors and
bipolar cells of the retina, and in hair cells of the inner ear.
Most studies employed fluorescence immunohistochemistry,
but several also used immuno-electron microscopy, molecular
biology, and Western blotting.
Synapsins, calcium-regulated ATP-binding proteins that
link vesicles to the actin cytoskeleton, are universally found on
vesicles at conventional synapses. Over 10 years ago, it was
discovered that retinal ribbon synapses lacked this family of
proteins in rat, salamander and monkey.(59,60) Additional
experiments have confirmed this finding at almost all other
ribbon synapses tested, including those in pinealocytes.(62)
Furthermore, transgenic expression of synapsin in mice
photoreceptors does not seem to alter photoreceptor
function.(61) These results led to the hypothesis that the ribbon
assumes synapsin's presumed role in controlling vesicle
clustering and mobilization.(59) This interpretation, though
intriguing, is incomplete because there are many vesicles at
ribbon synapses that are not tethered to the ribbon, but
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nevertheless concentrate in the nearby cytoplasm.(6) The
forces that aggregate these vesicles remain completely
unknown, and this argues against the view that vesicles move
freely in the cytoplasm. Synaptotagmin, another vesicle
protein, is proposed to be a calcium sensor for exocytosis.
While all ribbon synapses examined express this protein,
cochlear hair cells express the rarer types IV and VI±IX, and
photoreceptors from several species express types I and II,
which are the most abundant at conventional synapses.
Synaptophysin has also been found at almost all ribbon
synapses examined. Synaptoporin may or may not be
expressed in rat photoreceptors, but is differentially expressed
in the rabbit retina, appearing faintly in electron micrographs of
bipolar cell synapses, but not photoreceptors. Ribbon
synapses in every retina examined except cow express SV2,
another common vesicle protein. Studies have found Rab3a,
postulated to regulate synaptic vesicle fusion, to be present in
rodent and cow retina and inner ear, although studies using the
identical antibody have generated opposite results in mouse
photoreceptors. Rabphilin, a Rab3a binding-protein, is present
in cow but absent from mouse photoreceptors.
Vesicle docking at the plasma membrane is thought to be
mediated in part by assembly of a core SNARE-complex,
composed of synaptobrevin in the vesicle membrane and
syntaxin and SNAP-25 in the plasma membrane. Each of
these proteins is expressed at ribbon synapses, although their
subtypes vary. Synaptobrevin 1 occurs in cow photoreceptors
and guinea pig cochlear hair cells, while synaptobrevin 2 is
present in rat retina. There is less agreement on Snap-25
expression, although the majority of studies have found that it
is expressed in retinal ribbon synapses, including one using
immuno-EM. This protein is also expressed in cochlear hair
cells. Finally, a form of syntaxin is present at all ribbon
synapses examined. Syntaxin I appears in hair cells, while
syntaxin III is expressed in rat and cow retina.
Another plasma membrane protein of the active zone,
Munc13-1, has been found to be absent from retinal ribbon
synapses.(63) Ribbon synapses may therefore employ an
alternate mechanism of vesicle priming, the proposed role of
Munc13-1 at conventional synapses.
These studies show that, in general, ribbon synapses
express the conventional set of synaptic proteins, with the
exception of synapsins, which are almost always absent.
While some ribbon synapses contain rarer isoforms of some of
these molecules, and there are discrepancies between studies, the consensus that emerges is that most of the principal
components identified at other synapses are also in play at
ribbon synapses. Furthermore, whenever synaptic vesicle
protein expression has been examined by immuno-gold
electron microscopy,(59,62,64) vesicles bound to the ribbon
are indistinguishable from those in the cytoplasm or docked at
the active zone, arguing that they are biochemically similar.
Thus, expression patterns of synaptic vesicle proteins further
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Table 1.
Synaptic protein expression in cells making ribbon sympses
Photoreceptors and bipolar cells of the retina
Present
Synpatic vesicle proteins
Synapsin I, II
‡ cow(71)
Synaptotagmin
Synaptophysin I
Synaptogyrin
Synaptoporin/
Synaptophysin II
SV2
Rab3a
Rabphilin
Core complex proteins
Synaptobrevin
Snap-25
Syntaxin
‡ mouse(61, type I)
‡ rat(73, type I, II) (76)
‡ cow(71, type I)
‡ monkey(64)
‡ salamander(59)
‡ mouse(61) (72)
‡ rat(59) (72) (73) (75)
‡ rabbit(75)
‡ cow(54yy) (71)
‡ monkey(59) (72)
‡ cow(71)
‡ rat(73)
‡ rabbit(75yyy)
‡ salamander(59)
‡ rat(59)
‡ cow(71, type B)
‡ mouse(71)
‡ rat(71) (73)
‡ cow(71)
‡ cow(71)
‡ rat(73) (83,
‡ cow(71)
Hair cells of the inner ear
Absent
Present
ÿ salamander(59)
ÿ gerbil(62)
ÿ mouse(71) (72)
ÿ rat(59) (62) (71) (72) (73)
ÿ monkey(59) (64) (72)
ÿ guinea pig(74y)
ÿ cat(60)
‡ guinea pig(74y,
type IV, VI-IX)
‡ chicken(77)
‡ mouse(78)
‡ rat(78)
‡ human(79)
ÿ guinea pig(74y,
ÿ rat(80)
ÿ guinea pig(74y)
type I, II, III, V)
(80) (81)
ÿ rat(75)
ÿ cow(71, type
ÿ mouse(82)
A)
‡ mouse(78)
‡ rat(78)
ÿ mouse(71)
type 2)
‡ rat(73) (83) (84)
‡ cow(71)
‡ rat(83, type III)
‡ cow(71, type III)
Absent
‡ guinea pig(74y,
ÿ mouse(72) (82)
ÿ rat(72)
ÿ monkey(72)
ÿ rat(73, type I) (83,
ÿ cow(71, type 1b)
type 1)
ÿ guinea pig(74y,
type 2 )
type I, V, VI)
ÿ guinea pig(74y,
type II, III, IV)
‡ guinea pig(74)y
type I ) (84)
‡ guinea pig(74y,
The presence( ‡ ) or absence( ÿ ) of immunoreactivity for each protein is listed for each species and tissue tested. Studies (superscripts provide the
reference) employed light-level immunohistochemistry, unless indicated. When distinguished, synaptic protein subtypes are indicated in italics. In retina,
proteins were found in both photoreceptors and bipolar cells unless noted. In references 59, 64, 75, 80 and 82±84 antigens were detected by immuno-gold
electron microscopy. y, Gene expression was assessed by RT-PCR, in-situ hybridization, Western blots, and immunohistochemistry. yy, An outer plexiform
layer subcellular fraction was probed by Western blot. y y y, Synaptoporin was found in bipolar cells, but not photoreceptors of the rabbit retina by immunoelectron microscopy.
