Synaptic Ca2+ in Darkness Is Lower in Rods than Cones, Causing Slower Tonic Release of Vesicles

Article (PDF Available)inThe Journal of Neuroscience : The Official Journal of the Society for Neuroscience 27(19):5033-42 · June 2007with6 Reads
DOI: 10.1523/JNEUROSCI.5386-06.2007 · Source: PubMed
Abstract
Rod and cone photoreceptors use specialized biochemistry to generate light responses that differ in their sensitivity and kinetics. However, it is unclear whether there are also synaptic differences that affect the transmission of visual information. Here, we report that in the dark, rods tonically release synaptic vesicles at a much slower rate than cones, as measured by the release of the fluorescent vesicle indicator FM1-43. To determine whether slower release results from a lower Ca2+ sensitivity or a lower dark concentration of Ca2+, we imaged fluorescent indicators of synaptic vesicle cycling and intraterminal Ca2+. We report that the Ca2+ sensitivity of release is indistinguishable in rods and cones, consistent with their possessing similar release machinery. However, the dark intraterminal Ca2+ concentration is lower in rods than in cones, as determined by two-photon Ca2+ imaging. The lower level of dark Ca2+ ensures that rods encode intensity with a slower vesicle release rate that is better matched to the lower information content of dim light.
Cellular/Molecular
Synaptic Ca
2
in Darkness Is Lower in Rods than Cones,
Causing Slower Tonic Release of Vesicles
Zejuan Sheng,
1
Sue-Yeon Choi,
1
Ajay Dharia,
1
Jian Li,
2
Peter Sterling,
2
and Richard H. Kramer
1
1
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, and
2
Department of Neuroscience, University of
Pennsylvania, Philadelphia, Pennsylvania 19104
Rod and cone photoreceptors use specialized biochemistry to generate light responses that differ in their sensitivity and kinetics.
However, it is unclear whether there are also synaptic differences that affect the transmission of visual information. Here, we report that
in the dark, rods tonically release synaptic vesicles at a much slower rate than cones, as measured by the release of the fluorescent vesicle
indicator FM1-43. To determine whether slower release results from a lower Ca
2
sensitivity or a lower dark concentration of Ca
2
,we
imaged fluorescent indicators of synaptic vesicle cycling and intraterminal Ca
2
. We report that the Ca
2
sensitivity of release is
indistinguishable in rods and cones, consistent with their possessing similar release machinery. However, the dark intraterminal Ca
2
concentration is lower in rods than in cones, as determined by two-photon Ca
2
imaging. The lower level of dark Ca
2
ensures that rods
encode intensity with a slower vesicle release rate that is better matched to the lower information content of dim light.
Key words: retina; photoreceptor; neurotransmitter release; ribbon synapse; exocytosis; Ca
2
imaging
Introduction
Retinal rods and cones are specialized for transducing and trans-
mitting information about different ranges of light intensity.
Rods respond to dim light, generating a slow, prolonged signal,
whereas cones require brighter light, generating a rapid, more
transient signal (Dowling, 1987). The distinct electrophysiologi-
cal responses of rods and cones are attributable to differences in
the biochemical machinery of phototransduction in outer seg-
ments (Tachibanaki et al., 2001; Kefalov et al., 2003, 2005; Rebrik
and Korenbrot, 2004). However, it is unclear whether there are
also intrinsic differences in rod and cone synapses that contribute
to the distinct messages transmitted by the two cell types.
Rods and cones are depolarized in the dark, resulting in main-
tained activation of voltage-gated Ca
2
channels, continual
Ca
2
influx, and tonic neurotransmitter release. Increasing light
intensity induces a graded hyperpolarization that turns off these
events and suppresses neurotransmitter release. Because neuro-
transmitter is packaged in synaptic vesicles that are released in a
quantal manner, the precision of synaptic encoding is con-
strained by the ambient rate of release, which is maximal in the
dark. The greater the release rate in the dark, the more accurately
increments of light intensity can be represented by decrements in
the release rate. Rods respond to dimmer light which, because of
its quantal nature, includes fewer gradations of intensity. Hence,
the signal-to-noise ratio of rods is lower than cones. Therefore,
the rod light response conveys less information leading to the
prediction that fewer vesicles are necessary for it to be encoded
synaptically.
We previously measured dark release from photoreceptors in
the cone-only retina of anole lizards (Choi et al., 2005a). We
imaged the loss of the fluorescent synaptic vesicle dye FM1-43
and used electron microscopy (EM) to count synaptic vesicles in
the terminals to estimate the vesicle release rate. Here, we mea-
sure release from rods, using fluorescence imaging and EM on the
rod-only retina of geckos. We also estimate release rates from
rods and cones of tiger salamanders. For both interspecies and
intraspecies comparisons, we find that release in darkness is
much slower in rods than cones, in accord with phasic release
studies (Rabl et al., 2005), and consistent with the prediction that
rods transmit information at a slower rate.
Because vesicle release is Ca
2
dependent, slower release must
result from a lower intraterminal Ca
2
and/or a less Ca
2
-
sensitive release process. The Ca
2
dependence of phasic release
from photoreceptors was evaluated by measuring over the first
few seconds the change in membrane capacitance that occurs
after elevating cytoplasmic Ca
2
(Rieke and Schwartz, 1994,
1996; Kreft et al., 2003; Thoreson et al., 2004). However, capaci-
tance measurements are not ideal for studying tonic release be-
cause at longer times the capacitance increase from exocytosis is
confounded by a decrease caused by endocytosis. Thus, the Ca
2
dependence of tonic release is still unknown. Moreover, the dark
Ca
2
concentration is unknown in rod or cone terminals. Here,
we show that the Ca
2
dependence of tonic release is similar in
rods and cones, but in darkness, rods have a lower resting Ca
2
underlying their slower tonic release.
Materials and Methods
Retinal preparation
Procedures for the care and use of tokay gecko lizards (Gekko gecko),
anole lizards (Anolis segrei), and larval tiger salamanders (Ambystoma
Received Dec. 13, 2006; revised March 7, 2007; accepted April 1, 2007.
This work was supported by National Eye Institute Grant EY15514. We thank Skyler Jackman for helpful com-
ments on this manuscript.
Correspondence should be addressed to Dr. Richard H. Kramer, Department of Molecular and Cell Biology, 121
Life Sciences Addition, University of California, Berkeley, CA 94720-3200. E-mail: rhkramer@berkeley.edu.
DOI:10.1523/JNEUROSCI.5386-06.2007
Copyright © 2007 Society for Neuroscience 0270-6474/07/275033-10$15.00/0
The Journal of Neuroscience, May 9, 2007 27(19):5033–5042 5033
tigrinum) were approved by the University of California Berkeley Animal
Care and Use Committee. Animals were maintained on a 12 h light/dark
cycle. All procedures were performed in darkness using infrared illumi-
nation and night-vision goggles. To obtain retinas, animals were dark-
adapted overnight (1 h for anoles) and quickly killed by decapitation.
After enucleation, eyes were hemisected and the retinas were removed.
Retinas were bathed in normal saline for gecko [containing (in m
M) 160
NaCl, 3.3 KCl, 1.5 CaCl
2
, 1.5 MgCl
2
, 10 HEPES, 10 glucose, pH 7.4],
anole [containing (in m
M) 149 NaCl, 4 KCl, 1.5 CaCl
2
, 1.5 MgCl
2
,10
HEPES, 10 glucose, pH 7.4], or salamander [containing (in m
M) 110
NaCl, 2 KCl, 2 CaCl
2
, 1 MgCl
2
, 10 HEPES, 10 glucose, pH 7.4). All dye
solutions were made in normal saline unless otherwise stated. Ca
2
-free
saline replaced CaCl
2
with 1 mM EGTA. High-K
saline contained 50
m
M KCl, iso-osmotically replacing NaCl.
FM1-43 dye loading and unloading
Measuring the FM1-43 unloading rate from gecko rod. Procedures for
FM1-43 loading and unloading were modified from those described pre-
viously (Choi et al., 2005a). The retina was mounted onto filter paper and
loaded with 30
M FM1-43 (Invitrogen, Eugene, OR) for 2 h and washed
with 1 m
M Advasep-7 (CyDex, Lenexa, KS) for 10 min. FM1-43 unload-
ing was followed by imaging the outer plexiform layer (OPL) every 4 min
in darkness, bright light (10
7
photons/
m
2
/s), or high-K
saline. Fluo
-
rescence of FM1-43 was counted from the stack images through the OPL
and normalized to the value t 0. At the end of experiments of unloading
in darkness or under bright light, high-K
saline was added to release all
releasable vesicles, and remaining background fluorescence was sub-
tracted. Variability among data are expressed as mean SEM.
To test whether the two-photon scans altered the unloading rate of
gecko rods, the FM1-43-loaded gecko retina was cut into a few pieces and
placed in complete darkness. One piece of retina was taken out at t 0
min, t 10 min, and t 20 min and transferred to Ca
2
-free saline to
stop unloading, and then imaged. For each piece of retina, FM1-43 flu-
orescence was averaged over five areas and normalized to the value t 0.
Measuring FM1-43 loading rate from salamander photoreceptors. The
retina was bathed in 30
M FM1-43 for 0 –15 min, transferred to Ca
2
-
free saline, and dissociated as described previously (Rea et al., 2004). A
suspension of dissociated retinal cells was then transferred onto a cover-
slip for imaging. Total fluorescence from photoreceptor terminals was
measured and normalized to the value from cone terminals at t 15 min.
Forty optical sections (focused 0.5
m apart) were obtained for each cell
and total FM1-43 fluorescence from each terminal was calculated. Vari-
ability among data are expressed as mean SEM.
Fluorescence recovery after photobleaching. FM1-43-loaded retinal
slices were prepared as described previously (Rea et al., 2004). A 4
m
2
region of the photoreceptor terminal was photobleached by continually
scanning three times at high laser power. Images were acquired every 3 s
before and after photobleaching with a spatial resolution of 34 pixels per
micrometer. Variability among data is expressed as mean SEM.
Calcium imaging with fura-2
Measuring Ca
2
dependence of release. Retinas from gecko and anole were
loaded with FM1-43 in darkness in normal saline. FM1-43 loaded retinas
were sliced in Ca
2
-free saline and transferred to Ca
2
-free saline con
-
taining 5
M fura-2 AM (Invitrogen) for 30 min. The double-labeled
retinal slices were imaged using an upright Nikon (Tokyo, Japan) micro-
scope. Fluorescence was detected with an Imago Sensicam (TILL Pho-
tonics, Planegg, Germany) via a 40 0.7 numerical aperture water-
immersion objective (Olympus, Tokyo, Japan). The wavelength of
excitation light was controlled with a Lambda 10 –2 filter wheel (Sutter
Instruments, Novato, CA). FM1-43 was excited with 490 nm light.
Fura-2 was excited with 350 and 380 nm light for ratiometric measure-
ment. A 510 10 nm emission filter was used for both dyes. Images were
acquired and analyzed with Imaging Workbench software (Indec BioSys-
tems, Santa Clara, CA). Images of fura-2 and FM1-43 were acquired
every 10 s and binned 4 4. The in vitro fura-2 calibration of the pho-
tometric system was performed with a fura-2 calcium imaging calibra-
tion kit (Invitrogen). Variability among data is expressed as mean
SEM.
Measuring intraterminal Ca
2
concentration in darkness. The retina
was bathed in 125
M fura-2 AM in darkness for 60 min. The retina was
then washed and mounted on filter paper for imaging. The fura-2 ratio
was obtained using two-photon excitation wavelengths of 700 and 760
nm and a 500 –550 bandpass (BP) emission filter. To locate the photore-
ceptor terminals accurately, anole and salamander retina were preloaded
with FM4-64 in darkness before fura-2 loading. OPL was first identified
with FM4-64 fluorescence using a two-photon excitation wavelength of
880 nm and 575– 640 BP emission filter. The in vitro fura-2 calibration of
the two-photon imaging system was performed with a fura-2 calcium
imaging calibration kit (Invitrogen).
Electron microscopy
Gecko and salamander retinas were prepared and fixed for EM as de-
scribed previously (Rea et al., 2004; Choi et al., 2005a). Vesicle density in
photoreceptor terminals was calculated from ultrathin EM sections (70
nm). The average terminal volume of gecko rods was calculated from
three-dimensional (3D) reconstructions of EM sections using Recon-
struct software (Synapse Web, http://synapse.bu.edu; Boston University,
Boston, MA). The average terminal volume of salamander rods and
cones was calculated from 3D reconstructions of fluorescence images of
FM1-43-loaded terminals (Imaris software; Bitplane, Saint Paul, MN).
Results
Release of FM1-43 in the dark is slow in rods
We measured synaptic release from the rod-only gecko retina by
analyzing the loss of FM1-43. We then compared this data with
previous measurements of dye release from anole cones (Choi et
al., 2005a). Gecko retina is advantageous because the rod termi-
nals are large (5–10
m diameter), facilitating optical measure-
ments from an individual cell, and they are also homogenous,
allowing simultaneous fluorescent measurement from an array of
terminals in the OPL. Because the tokay gecko is nocturnal, its
visual system is normally operating only in the scotopic range of
light intensity. Gecko rods are thought to be transmuted from an
ancestral cone photoreceptor and they still have some cone-like
properties, including cone-like photopigments and a large
pedicle-shaped terminal with multiple synaptic ribbons (Kojima
et al., 1992; Ro¨ll, 2000). However, the intensity–response rela-
tionship and kinetics of the gecko rod light response are very
similar to those of rods from other species (Kleinschmidt and
Dowling, 1975; Rispoli et al., 1993), strongly suggesting that the
gecko rod synapse encodes light information in a manner that is
similar to rod synapses in other species.
Intact retinas were first loaded with FM1-43 in the dark,
which, in the OPL, results in selective labeling of photoreceptor
terminals (Choi et al., 2005b). To monitor dye release in the dark,
we used two-photon microscopy, which uses infrared light to
excite fluorescence while minimizing retinal phototransduction.