support the hypothesis that ribbon-tethered vesicles are in the
release pathway.
Finally, some of the molecular components of ribbons
themselves are beginning to be identified. Due to the paucity of
starting material, ribbons have been difficult to purify, and
progress has been slow. Nevertheless, several proteins
appear to contribute to the ribbon's structure. The B16 antibody binds to retinal ribbons in all species tested, and labels
the matrix of ribbons using immuno-EM.(65) This antibody
immunoprecipitates a clathrin-adaptor-like protein,(66) perhaps also implicating ribbons in endocytosis. Another ribbonspecific antibody has been used to clone a novel component of
ribbons, RIBEYE.(67) This protein of 120 kDa is composed of
two domains: a novel A domain specific for ribbons, and a B
domain identical to CtBP2, a transcriptional repressor.
Schmitz et al. propose a model where one domain may act
as an aggregating module, while the other may act as an
enzyme and NAD-binding factor. The function of this putative
enzymatic activity is however unclear. In addition to KIF3A
mentioned above, one other ribbon protein has been
identified. RIM, a putative Rab3-interacting protein, localizes
to active zones at conventional synapses, but is also found in
photoreceptor ribbons.(68) Immuno-EM revealed distinct labeling of only the ribbon and its active zone. RIM also has a PDZ
protein±protein interaction domain, which suggests it also has
a structural role at the synapse. Another structural protein,
BioEssays 23.9
837
Review articles
bassoon, a large presynaptic protein which is highly concentrated at the active zone of conventional synapses, probably
serves as a scaffolding protein of the presynaptic terminal.
This protein has recently been found at the large ribbons of rat
photoreceptors, but surprisingly not in the smaller ribbons of
bipolar cells.(69) Immuno-EM revealed extremely localized
labeling of the active zones only, but only in rods and cones.
Zebrafish genetics has also recently pointed to heterogeneity
among ribbon synapses in the retina.(70) The nrc mutation
disorganizes cone but not bipolar cell ribbon synapses by
disrupting the invagination of the presynaptic terminal by the
postsynaptic cells, and dissociating ribbons from the active
zone. Thus, equipped with different synaptic machinery,
ribbon synapses may be tuned to different performance
specifications, and this may explain physiological differences
observed among ribbon synapses.(39)
In summary, there appear to be several species and
sensory organ differences in the expression patterns of some
of the above proteins, and there are also differences within
tissues, suggesting that ribbon synapses are not built to a
strictly identical specification. Thus, the basic bauplan of
ribbons may be common, but the modus operandi of
morphologically distinct ribbons may differ significantly.
Conclusions
Ribbon synapses are probably optimized for their roles in
cells that release transmitter continuously and in response
to graded signals, and may do so copiously under strong
stimulation. While their physiology and anatomy suggest
that ribbons act to control vesicle traffic, perhaps by guiding
vesicles towards the active zone, they probably do so using
standard molecular components. Apart from some of the
components of the ribbon itself, such as the B16 antigen and
RIBEYE, all the proteins, or their close relatives, described at
ribbon synapses also occur at conventional synapses.
Many questions remain about the nature and physiology of
ribbon synapses. Still lacking, for example, is a clear understanding of how ribbons actually function during exocytosis,
and perhaps endocytosis. There is no direct evidence yet for
vesicle mobility on the ribbon, or dynamic exchange of
tethered and cytoplasmic vesicles in live cells. Improvements
in imaging technology may make these sorts of experiments
feasible in the future.(31) Also unexplained is the great variance
in ribbon size and shape. How does the ribbon's shape finetune synaptic performance, and why do ribbons vary in size
over an order of magnitude? There are also many questions
about ribbon behaviors on longer timescales. For example,
why and how do ribbons disappear and reappear in some
tissues but not others, and why does synaptic morphology
seem to change so dramatically in a stimulus-dependent
fashion. Advances in imaging, molecular biology, and genomics should help solve these challenging but fascinating
questions in the near future.
838
BioEssays 23.9
Acknowledgments
We thank Miriam Goodman for comments on the manuscript,
and our colleagues for permission to reprint figures. We
apologize for our inability to reference all the relevant literature
due to space limitations.
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