Because infrared light penetrates deeply into tissue, two-photon
imaging enables visualization of photoreceptor terminals
through the thickness of a flat-mount retina (200
m). Retinas
were imaged every 2– 4 min in the dark. We found that FM1-43
was released much more slowly from rods than cones (Fig. 1A).
Dye loss from both could be fit with a single exponential function,
but with very different time constants (69 min for rods and 10
min for cones) (Fig. 1B).
The different rates of release suggest that rods and cones have
intrinsic differences in synaptic function. However, it is also pos-
sible that the infrared laser inadvertently excited photopigments
in the outer segments of rods and cones and triggered photo-
transduction, which would inhibit vesicle release. We had estab-
lished previously that FM1-43 release from cones is unaffected by
altering the duration of infrared illumination (Choi et al., 2005a).
However, rods are 50 –100 times more sensitive to light than
5034 J. Neurosci., May 9, 2007 27(19):5033–5042 Sheng et al. Synaptic Vesicle Release and Ca
2
in Rods and Cones
cones (Dowling, 1974), perhaps accentuating the problem of
stimulating them inadvertently. To test for this, we measured
FM1-43 release from preloaded pieces of gecko retina that were
allowed to unload the dye in complete darkness for various times.
To terminate unloading, the tissue was placed in Ca
2
-free sa
-
line, and only then was the tissue visualized with two-photon
microscopy. Dye loss measured with this protocol (Fig. 1B,R
2
)
involved no exposure to infrared light, yet the rate of dye loss was
the same as that measured with repeated two-photon scanning
(Fig. 1B,R
1
)(p 0.001, Student’s t test). Hence, under these
experimental conditions, two-photon scanning does not perturb
the dark rate of FM1-43 release from rods.
FM1-43 release from gecko rods was completely suppressed
by exposure to bright light (Fig. 1C), similar to results obtained
from anole cones (Choi et al., 2005a). Depolarizing rods with
high-K
saline (Fig. 1 D) triggered a rapid rate of release (
7.7
min; n 4 retinas) that was similar to that observed in cones (5.9
min; n 5 retinas) (Choi et al., 2005a). Approximately 94% of
the dye in rods was released after 20 min in high K
, suggesting
that labeling of unreleasable (i.e., reserve) vesicles and/or other
internal membranes accounts for at most 6% of the fluorescence.
Release from both rods (Fig. 1 D) and cones (Choi et al., 2005a)
was blocked by removing extracellular Ca
2
, consistent with
Ca
2
-dependent exocytosis. Hence, given sufficient depolariza
-
tion, both rods and cones are capable of faster release, indicating
that in the dark their release machinery is not operating at full
speed. For rods, the release rate in high K
is approximately ninefold higher than the
dark rate, and for cones it is 1.6-fold
higher.
Quantifying synaptic vesicle release
rates from gecko rods and anole cones
To quantify the rate of synaptic vesicle re-
lease, we needed the rate of FM1-43 release
and the number of vesicles in the releas-
able (active) vesicle pool. This pool size
equals the fraction of synaptic vesicles that
is actively participating in release times the
total number of vesicles in the terminal
(which we determine by EM). In a phasic
synapse, only a small fraction of synaptic
vesicles can move to the active zone and be
released (15–25% in hippocampal syn-
apses) (Pyle et al., 2000; Richards et al.,
2003); the rest are considered “reserve ves-
icles.” However, in a cone photoreceptor,
most of the vesicles are releasable and
there is little or no reserve. We knew that
86% of vesicles in a cone terminal accu-
mulate FM1-43 within 10 min of loading
(Choi et al. 2005a), closely matching the
percentage of vesicles that remain mobile
in the cytoplasm (87%), as determined by
fluorescence recovery after photobleach-
ing (FRAP) measurements (Rea et al.,
2004).
To estimate the fraction of mobile ves-
icles in the gecko rod terminal, we again
used FRAP. After applying high-K
saline
to saturate the loading of FM1-43 in gecko
rods and then applying Ca
2
-free saline to
prevent release, retinal slices were imaged
to find individual labeled rod terminals. A small area (4
m
2
)in
the center of a terminal was scanned with the two-photon laser at
high power for 1 s (Fig. 2A). This bleached the FM1-43 such that
vesicles within this region were no longer fluorescent. Subse-
quent low-intensity scans showed that the fluorescence within
the bleached region rapidly recovered and that the fluorescence
in the remainder of the terminal decreased as vesicles redistrib-
uted. To assess the fraction of vesicles that are mobile, we mea-
sured the recovery of fluorescence in the bleached region com-
pared with the average fluorescence in the remainder of the
terminal (FRAP index) (Fig. 2B). At steady state (21–30 s after the
bleach) the FRAP index was 90%. This indicates that 90% of
those vesicles that had accumulated dye during endocytosis re-
main mobile in the cytoplasm, similar to the releaseable fraction
of vesicles in anole cones (86%). This similarity also suggests that
few, if any, vesicles failed to accumulate dye during the initial
loading of gecko rods. Indeed, neither rods nor cones contain
synapsin (Mandell et al., 1990), the key protein that immobilizes
vesicles and therefore holds them in reserve at conventional syn-
apses (Huttner et al., 1983; Ba¨hler and Greengard, 1987; Benfe-
nati et al., 1989), suggesting the lack of a reserve pool in
photoreceptors.
We used EM to visualize and quantify synaptic ribbons and
synaptic vesicles in the gecko rod terminal. Photoconversion of a
fixable FM1-43 analog shows that the dye is localized to synaptic
vesicles in gecko rod terminals (data not shown), as demon-
Figure 1. Gecko rods release FM1-43 slower than anole cones in darkness. A, Fluorescence images showing the gradual
unloading of FM1-43 in darkness from synaptic terminals of gecko rods (top) and anole cones (bottom). Images were obtained by
two-photon scans at 4 min intervals of a z section through the OPL in each retinal flat mount. Scale bars: top, 20
m; bottom, 10
m. B, Time course of unloading from rod (R) and cone (C) terminals in darkness, measured by repeated two-photon scans.
Fluorescence was measured from the stack images through the OPL. Data for cones (triangles) in B–D were reported previously
(Choiet al.,2005a).Continuous linesshow fitstosingle exponentialfunctions withrelease timeconstantsof 69and 10minfor rods
(n 8)andcones (n 9), respectively. R
1
(open squares) isforthe rod unloading procedure with repeated two-photonscans;R
2
(closed squares) is for unloading without scans (n 3). At the end of experiments, high-K
saline was added to release all
releasable vesicles and remaining background fluorescence (6%) was subtracted (D). C, Time course of unloading from rod and
cone terminals with the retina exposed to bright light between repeated scans. D, Time course of unloading in high-K
saline
fromrods andcones. Remainingbackgroundfluorescence afterunloading wasnot subtracted.Data wasfit withsingleexponential
function with release time constant of 7.7 and 5.9 min in rods (n 4) and cones (n 5), respectively. R
0
(closed squares) is for
gecko rod unloading in high K
,Ca
2
-free saline (n 3).
Sheng et al. Synaptic Vesicle Release and Ca
2
in Rods and Cones J. Neurosci., May 9, 2007 27(19):5033–5042 5035
strated previously in anole cone terminals (Choi et al., 2005a).
We calculated the total number of vesicles in gecko rods
(156,000) by measuring the average number of vesicles per unit
volume (1525 69 per
m
3
; n 5) and the average volume of
terminals (102 15
m
3
; n 5). Taking into account the per
-
cent that are mobile, gecko rod terminals contain 140,000 re-
leasable vesicles (Table 1), similar to the 146,000 measured pre-
viously in anole cones (Choi et al., 2005a). By multiplying these
values by the release rate determined from the initial FM1-43
unloading rate, we estimate that, in darkness, gecko rods release
35 vesicles/s, sevenfold fewer than we reported for anole cones
(246 vesicles/s). Gecko rods have fewer ribbons than anole cones
(14 vs 25), so the release rates per ribbon (2.5/s for rods and 10/s
for cones) differ by approximately fourfold. Nevertheless, in
darkness, the rods clearly release much more slowly than cones.
However, in high K
, the situation is different; both rods and
cones release at similar rates (300 400 vesicles/s). The release
rate per ribbon was also similar in rods and cones (15–21 vesicles/
s). Apparently, both rods and cones are capable of rapid tonic
release, but in darkness, rods use this capability to a much smaller
extent.
Quantifying synaptic vesicle release rates from salamander
rods and cones
To confirm the difference between rod and cone release rates in
the same species, we turned to the duplex retina of the tiger
salamander. The terminals of salamander rods and cones are in-
terspersed in the OPL and adhere to one another, making it dif-
ficult to separately measure FM1-43 fluorescence in the two cell
types. We tried to avoid this problem by isolating rods and cones
with intact terminals by enzymatic dissociation (Townes-
Anderson et al., 1985), but their dye release in darkness was very
slow or absent (our unpublished observations), suggesting dis-
ruption of physiological function. Therefore, we chose an alter-
nate approach, measuring the rate of endocytosis (by FM1-43
loading), which at steady-state should equal the rate of exocyto-
sis. The advantage was that loading could be performed on the
intact retina and measurements of dye accumulation could be
done after retinal dissociation.
To determine the rate of dye loading, salamander retina was
incubated in FM1-43 for 0 –15 min in the dark. The tissue was
then washed in Ca
2
-free saline to prevent additional loading or
unloading. After enzymatic dissociation (Rea et al., 2004), indi-
vidual large-type rods and large-type single cones were identified
by morphological criteria (Sherry et al., 1998). In rods, the termi-
nal is connected to the inner segment by a long thin axon,
whereas in cones, the terminal emanates directly from the inner
segment (Fig. 3A). Fluorescence images show that FM1-43 accu-
mulates in the rod and cone terminals. The dye subsequently
could be released after depolarization with high-K
saline (Fig.
3B), confirming that, in the terminals, the dye had specifically
accumulated in releaseable synaptic vesicles.
Dye accumulated more rapidly in cone than in rod terminals
(Fig. 3C). Over the initial 1 min of loading, cones incorporated
dye approximately fourfold faster. Even after dye loading had
reached steady state in cones (5–7 min), it continued to accumu-
late in rods (10 min). Assuming that each endocytosed synaptic
vesicle captures the same amount of fluorescent dye, rods must
endocytose synaptic vesicles more slowly than cones.
To estimate dye uptake in terms of vesicle endocytosis rate, we
first assumed that prolonged incubation of retina in high-K
saline resulted in the loading of all active vesicles in rods and
cones with FM1-43, providing a measure of the maximal fluores-
cence in the terminals. The maximal dye uptake in cones is 2.5-
Figure 2. FRAP experiments show that most of the vesicles in the gecko rod terminal are mobile. A, Series of images taken from a single rod terminal in a slice preloaded with FM1-43, showing
fluorescence before and after photobleaching of a 4
m
2
region (white square). Scale bar, 2
m. B, Time course of FRAP in Ca
2
-free saline. Photobleaching was triggered at time 0 and recovery
was measuredat various times thereafter.The FRAP index istheratio of the fluorescencewithin the bleached regionand the average fluorescenceofthe entire terminal, normalizedto the prebleach
ratio. The continuous line shows fit to a double-exponential function with time constants of 1.1 and 18 s. n 3 terminals.
Table 1. Synaptic vesicle release rates in rods and cones
Releasable vesicles per terminal Ribbons per terminal
Vesicle release rate (SV/s)
In darkness In high K
ringer
per term per rib per term per rib
Gecko rod 140,400 14 35 2.5 304 21
Anole cone 146,200 25 246 10 389 15
Salamander rod 80,200 7
a
127 18 ND ND
Salamander cone 194,000 ND 501 ND ND ND
Data were obtainedfrom initial FM1-43release rates and EM determinationsof total synapticvesicles per terminal.The releasablevesicle pool sizewas calculated bymultiplying thetotal vesicle poolby the mobilefraction. rib, Ribbon; term,
terminal; SV, synaptic vesicle; ND, not determined.
a
From Townes-Anderson et al., 1985
5036 J. Neurosci., May 9, 2007 27(19):5033–5042 Sheng et al. Synaptic Vesicle Release and Ca
2
in Rods and Cones
fold that observed in rods, consistent with their having 2.4-fold
more vesicles (Table 1), supporting the notion that all of the
internalized dye remained localized to the terminals and was re-
stricted to synaptic vesicles. Over a brief initial time period (1
min), the fractional accumulation of fluorescence represents the
percentage of vesicles that were endocytosed during this time.
Thus, each minute in darkness, cone and rod terminals endocy-
tose 15.5 and 9.5% of their active vesicle pools.
The total number of vesicles was determined by measuring the
terminal volume of rods (32.82 6
m
3
; n 6) and cones
(82.07 12
m
3
; n 6) from z-stacks of dye-loaded terminals,
and the average density of vesicles within rods (2714 300 ves-
icles/
m
3
; n 5) and cones (2628 271 vesicles/
m
3
; n 5),
determined from EM analysis. These calculations indicate that
rods and cones have, respectively, 89,100 and 215,600 vesicles.
This is consistent with the smaller volume of the rod terminal.
Assuming that the fraction of mobile vesicles is similar to that in
gecko rods and anole cones (90%), the size of the cycling synaptic
vesicle pool in salamander rods and cones is 80,200 and 194,000,
respectively (Table 1).
Multiplying the cycling pool by the rate of endocytosis, we
estimate that, in darkness, the synaptic vesicle uptake rate is
30,070 vesicles/min (501 vesicles/s) in cones and 7619 vesicles/
min (127 vesicles/s) in rods, only 25% of the cone release rate.
Our estimation of rod release rate is lower than previous esti-
mates [400 vesicles/s (Rieke and Schwartz, 1996)], but these
relied on capacitance measurements that report transient rather
than tonic release. Hence, within a species as well as across spe-
cies, rods exhibit slower tonic release than cones.
The Ca
2
dependence of release is similar in rods and cones
To understand what mechanisms cause rods to release vesicles
more slowly than cones, we considered the role of Ca
2
. The
release rate in darkness depends both on the relationship be-
tween Ca
2
and release and the dark concentration of intra
-
cellular Ca
2
.
To determine which of these parame-
ters differ between rods and cones, we re-
turned to the gecko and anole retinas to
measure the Ca
2
dependence of release.
This required simultaneous measurement
of intracellular Ca
2
(with the fluorescent
indicator fura-2) and vesicle release (with
FM1-43). Our strategy was to monitor
vesicle release while “clamping” intracel-
lular Ca
2
at different levels by applying
the Ca
2
ionophore ionomycin with dif
-
ferent extracellular Ca
2
concentrations.
Gecko or anole retinas were loaded
with FM1-43 and then placed in Ca
2
-
free saline to prevent unloading. They
were then sliced and bathed in fura-2 AM,
which entered cells, where it was con-
verted into fura-2. To separately monitor
Ca
2
and vesicle release, we used excita
-
tion wavelengths selective for fura-2 (350
and 380 nm) and FM1-43 (490 nm). In
Ca
2
-free saline, the fura-2 ratio was low
in both gecko rod and anole cones, indi-
cating low intraterminal Ca
2
, but the
FM1-43 signal was high, indicating many
labeled vesicles (Fig. 4A). We then treated
the slices with ionomycin and added ex-
tracellular Ca
2
. This increased intracellular Ca
2
throughout
the tissue samples (Fig. 4A). The rise in Ca
2
triggered a decrease
in FM1-43 fluorescence, but this was selective for the OPL, where
the photoreceptor terminals reside. Hence, ionomycin-
facilitated Ca
2
entry caused synaptic vesicle release from both
rod and cone terminals.
Figure 4B shows recordings of Ca
2
and release, using the
gecko rods as an example. Each measurement was from a differ-
ent retinal slice that was preincubated with the same concentra-
tion of ionomycin in Ca
2
-free saline, and then exposed to saline
with different concentration of extracellular Ca
2
. The fura-2
ratio and the FM1-43 unloading rates were measured after inter-
nal Ca
2
had reached steady state (3 min after perfusion). A
higher fura-2 ratio was always correlated with faster FM1-43
unloading.
By plotting data from many such experiments we could ob-
serve the relationship between the fura-2 ratio and the FM1-43
release rate (Fig. 4C). For both rods and cones, the release rate
accelerated with increasing intracellular Ca
2
. Least-squares fits
to the data show that the slopes of the Ca
2
versus release plots
are nearly the same in rods and cones, consistent with the two cell
types having similar release machinery. Hence, the difference in
the dark release rate of rods and cones is not caused by a differ-
ence in the Ca
2
dependence of exocytosis.
Rods have a lower intraterminal Ca
2
concentration
than cones
We next compared the dark concentration of Ca
2
in rod and
cone terminals. To avoid triggering phototransduction, we began
by using a nonratiometric Ca
2
dye that can be excited with
two-photon infrared illumination (Oregon-green BAPTA, which
is excited by 800 nm light). However, the amount of dye loading
varied greatly among terminals in the intact retina, leading to
variability in fluorescence. To circumvent this, we returned to
fura-2, which can be imaged ratiometrically, thereby correcting
for differences in dye loading. We used two-photon excitation of
Figure 3. Dark loading of FM1-43 in salamander retina is slower in rods than in cones. A, Dissociated rod and cone obtained
from a salamanderretinapreloaded with FM1-43 for 30 min. Bright field (left),FM1-43fluorescence (center), and merged images
(right)are shown.Note theFM1-43 accumulationin thebipartiterodspherule(arrowheads)andcone pedicleat thebase ofthe cell
body (arrow). Scale bar, 2
m. B, FM1-43 fluorescence images of salamander retina slices. Transmitted light (red) and FM1-43
fluorescence (green) images are overlaid. Retina was preloaded with FM1-43 in the dark for 30 min. Before slicing, the retina was
incubated in Ca
2
-free saline (left) or high-K
saline for 20 min. The positions of OPL are indicated with arrows. C, Time course
of FM1-43 fluorescence uptake for rods (R) and cones (C) in darkness. F
arb.
is the measured fluorescence taken up during FM1-43
preloading of various durations, normalized to the fluorescence of saturation-loaded cone terminals (15 min FM1-43 loading in
high-K
saline).Loadingtime constant is10 min forrods,6 min forcones. n 6–12 terminals foreachdata point. Opensymbols
represent the fluorescence of saturation-loaded rod (n 11) and cone terminals (n 12) (15 min FM1-43 loading in high-K
saline).
Sheng et al. Synaptic Vesicle Release and Ca
2
in Rods and Cones J. Neurosci., May 9, 2007 27(19):5033–5042 5037
fura-2 at 700 and 760 nm instead of the
usual excitation wavelengths (e.g., 350 and
380 nm). This confined excitation to a
narrow focal plane within the OPL and
minimized two-photon excitation of pho-
topigment in outer segments, which are
50
m deeper into the flat-mount
retina.
Treatment of gecko retina with fura-2
AM led to greater dye accumulation in rod
terminals than in surrounding structures,
allowing clear identification of the termi-
nals in the intact retina (Fig. 5A). The ratio
image showed that rod terminals have
slightly lower Ca
2
than the surrounding
tissue. In contrast, anole cones accumu-
late much less dye than the neighboring
Mu¨ller glia cells (Fig. 5B), which appear
bright when illuminated with a single
wavelength (700 or 760 nm). This made it
difficult to identify cone terminals to se-
lectively measure their fluorescence. To
visualize cone terminals selectively, we
preloaded the retina with FM4-64, a syn-
aptic vesicle dye whose peak excitation
and emission wavelengths do not overlap
with those of fura-2. The ratio image
shows that the cone terminals identified
by FM4-64 have a higher 700/760 ratio
and, therefore, a higher Ca
2
concentra
-
tion than the surrounding tissue. Analysis
of 41 gecko rods and 24 anole cones
showed that rod terminals have signifi-
cantly lower Ca
2
by 50% than cone terminals (157 5nM in
rods, 317 13 nM in cones; p 0.05, Student’s t test). In fact,
there is almost no overlap in the ranges of Ca
2
concentrations
observed in the two cell types.
We were again concerned that the light used for ratio imaging
might have triggered phototransduction and therefore lowered
intraterminal Ca
2
, especially in rods. Our standard ratio image
was obtained by scanning a two-dimensional area (0.01 mm
2
)of
the OPL for a total of 970 ms with each excitation wavelength. To
estimate the upper limit of Ca
2
perturbation that this illumina
-
tion might cause, we repeatedly line-scanned an individual rod
terminal with 700 or 760 nm light for 2 s. We found that the
700/760 ratio decreased by 3% over the first second of scanning
and 5% over 2 s. During a line-scan, the light is concentrated on
a single terminal, whereas during a two-dimensional scan, the
light is spread over 100 terminals, with each receiving a small
fraction of the total photon flux. Even disregarding this point, the
decrease in the 700/760 ratio is much too small to account for the
50% difference of intraterminal Ca
2
that we observed between
gecko rods and anole cones.
To test whether unhydrolyzed fura-2 AM was sequestered in
organelles, which could distort cytoplasmic Ca
2
measurements,
we substituted Ca
2
with Mn
2
(1.5 mM), a quencher of fura-2
fluorescence. We applied high-K
saline to stimulate Mn
2
in
-
flux into terminals. We found that fura-2 fluorescence was re-
duced by up to 95% in both gecko rods and anole cones, confirm-
ing nearly complete hydrolysis and consistent with results from
salamander (Szikra and Krizaj, 2006).
Finally, we compared the dark level of Ca
2
in salamander
rods and cones. We used FM4-64 to identify photoreceptor ter-
minals in the OPL and fura-2 to indicate intraterminal Ca
2
. Rod
terminals are spherical or ovoid and smaller than the adjacent
cone terminals (Fig. 6A, top). The 700/760 fura-2 ratio was con-
sistently lower in rod terminals, which appear as relatively dark
spots in pseudocolored ratio images (Fig. 6A, bottom). Analysis
of 36 rods and 19 cones showed that the intraterminal Ca
2
concentration is significantly lower by 29% in rods than in
cones (236 10 n
M in rods, 332 13 nM in cones; p 0.05,
Student’s t test) in the dark. We tested the extent of inadvertent
phototransduction by continuously line scanning individual ter-
minals with 700 or 760 nm light for 2 s (Fig. 6C). For rods, the
700/760 ratio decreased by 8% over the first second of scanning
and 10% over 2 s, but the ratio decreased by the same extent in
cones. Hence, inadvertent phototransduction decreased Ca
2
to
the same extent in rods and cones and, therefore, cannot account
for the difference of dark intraterminal Ca
2
in the two cell types.
Discussion
Functional imaging of photoreceptor terminals
Fluorescent indicator dyes enable the functional imaging of vi-
sual system activity in vitro (MacLean et al., 2006) and in situ
(Ohki et al., 2005). However, optical imaging of the retina pre-
sents a problem: light used to excite fluorescent indicators and
the resulting emitted light can inadvertently activate phototrans-
duction in rod and cone outer segments, altering activity in their
synapses and in downstream neurons. Two-photon microscopy
helps alleviate this problem. First, it uses infrared light, which is
relatively ineffective in triggering phototransduction. Second,
two-photon excitation is confined to a narrow focal plane (1
m thick), minimizing photopigment activation in off-target fo-
cal planes.
Figure 4. Simultaneous FM1-43 and fura-2 imaging shows that the Ca
2
dependence of release is similar in gecko rods and
anole cones.A, Pseudocolor images of geckoand anole retinal slices preloadedwith FM1-43 to measure vesiclerelease, and fura-2
to measureintracellular Ca
2
. Thefura-2 ratio and theintensityof FM1-43 fluorescence andarecolor-coded as shown below.The
circled areas indicate the photoreceptor terminals. In Ca
2
-free saline, the fura-2 ratio is low and FM1-43 fluorescence is high.
After treatment with ionomycin plus extracellular Ca
2
, the fura-2 ratio rises and FM1-43 fluorescence decreases. B, Time course
of changesinCa
2
and vesiclereleasetriggered by adding extracellular Ca
2
to ionomycin-treatedgeckoretinal slices. Slices are
firstincubatedin Ca
2
-freesalineand extracellularCa
2
isaddedat time0 (indicated withdash line).Beforetime 0,there is little
FM1-43 unloading and FM1-43 fluorescence is normalized to the average FM1-43 fluorescence (t 0; F
initial
F
average
). When
Ca
2
risereaches steadystate afteradding extracellularCa
2
(3min), FM1-43fluorescence isnormalized tothe firstpoint over
the measurement timeperiod(t 200 s; F
initial
F
t200s
). The averagefura-2ratios and the FM1-43 unloading rates from OPLare
then measured. C, Group data from gecko rods and anole cones showing similar relationship between Ca
2
and release. Intra
-
terminal Ca
2
concentration and FM1-43 release rates were obtained as in B. FM1-43 release rates at similar intraterminalCa
2
levels are binned together and then plotted. Continuous lines show least square fits to rod (black) and cones (gray) data.
5038 J. Neurosci., May 9, 2007 27(19):5033–5042 Sheng et al. Synaptic Vesicle Release and Ca
2
in Rods and Cones
Despite these advantages, two-photon scanning can trigger
inadvertent phototransduction, as reported in previous studies
monitoring fast Ca
2
transients in response to light stimuli in
ganglion (Denk and Detwiler, 1999) and amacrine cells (Euler et
al., 2002). Therefore, it was important to gauge how much inad-
vertent phototransduction might confound our measurements.
Eliminating exposure to two-photon illumination in rods (Fig.
1B) or changing its duration in cones (Choi et al., 2005a) had no
effect on vesicle release rate, measured with FM1-43. However,
continuous line scans of rod or cone terminals did trigger photo-
transduction, leading to a 5–10% drop in the fura-2 ratio over 2 s
(Figs. 5D, 6C). If we had wanted to monitor Ca
2
continuously,
for example, to track transients in response to light flashes, this
would have been a problem. However, because we were mon-
itoring only steady-state Ca
2
and tonic vesicle release, the
scan could be applied infrequently, or even only once. By
sacrificing temporal resolution, we obtained reasonably accu-
rate measurements with little interference from inadvertent
phototransduction.
To quantify Ca
2
in photoreceptor terminals without trigger
-
ing phototransduction in outer segments, we used the novel ap-
proach of two-photon ratiometric fura-2 imaging. Our results
provide information about average levels of Ca
2
in the termi
-
nals. However, because the Ca
2
channels are localized near the
synaptic ribbons (Nachman-Clewner et al., 1999; Morgans et al.,
2005), a gradient of Ca
2
is likely in darkness. Observing this
gradient and accurately measuring sub-
membrane Ca
2
at the release sites are
technical challenges, but such measure-
ments could help elucidate the mecha-
nisms underlying dark release.
Our studies involve an interspecies
comparison between gecko rods and anole
cones and an intraspecies comparison be-
tween rods and cones of tiger salamander.
Presumably, two-photon FM1-43 and
Ca
2
imaging can also be applied to mam
-
malian photoreceptors, but the small size
of rod terminals ( 1
m diameter) makes
them difficult to unambiguously distin-
guish from surrounding tissue. Moreover,
we found that isolated mouse and rat ret-
inas begin to deteriorate during the long
time course required for dark loading of
FM1-43 into rods, such that subsequent
release is impaired, even in high K
.Ge
-
netically expressed indicators of vesicle fu-
sion (e.g., synaptopHluorin) (Miesenbo¨ck
et al., 1998; Bozza et al., 2004; Li et al.,
2005) may provide a more effective way to
study release from mammalian rod and
cone terminals.
Mechanisms underlying slower dark
release from rods
The synaptic vesicle release rate from pho-
toreceptor terminals is determined by the
nature of the Ca
2
-dependent release ma
-
chinery and the dark concentration of
Ca
2
. Rods and cones have the same sub
-
types of exocytotic and endocytotic pro-
teins, although there are differences in
their relative abundance (Sherry and Hei-
delberger, 2005). Also, synaptic vesicle protein 2B is found in rods
and not cones (Wang et al., 2003), but its function is unclear.
Despite these differences, we find the same Ca
2
dependence of
release from rods and cones.
In contrast, we show direct evidence for lower dark intrater-
minal Ca
2
in rods. This could reflect differences in Ca
2
entry
or removal from the cytoplasm. Although rods and cones exhibit
some differences in L-type Ca
2
-channel subtypes (Morgans et
al., 2005), the voltage dependence of activation is similar in dis-
sociated rods and cones (Corey et al., 1984; Maricq and Koren-
brot, 1988; Barnes and Hille, 1989; Rieke and Schwartz, 1994;
Wilkinson and Barnes, 1996; Savchenko et al., 1997; Hosoi et al.,
2005). However, the Ca
2
current in cone, but not rod terminals
is modulated by horizontal cell feedback (Verweij et al., 1996;
Cadetti and Thoreson, 2006), so in the intact retina, the voltage-
dependent activation of rod and cone Ca
2
current may differ
substantially. Moreover, group III metabotropic glutamate re-
ceptors and exocytosed protons can modulate the Ca
2
current
of cones, but not rods (Hosoi et al., 2005). L-type Ca
2
channels
are the sole pathway for extracellular Ca
2
entry in rods, but
cyclic nucleotide-gated channels also contribute to Ca
2
influx
in cones (Rieke and Schwartz, 1994; Savchenko et al., 1997). Also,
Ca
2
-induced Ca
2
release from intracellular stores is more pro
-
nounced and contributes to tonic release in rods, but not cones
(Krizaj et al., 2003; Suryanarayanan and Slaughter, 2006). Thus,
there are several differences in the Ca
2
entry pathways and in
Figure5. ThedarkCa
2
concentrationis lower interminals ofgeckorods thananole cones.A,Gecko retinaloaded withfura-2.
The OPL was imaged by two-photon excitation of fura-2 with 700 and 760 nm light. Rod terminals preferentially accumulate the
dye (e.g., boxed areas). Ratio image shows slightly lower Ca
2
in the terminals than in surrounding tissue. B, Anole retina
coloaded with fura-2 and FM4-64. Fura-2 fluorescence excited by single wavelengths (700 or 760 nm) is bright in Mu¨ller cells (M)
and dim in cone terminals (C), indicating relatively little dye accumulation. However, the 700/760 fura-2 excitation ratio is higher
in cone terminals than Mu¨ller cells, indicating higher Ca
2
. Cone terminals were unambiguously identified by their loading with
FM4-64, imaged with 880 nm light. Merged imaged shows overlap between FM4-64 and fura-2 ratio images. C, Box plot of
intraterminal Ca
2
concentration in gecko rod (n 41 terminals) and anole cone terminals (n 24 terminals). The centerline is
the median value, the edges of the boxes are the 25th and 75th percentiles, and the extremes are the range of the data. D, Time
course of changes in the excitation ratio of fura-2-loaded rod terminals elicited by continuous two-photon line scans. Terminals
were continuouslyscannedwith 700 and 760 nm light for2s. The fura-2 ratio drops by5%over 2 s. n 6terminals.Scale bars:
A,10
m; B,5
m.
Sheng et al. Synaptic Vesicle Release and Ca
2
in Rods and Cones J. Neurosci., May 9, 2007 27(19):5033–5042 5039
the modulation of voltage-gated Ca
2
channels that could contribute to the
lower dark level of Ca
2
in rods.
The main route of Ca
2
extrusion
from photoreceptor terminals is through
the plasma membrane Ca
2
-ATPase
(PMCA) (Krizaj and Copenhagen, 1998).
The most abundant isoform, PMCA1, is
found in both rods and cones (Krizaj et al.,
2002). However, the PMCA2 isoform,
which has a higher Ca
2
affinity, is ex
-
pressed only in rods (Duncan et al., 2006).
This could contribute to rods having a
lower intraterminal Ca
2
concentration
than cones and, therefore, a slower release
rate in the dark. Indeed, knock-out mice
lacking PMCA2 exhibit impaired rod syn-
aptic transmission and reduced visual sen-
sitivity to scotopic light (Duncan et al.,
2006), suggesting that low Ca
2
in rod
terminals is essential for normal visual
function.
Synaptic encoding of light intensity in
rods and cones
Rods respond to dim light, which, because
of photon fluctuations, has a lower signal-
to-noise ratio than bright light (Rose,
1973). We have shown that rods release
synaptic vesicles more slowly than cones
by fourfold to sevenfold. Release of vesi-
cles from rods may occur at regular inter-
vals (Schein and Ahmad, 2005), or by a
stochastic process that follows Poisson
statistics (van Rossum and Smith, 1998;
Berntson et al., 2004). In either case,
slower release would make the rod synapse
less precise in encoding increments of light intensity than the
cone synapse. We reported previously that the dark release rate
from anole cones allows reliable distinction of 10 steps of light
intensity, assuming Poisson release (Choi et al., 2005a). Applying
the same statistical analysis, the dark release rate from gecko rods
would allow distinction of only four steps. Thus, the slow release
from rods has evolved to match the precision of signal output to
the information content of light input, which optimizes use of the
limited resource of continuously cycling vesicles (Laughlin, 1994;
Sterling, 2004). For the same reason, one might expect chromatic
cones types with higher absolute light sensitivity and, therefore,
less precise encoding of light information, to have a lower tonic
vesicle release rate. Selective genetic expression of a fluorescent
indicator of vesicle release (i.e., synaptopHluorin) could confirm
whether the correlation between high sensitivity and low release
rate is a general phenomenon.
Our estimate of the dark release rate from gecko rods (35
vesicles/s) is surprisingly slow, even when compared with
salamander rods (127 vesicles/s). Our only assumption in these
estimations was that 90% of vesicles are available for release.
Both the lack of synapsin (Mandell et al., 1990) and our FRAP
results suggest that few vesicles are held immobilized in a reserve
pool. The presence of any vesicles that are unavailable for release
would result in an overestimate of the release rate; thus, 35 vesi-
cles/s is actually an upper limit of the dark release rate from gecko
rods. If transmission from gecko rods were similar to that from
mouse rods (Field and Rieke, 2002; Sampath and Rieke, 2004),
this slow rate of release would be sufficient to saturate glutamate
receptors on On-bipolar cells. Perhaps the particularly slow re-
lease from gecko rods is coupled with particularly slow glutamate
reuptake to ensure saturation of metabotropic glutamate
receptors.
Although rods conserve energy by releasing slowly in the dark,
they are capable of tonic release at an approximately ninefold
higher rate, given sufficient depolarization with high K
. Cones
have an excess release capability of 1.6-fold higher than the
dark rate, which may be important for horizontal cell feedback to
expand the temporal and spatial contrast sensitivity of synaptic
transmission (Choi et al., 2005a). The role of the enormous excess
release capability of rods is unknown, but it coincides with other
aspects of rod physiology. A large excess of the phototransduc-
tion machinery, including the number of rhodopsin molecules
and cyclic nucleotide gated channels, is important for maximiz-
ing light sensitivity (Yau and Baylor, 1989). Likewise, the excess
magnitude of the Ca
2
conductance, synaptic vesicle number,
and synaptic vesicle release rate may be crucial for maximizing
the speed and reliability of signal transfer at the rod synapse.
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J. Neurosci., May 9, 2007 27(19):5033–5042 Sheng et al. Synaptic Vesicle Release and Ca
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    • "Other factors such as vesicle descent down the ribbon may contribute to slower kinetics of replenishment, but if vesicle delivery to the ribbon is the rate-limiting step in replenishment, then this suggests that the probability of a single vesicle attaching to the ribbon upon collision is likely to be significantly <1. Although we used vesicle density measurements from salamander cones (Sheng et al., 2007), vesicles appear less concentrated in mouse rod terminals, 580–750 v/µm 3 (Zampighi et al., 2011 ), implying there may be cell-tocell or species-to-species differences in the kinetics of vesicle resupply. We developed two variations of the model to test possible sites of Ca 2+ /CaM regulation of replenishment. "
    [Show abstract] [Hide abstract] ABSTRACT: At the first synapse in the vertebrate visual pathway, light-evoked changes in photoreceptor membrane potential alter the rate of glutamate release onto second-order retinal neurons. This process depends on the synaptic ribbon, a specialized structure found at various sensory synapses, to provide a supply of primed vesicles for release. Calcium (Ca(2+)) accelerates the replenishment of vesicles at cone ribbon synapses, but the mechanisms underlying this acceleration and its functional implications for vision are unknown. We studied vesicle replenishment using paired whole-cell recordings of cones and postsynaptic neurons in tiger salamander retinas and found that it involves two kinetic mechanisms, the faster of which was diminished by calmodulin (CaM) inhibitors. We developed an analytical model that can be applied to both conventional and ribbon synapses and showed that vesicle resupply is limited by a simple time constant, τ = 1/(Dρδs), where D is the vesicle diffusion coefficient, δ is the vesicle diameter, ρ is the vesicle density, and s is the probability of vesicle attachment. The combination of electrophysiological measurements, modeling, and total internal reflection fluorescence microscopy of single synaptic vesicles suggested that CaM speeds replenishment by enhancing vesicle attachment to the ribbon. Using electroretinogram and whole-cell recordings of light responses, we found that enhanced replenishment improves the ability of cone synapses to signal darkness after brief flashes of light and enhances the amplitude of responses to higher-frequency stimuli. By accelerating the resupply of vesicles to the ribbon, CaM extends the temporal range of synaptic transmission, allowing cones to transmit higher-frequency visual information to downstream neurons. Thus, the ability of the visual system to encode time-varying stimuli is shaped by the dynamics of vesicle replenishment at photoreceptor synaptic ribbons.
    Full-text · Article · Oct 2014
    • "Ca 2+ -flux through VGCC as well as the subsequent cross-talk to other Ca 2+ -regulating systems in the presynaptic terminal affect structural plasticity and developmental maturation of the synaptic ribbon in photoreceptor synapses. In the light, when Ca 2+ is low in presynaptic photoreceptor terminals (Thoreson et al., 2004; Choi et al., 2005 Choi et al., , 2008 Sheng et al., 2007; Jackman et al., 2009), synaptic ribbons appear to be smaller than in the dark (Adly et al., 1999; Balkema et al., 2001; SpiwoksBecker et al., 2004; Hull et al., 2006; Regus-Leidig et al., 2010a). Similarly, in knockout mouse models in which extrusion of Ca 2+ is disturbed and presynaptic Ca 2+ increased, synaptic ribbons were longer than in control animals (Yang et al., 2007; Aartsen et al., 2009). "
    [Show abstract] [Hide abstract] ABSTRACT: Changes in intracellular calcium ions [Ca(2+)] play important roles in photoreceptor signaling. Consequently, intracellular [Ca(2+)] levels need to be tightly controlled. In the light-sensitive outer segments (OS) of photoreceptors, Ca(2+) regulates the activity of retinal guanylate cyclases thus playing a central role in phototransduction and light-adaptation by restoring light-induced decreases in cGMP. In the synaptic terminals, changes of intracellular Ca(2+) trigger various aspects of neurotransmission. Photoreceptors employ tonically active ribbon synapses that encode light-induced, graded changes of membrane potential into modulation of continuous synaptic vesicle exocytosis. The active zones of ribbon synapses contain large electron-dense structures, synaptic ribbons, that are associated with large numbers of synaptic vesicles. Synaptic coding at ribbon synapses differs from synaptic coding at conventional (phasic) synapses. Recent studies revealed new insights how synaptic ribbons are involved in this process. This review focuses on the regulation of [Ca(2+)] in presynaptic photoreceptor terminals and on the function of a particular Ca(2+)-regulated protein, the neuronal calcium sensor protein GCAP2 (guanylate cyclase-activating protein-2) in the photoreceptor ribbon synapse. GCAP2, an EF-hand-containing protein plays multiple roles in the OS and in the photoreceptor synapse. In the OS, GCAP2 works as a Ca(2+)-sensor within a Ca(2+)-regulated feedback loop that adjusts cGMP levels. In the photoreceptor synapse, GCAP2 binds to RIBEYE, a component of synaptic ribbons, and mediates Ca(2+)-dependent plasticity at that site. Possible mechanisms are discussed.
    Full-text · Article · Feb 2014
    • "The exocytotic Ca 2 sensor in rods and cones shows an unusually high Ca 2 sensitivity (Rieke and Schwartz, 1996; Thoreson et al., 2004; Sheng et al., 2007; Duncan et al., 2010). In cones, [Ca 2 ] i at the base of the ribbon in darkness is therefore sufficient to stimulate release of a vesicle as soon as it is available for fusion (Jackman et al., 2009). "
    [Show abstract] [Hide abstract] ABSTRACT: Vesicle release from rod photoreceptors is regulated by Ca(2+) entry through L-type channels located near synaptic ribbons. We characterized sites and kinetics of vesicle release in salamander rods by using total internal reflection fluorescence microscopy to visualize fusion of individual synaptic vesicles. A small number of vesicles were loaded by brief incubation with FM1-43 or a dextran-conjugated, pH-sensitive form of rhodamine, pHrodo. Labeled organelles matched the diffraction-limited size of fluorescent microspheres and disappeared rapidly during stimulation. Consistent with fusion, depolarization-evoked vesicle disappearance paralleled electrophysiological release kinetics and was blocked by inhibiting Ca(2+) influx. Rods maintained tonic release at resting membrane potentials near those in darkness, causing depletion of membrane-associated vesicles unless Ca(2+) entry was inhibited. This depletion of release sites implies that sustained release may be rate limited by vesicle delivery. During depolarizing stimulation, newly appearing vesicles approached the membrane at ∼800 nm/s, where they paused for ∼60 ms before fusion. With fusion, vesicles advanced ∼18 nm closer to the membrane. Release events were concentrated near ribbons, but lengthy depolarization also triggered release from more distant non-ribbon sites. Consistent with greater contributions from non-ribbon sites during lengthier depolarization, damaging the ribbon by fluorophore-assisted laser inactivation (FALI) of Ribeye caused only weak inhibition of exocytotic capacitance increases evoked by 200-ms depolarizing test steps, whereas FALI more strongly inhibited capacitance increases evoked by 25 ms steps. Amplifying release by use of non-ribbon sites when rods are depolarized in darkness may improve detection of decrements in release when they hyperpolarize to light.
    Full-text · Article · Jan 2013
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